WO2018150260A1

WO2018150260A1 - Method for the identification of mutated proteins having modified thermal stability

Info

Publication number: WO2018150260A1
Application number: PCT/IB2018/000194
Authority: WO
Inventors: Nicolas REYES
Original assignee: Institut Pasteur
Priority date: 2017-02-16
Filing date: 2018-02-15
Publication date: 2018-08-23

Abstract

The invention relates to a method for the identification of mutated proteins having modified thermal stability and in particular having improved thermal stability. The amino acid sequence of the mutated protein results from the substitution of amino acid residues by consensus amino acid residues determined following MSA and selection of the consensus residue based on sequence comparison of homologues.

Description

METHOD FOR THE IDENTIFICATION OF MUTATED PROTEINS HAVING

MODIFIED THERMAL STABILITY

[1] The invention relates to a method for the identification of mutated proteins having modified thermal stability and in particular having improved thermal stability. The amino acid sequence of the mutated protein results from the substitution of amino acid residues by consensus amino acid residues determined following MSA and specific selection of each consensus residue based on sequence comparison of homologues.

[2] The identification of mutant amino acid sequences for a target protein using the process of the invention enables the production of mutant proteins having modified thermal stability, in particular improved thermal stability.

[3] Thermal stability of proteins is a key issue for their production and purification in cellular systems when said production and purification involves the use of detergent solutions. This challenge exists especially for membrane proteins, in particular for Integral Membrane Proteins such as transport proteins that may be active as drug targets or more generally in drugs pharmacokinetics.

[4] Methods are available in the prior art that improve protein stability, in particular methods that have been applied to Integral Membrane Proteins (IMP). These methods include alanine-Scanning (AS), Directed Evolution (DE) or Rational

Design (RD).

[5] AS is currently the most established method, and relies on screening the thermal stability of single alanine-mutants at each position in the target protein. This feature makes AS a labour-intensive and costly method, and difficult to apply to large IMP (>500 amino acids residues) or multi-protein complexes. Moreover, AS requires the use of radioactive compounds with high affinity (nano molar) for the target protein, which are not available for most membrane proteins.

[6] The DE method relies on a key selection assay for protein expression and stability that requires a small fluorescent compound with high affinity for the protein target, which are not available for most IMP. Moreover, DE in its current stage can only be done using bacterial expression systems, which have been found problematic for most human membrane proteins. [7] The RD approach relies on previous knowledge of atomic-resolution structures of the protein target, which are unknown for most IMP, and requires a great deal of expertise to design the mutagenesis.

[8] The process of the invention is based on the stabilizing effect of consensus mutagenesis, and provides major improvements on protein stability with minimal changes of the amino acid sequence compared to the target protein, and without affecting critical residues for function as illustrated for Integral Membrane Protein function.

[9] As a proof of concept, the inventor applied the method to a family of human neurotransmitter transporters, which are membrane proteins whose biochemical instability has previously precluded protein purification and in vitro studies, besides their importance as drug targets.

[10] Membrane proteins account for ~30% of all open reading frames in the human genome ¹⁸ They play essential roles in human physiology and pathology that range from synaptic transmission to viral infection ^{19 20}. Despite the paramount importance of human membrane proteins for life, their molecular mechanisms or function and pharmacology remain largely unknown. This is mainly due to the tremendous experimental challenges associated to their production, purification and analysis by high-resolution structural and functional approaches ^{21 -24} Clearly, new approaches and efforts are needed to understand the molecular mechanisms of this important class of proteins.

[11] The invention defines a process which is a solution to at least part of the drawbacks of the method disclosed in the art to improve thermal stability of proteins. Accordingly, mutant proteins were designed and obtained that showed improved thermal stability and that could be purified, and proved to be amenable for in vitro structural and functional studies, both of which have been performed successfully. In a particular embodiment, the invention accordingly provides a method to design mutated proteins that harbor improved thermal stability in detergents, wherein said proteins are membrane proteins, especially integral membrane proteins (IMP). The invention also relates to mutant proteins, in particular membrane proteins, especially IMP, designed and recovered accordingly.

[12] Importantly, the mutant protein, in particular mutant IMP, for example mutant transporter, retained the functional and pharmacological properties of the Wild Type human protein, in particular IMP, for example transporter, and shares at least 85% amino acid sequence identity with it, more preferably at least 90% amino acid sequence identity with it, and most preferably at least 95% amino acid sequence identity with it. Therefore, it constitutes an excellent structural and pharmacological model of protein, as illustrated for human protein.

[13] The examples provided therein show that it is the first time that a human IMP could be stabilized for in vitro studies by protein engineering with such a small change in its amino acid sequence and without the need for screening a wide variety of mutants. The method according to the invention requires the production of a reduced number of mutant proteins to be assessed for thermal stability property. Therefore, the method according to the invention is faster and requires far less mutant proteins production. The method herein defined accordingly provides a reliable tool for the production of functional proteins suitable for use in detergent solutions, without the need to produce a wide variety of constructs. Finally, ongoing experiments disclosed in the examples of the invention, show that the method can be successfully applied to other families of IMP, known for being very unstable proteins in detergent solutions, or even other proteins, eukaryotic or prokaryotic, in particular human or bacterial, with similar minimal changes of their amino acid sequence.

[14] Hence, the method that was developed according to the invention should contribute to better understanding of the molecular basis or function and pharmacology of proteins such as IMP, including those of human origin, and to develop efficient drugs that target them.

[15] Moreover, the method can also be applied to water-soluble proteins, in particular to those used in pharmaceutical and non-pharmaceutical industries.

[16] This new approach overcomes major problems associated to the existing methods:

I. It can be applied to any membrane protein or even membrane protein complexes,

II. it can be used with any protein expression system,

III. it does not require specific small compounds (radioactive or fluorescent) with high affinity for the target protein, IV. it relies only on knowledge of the amino acid sequence of the target and not of its three-dimensional structure,

V. it does not require special training skills, and

VI. it is both labour- and cost-effective.

[17] Proteins thermostable in detergent solutions may be useful in various applications, in particular structural analysis and biological analysis. Structural and biological analysis of proteins that requires proteins to be stable in a detergent solution encompass methods such as crystallography (i.e. X-ray crystallography), electron microscopy (i.e. cryo-electron microscopy), protein NMR, X-ray scattering (Small-angle X-ray scattering), circular dichroism, and the like. Methods for the biological analysis or pharmacological analysis encompass ligand-binding screening, affinity/kinetic screening, functional analysis of the proteins, drug targets screening, antagonist/agonist screening, and the like. As an example, production of proteins in detergents for crystallization requires a well- ordered crystal. Disposing stable proteins in detergents remains both challenging and necessary in order to have proteins suitable for study of their properties, in particular of their structural or pharmacological properties.

[18] The invention accordingly relates to a method of designing a protein with modified thermal stability in a detergent solution with respect to the thermal stability of a target protein comprising: a. providing a multiple sequence alignment (MSA) wherein at least a portion comprising at least 50 % of the full-length amino acid sequence of a target protein is aligned, in particular the full-length amino acid sequence of the target protein is aligned, with the amino acid sequence of homologous sequences of said protein present (i) in other species and/or (ii) in protein variants of the same species, using a determined alignment tool; wherein the homologous sequences are each different from the amino acid sequence of the target protein by at least one amino acid residue in their amino acid sequence, in particular wherein each homologous sequence shares at least 50% identity, more preferably at least 70% identity, with the amino acid sequence of the target protein or with the aligned portion thereof; b. defining for at least part of the positions of the amino acid residues in the target protein, in particular for each amino acid position of the aligned amino acid sequences, a consensus amino acid residue wherein the consensus amino acid residue has the following characteristics:

i. a frequency of occurrence among the aligned amino acid sequences which is higher than 20%,

ii. a position in the alignment of the amino acid sequences wherein gaps constitute less than 30% of the entries,

iii. a frequency of occurrence which is higher than 10% with respect to the frequency of occurrence of the residue at the same position in the target amino acid sequence when said frequency is evaluated among the aligned amino acid sequences;

c. providing a mutated amino acid sequence defining a consensus mutant protein of the target sequence by substituting one or more amino acid residues in the amino acid sequence of the target protein by its(their) respective consensus amino acid residue(s) defined for said each amino acid residue according to step b.

In a particular embodiment of the invention, the target protein is a membrane protein, in particular an integral membrane protein.

[19] The invention also relates to a process to produce a protein with modified thermal stability in a detergent solution with respect to the thermal stability of a target protein comprising: a. providing a multiple sequence alignment (MSA) wherein at least a portion comprising at least 50 % of the full-length amino acid sequence of a target protein is aligned, in particular the full-length amino acid sequence of the target protein is aligned, with the amino acid sequence of homologous sequences of said protein present (i) in other species and/or (ii) in protein variants of the same species, using a determined alignment tool; wherein the homologous sequences are each different from the amino acid sequence of the target protein by at least one amino acid residue in their amino acid sequence, in particular wherein each homologous sequence shares at least 50% identity, more preferably at least 70% identity, with the amino acid sequence of the target protein or with the aligned portion thereof;

b. defining for at least part of the positions of the amino acid residues in the target protein, in particular for each amino acid position of the aligned amino acid sequences, a consensus amino acid residue wherein the consensus amino acid residue has the following characteristics:

ii. a position in the alignment of the amino acid sequences wherein gaps constitute less than 30% of the entries, iii. a frequency of occurrence which is higher than 10% with respect to the frequency of occurrence of the residue at the same position in the target amino acid sequence when said frequency is evaluated among the aligned amino acid sequences;

c. providing a mutated amino acid sequence defining a consensus mutant protein of the target sequence by substituting one or more amino acid residues in the amino acid sequence of the target protein by its(their) respective consensus amino acid residue(s) defined for said each amino acid residue according to step b.;

d. obtaining a polynucleotide encoding the mutant protein of step c. for expression in a cellular expression system;

e. producing the mutant protein from the polynucleotide encoding the mutated amino acid sequence provided in step d. in a cellular expression system;

f. evaluating thermal stability in a detergent solution of the mutant protein recovered from the production cells in step e.

A portion of an amino acid sequence corresponds to at least 50% of the full-length amino acid sequence of the target protein, more preferably at least 75% of the full length amino acid sequence, more preferably at least 90% of the full-length amino acid sequence. A portion of amino acid sequence may also correspond to a plurality of non-contiguous portions of the full-length amino acid sequence, said plurality representing at least 50% of the full-length amino acid sequence. [20] Thermal stability in detergent solution is a parameter that is essential for the production of thermostable variants of proteins as it enables their purification in detergent solutions while preserving the biological function(s) of the wild type (or target) protein. It also enables production of variants of proteins (by oriented mutations) that are stable for biophysical analysis or structural analysis, including for the determination of their three-dimensional structure (by crystallization or electron microscopy for example) and for the determination of their biological function (ligand-binding assay for example). Thermal stability is accordingly the property of the target protein that is primarily altered when performing the process of the invention, in particular that is increased when providing or selecting a mutated sequence of the target protein. Thermal stability may be measured as disclosed hereafter in a denaturation assay assessing resistance to denaturation of the produced mutant protein. The invention thus provides a process for the identification and for the preparation of thermostable mutant proteins and in particular provides a thermostable variant of a target protein.

[21] In a particular embodiment other properties may additionally be modified in relation or independently of thermal stability of the target protein in accordance with the process of the invention. Such properties include the capability of the protein to efficiently keep their three-dimensional structure in detergent solutions and therefore keep their function. As an example, production crystal that are well- ordered may involve determination of the capability of said crystal to diffract X- rays to high resolution and comparison to a control. It has been shown indeed that there is a measurable correlation between the stability of a protein, in particular a membrane protein in a detergent and the capability of proper crystallization.

[22] The target protein for use in the invention is any protein present in any species.

In particular it is a eukaryotic protein and in particular belongs to animal, such as to vertebrates, especially to human, or it is a prokaryotic protein and belong to virus, bacteria, parasites species, in particular to a pathogenic species. According to a particular embodiment of the invention, the target protein is membrane protein of such organism, especially an IMP and in particular is a viral envelope protein, or a virus membrane receptor on a cell, especially a human cell. According to a particular embodiment, the target protein is a wild-type protein. According to another embodiment the target protein is a mutant, in particular a naturally occurring mutant of a wild-type protein, in particular a mutant selected or designed to alter properties with respect to the wild-type protein, possibly in respect of thermal stability or crystallization capability.

Accordingly, the target protein may have any length determined in number of amino acids residues. Advantageously, the protein has at least 300 amino acid residues, in particular at least 400 or 500 amino acid residues, but the method can also be performed with a target protein having less than 300 amino acid residues. In a particular embodiment of the invention, the protein has at least 1300 amino acid residues. Advantageously, the protein comprises between 300 amino acid residues and 1500 amino acid residues, and more advantageously, the protein comprises between 350 and 1400 amino acid residues. It should be noted that the methods of the prior art are increasingly cumbersome with the length of the amino acid sequence of the target protein. The method disclosed therein allows the efficient production of thermal stable proteins, irrespective of their number of amino acid residues.

For the purpose of the invention, the target protein is one the amino acid sequence of which is known and especially disclosed in databases such as GenBank. Similarly, the amino acid sequences of the homologous sequences of the target protein are known and in particular disclosed in databases such as GenBank.

In a particular embodiment, the amino acid sequences used for the performance of the alignment do not include a defined consensus sequence i.e., an artificial archetypal sequence defined using sequence data obtained from various known natural products but are rather representatives of isolated proteins. According to this embodiment, the target protein and the homologous proteins are wild-type sequences.

In another particular embodiment, especially the multiple sequence alignment (MSA) follows a first round or mutations in the amino acid sequence of the target protein, such as function-improving mutation(s) and/or structure-improving mutation(s). In such a case, the amino acid sequence of the target protein may be an artificial sequence, i.e., a sequence that has been mutated with respect to a naturally occurring protein in order to modify properties of the target protein. In another embodiment of the process of the invention, step c. of providing a mutated amino acid sequence defining a mutant protein of the target is followed by a further step of mutation in the sequence in order to alter other properties of the target protein.

[28] The alignment performed according to the process of the invention is a (MSA) which as such is well known to the person skilled in the art. It may be performed through available tools including a suitable algorithm (such as Clustal W) and software, especially using an alignment software. Many software suitable to perform alignments are available such as JALVIEW software or others listed on https://en.wikipedia.org/wiki/List_of_sequence_alignment_software. In a particular embodiment of the invention, the determined alignment tool used for the MSA is JALVIEW software. https://en.wikipedia.org/wiki/List_of_sequence_alignment_software

Multiple sequence alignment

*Sequence type: protein or nucleotide. **Alignment type: local or global

[29] Homologous sequences of the target protein may be found in species different from the species providing the target sequence. They may additionally encompass or alternatively consist in variants of the target sequence available in the same species. In particular, when the target protein is a human protein, the homologous sequences may originate from other vertebrate species, in particular from other mammal species. They may also encompass variants of the target sequences disclosed in human or in other vertebrate species. A homologous sequence may also be defined as a variant of the target protein having at least 50% identity with the target protein, in particular at least 70% identity, whether wild-type or mutated as disclosed above.

It is preferable but not necessary that each selected homologous sequence for the MSA has a difference with the target sequence such that both sequences share less than 95% sequence identity when aligned with the MSA tool.

Although there is no absolute requirement regarding the number of homologous sequences used in order to perform the MSA, having at least 7 and preferably more than 10 sequences in the alignment or more than 13 or more than 20 is advantageous for the performance of the process of the invention.

In step b. of the process the consensus amino acid residue may be defined for each amino acid position in the amino acid sequence of the target protein (also designated target sequence).

Alternatively the consensus amino acid residue may be defined for at least part of the amino acid residues in the target sequence and in particular for at least 25% or at least 50% or at least 75% or at least 90% of the amino acid positions in the target sequence.

In a particular embodiment of the process, the amino acid residues targeted for substitution by the consensus amino acid residues are contained in secondary structural elements of the target protein, such as in predicted or known (characterized) a-helices or in β-turns. Alternatively or in addition, the amino acid residues targeted for substitution by the consensus amino acid residues are contained in loops, in particular in unstructured loops connecting the other structured regions. In a particular embodiment such secondary structural elements and/or loops are involved in the alignment step performed on portion(s) of the amino acid sequence of the target protein.

When the consensus amino acid residue has been defined for the determined position of the target amino acid sequence, in accordance with steps i.to iii. of the process, a mutated amino acid sequence may be defined that results from the substitution of all amino acid residue(s) of the target sequence for which a consensus residue has been identified. The thus obtained amino acid sequence defines a mutant protein of the target protein. In a particular embodiment of the process the mutated protein has a sequence identity with the target protein which is at least 80%, more preferably at least 85%, more preferably at least 90%, most preferably at least 95%, in order to minimize the effects of the mutations on the properties of the target protein. Identity means to the extent to which two amino acid sequences have the same residue at the same position in an alignment. Identity could be defined as the number of exact matches in the alignment of two sequences (the target protein and a mutated protein), divided by the length of the target protein or the length of the aligned portion of the target protein. An exact match occurs when the target protein and the mutated protein have identical amino acid residues in the same position.

[36] From the identified mutant amino acid sequence, a polynucleotide can be designed, especially by mutation of the polynucleotide encoding the target protein, for the expression of the mutant protein in a cellular expression system.

The polynucleotide encoding the mutant protein having the mutated amino acid sequence may further be mutated for codon optimization in accordance with methods well known from the person skilled in the art for its expression in the selected cellular expression system. This polynucleotide may further be modified to add a sequence that encodes a purification handle at the terminus of the amino acid sequence of the mutant protein.

[37] The invention thus relates to the mutated protein which is produced and whose amino acid sequence consists in the consensus mutated sequence of the target protein.

[38] The invention also relates to the polynucleotide encoding the consensus mutated protein and to cells transformed with such polynucleotides in conditions enabling the expression of the polynucleotide as a mutated protein with respect to the target protein.

[39] According to another aspect of the invention, the process may additionally comprise, after step c. a further step (c(i)) of determination according to an additional parameter of the amino acid residues in the target sequence that may be mutated. In accordance with this embodiment step c(i) is a step of calculating pairs of residues that co-evolve in the target protein and substituting these pairs of residues in the target sequence.

[40] Step c(i) may be carried out using co-evolution software suitable for the determination of amino acid pairs that co-evolve such as

[41] EVcouplings software (website: http://evfold.org/evfold- web/evfold.do; reference: https://www.ncbi.nlm.nih.gov/pubmed/22579045) or others such as [42] GREMLIN (website: http://gremlin.bakerlab.org/index.php) (reference: https://www.ncbi. nlm.nih.gov/pubmed/?term=Assessing+the+utilitv+of+coevoluti on-based+residue-residue+contact+predictions+in+a+seguence- +and+structure-rich+era):

DCA (website: http://dca.rice.edu/portal/dca/)

(reference: https://www.ncbi. nlm.nih. gov/pubmed/?term=Direct- coupling+analvsis+of+residue+coevolution+captures+native+contacts+across+ manv+protein+families):

PSICOV

website: http://bioinfadm in. cs. ucl. ac. uk/downloads/PS ICON//) (reference: https:/ /www.ncbi.nlm.nih.gov/pubmed/?term=PSICOV%3A+precise+structural+contac t+prediction+using+sparse+inverse+covariance+estimation+on+large+multiple+ seguence+alignments)

[43] The additional but optional c(i) step has particular interest in situations where at the end of step c the mutant protein obtained has 80% or less sequence identity with the target sequence. The co-evolution determination brings the advantage of pointing to amino acid residues that co-evolved in the 3-dimensional structure of the protein rather than in its amino acid sequence. In a particular embodiment of the invention, this step c(i) may be performed when the number of homologous sequences available for the MSA is at least 5 times the number of amino acid residues of the target sequence. Because of that the homologous sequences may have to be found in species which are remote from the species of the target sequence, such as in prokaryotic homologues when the target protein is found in a eukaryotic species such as in human, and/or in divergent branches of the family protein of the target protein. When step c(i) is performed the identity of the mutations to be introduced in the target sequence is still based on step c. but the additional step ci enables to lessen the number of residues to be mutated. The obtained mutant sequence may thus be in the range of sequence identity of at least 90%, preferably at least 95% with the target sequence.

[44] Accordingly a variant (i.e. consensus mutant) protein obtained after step c and optionally c(i) may be prepared or produced in a cellular expression system and recovered for testing, in particular for thermal stability testing in detergent solution. [45] In a particular embodiment of the process of the invention, the target protein is a membrane protein, in particular a eukaryotic, such as a mammalian, especially a human, membrane protein. In a particular embodiment, the membrane protein is an integral membrane protein.

[46] In a particular embodiment of the process, the membrane protein is selected among channels, enzymes, and primary or secondary active transporters. Transporters may in particular be of the SLC1 transporters family which are ion- coupled amino acid transporters, and may comprise divergent branche(s) of this family, like sodium-dependent neutral amino acid transporters (ASCT1 -2).

[47] In another particular embodiment of the process of the invention, the target protein is a soluble protein, especially a globular soluble protein.

[48] The study of thermal stability of the mutant protein aims at determining whether mutations have improved, i.e., increased thermal stability in detergent solution. According to the invention, a produced, especially crystallized mutant protein having improved thermal stability shows resistance to denaturation when the melting temperature is raised for at least 2°C, advantageously for at least 3 to 10°C and in a particular embodiment up to 20°C with respect to the melting temperature of the target protein.

[49] The detergent solution is one known from the person skilled in the art in biochemistry, in particular one suitable for use in production, purification or analysis, especially by high-resolution structural and functional approaches. Detergent solutions may in particular be those used for preparation of protein for structure determination or biological function analysis; for example detergent used for X-ray structure or crystallization. For illustration such detergent solutions encompass small micelle detergents, dodecyl-p-D-maltopyranoside (12M), n- dodecyl-N,N-dimethylamine-N-oxide (LDAO), n-nonyl-p-D-maltopyranoside (9M), nonyl-p-D-glucopyranoside (NG), n-decyl-p-D-maltopyranoside. Detergent solutions may in particular be those used for preparation of protein for biophysical analysis and functional analysis using fluorescence spectroscopy (dodecyl-p-D- maltopyranoside, dodecyl-p-D-glucopyranoside, 7-Cyclohexyl-1 -Heptyl-p-D-

Maltoside), as well methods for determination of high-resolution three- dimensional structures in particular X-ray crystallography and single-particle electron microscopy (dodecyl- decyl-, nonyl-, or octyl-p-D-maltopyranoside; dodecyl-, decyl-, nonyl-, or octyl-p-D-glucopyranoside; Neopentyl glycol based detergents like 2,2-dioctylpropane-1 ,3-bis-p-D-glucopyranoside).

[50] Thermal stability of proteins may in general be characterized by the difference between the folded and unfolded states of the protein measured in kcalmol^"1. Measurement of stability may encompass thermal denaturation of the protein in a detergent solution and measurement by the increase of the apparent melting temperature of the mutant protein which is then compared to the melting temperature of the target protein by chromatographic observation, in particular by size-exclusion chromatography (SEC-TS).

[51] According to SEC-TS, protein stability may be determined with or without ligand.

A sample of the purified protein is heated and then cooled and centrifuged to remove aggregated proteins and run on analytical HPLC. When the melting temperature is reached and proteins precipitates or aggregates, pic height decreases and void pic height increases.

[52] In a particular embodiment of the process when expressed in a cellular system, the mutant protein is fused to a detectable marker, such as a fluorescent marker, for the evaluation of the melting temperature of the mutant protein.

[53] The expression of the mutant protein may be performed in any available cellular system. When the protein is a eukaryotic protein, it is advantageously produced in a eukaryotic cell.

[54] The process of the invention enables selecting and producing proteins having improved thermal stability with respect to the target protein.

[55] Accordingly, the invention also relates to a process for modifying, in particular for improving thermal stability of a protein comprising carrying out consensus mutagenesis in the amino acid sequence of a target protein using the process defined herein in accordance to its embodiments and recovering the mutant protein having a modified, in particular improved thermal stability.

[56] According to another aspect, the invention relates to a process to identify an amino acid sequence which is mutated with respect to the amino acid sequence of a target protein said process comprising the steps of: a. providing a multiple sequence alignment (MSA) wherein at least a portion comprising at least 50% of the full-length amino acid sequence of a target protein is aligned, in particular wherein the full- length amino acid sequence of a target protein is aligned, with the amino acid sequence of homologous sequences of said protein present (i) in other species and/or (ii) in protein variants of the same species, using a determined alignment tool; wherein the homologous sequences are each different from the amino acid sequence of the target protein by at least one amino acid residue in their amino acid sequence, in particular wherein each homologous sequence shares at least 50% identity, more preferably at least 70% identity, with the amino acid sequence of the target protein or with the aligned portion thereof,

c. providing a mutated amino acid sequence defining a mutant protein of the target sequence by substituting one or more amino acid residues in the amino acid sequence of the target protein by its(their) respective consensus amino acid residue(s) defined for said each amino acid residue according to step b.

[57] The invention also relates to a process for the preparation of a protein with modified crystallization capability comprising preparing a protein having modified thermal stability in a detergent solution according to any of the embodiments of the process of the invention. An optional step of further mutation may be performed after step c. and in particular before step d. of the process to prepare the mutant protein to further improve crystallization capability, such optional step of further mutation being in particular a step of mutation in the extracellular region of the target protein.

[58] In a particular embodiment of the invention, the process is performed, wherein the target protein is the Excitatory Amino Acid Transporter 1 (EAAT1 ) and the obtained mutant protein is EAAT1 of sequence SEQ ID No.1 SEQ ID No.2, SEQ ID No.3 or SEQ ID No.4. When the target protein is human EAAT1 , the homologous sequences aligned within the MSA may comprise at least one sequence selected from the group comprising SEQ ID No: 5, SEQ ID No: 6, SEQ ID No: 7, SEQ ID No: 8, SEQ ID No: 9, SEQ ID No: 10 and SEQ ID No: 1 1 . In a particular embodiment of the invention, when the target protein is human EAAT1 , the homologous sequences alignment within the MSA comprise at least the following sequences: SEQ ID No: 5, SEQ ID No: 6, SEQ ID No: 7, SEQ ID No: 8, SEQ ID No: 9, SEQ ID No: 10 and SEQ ID No: 1 1 .

[59] The invention thus also concerns a mutant protein of Excitatory Amino Acid Transporter 1 (EAAT1 ) the amino acid sequence of which is SEQ ID No.1 SEQ ID No.2, SEQ ID No.3 or SEQ ID No.4.

[60] In a particular embodiment of the invention, the process is performed, wherein the target protein is NPC1 L1 (Niemann-Pick C1 -Like 1 ), in particular human NPC1 L1 , in particular NPC1 L1 of Accession number Q9UHC9.2 (SEQ ID No: 12). Various isoforms of human NPC1 L1 , or ortholog proteins may be used in step a. of the method as homologous sequences, for example Niemann-Pick C1 - like protein 1 from Canis lupus familiaris (Accession number NP_001091019.1 - SEQ ID No: 14), from Bos Taurus (Accession number XP_588051 .4 - SEQ ID No: 15), from Loxodonta Africana (Accession number XP_003418622.1 - SEQ ID No: 16), from Mus musculus (Accession number Q6T3U4.1 - SEQ ID No: 17), from Monodelphis domestica (Accession number XP_001379744.2 - SEQ ID No: 18), from Dasypus novemcinctus (Accession number XP_004467651 .1 - SEQ ID No: 19), from Xenopus tropicalis (Accession number XP_002932620.2 - SEQ ID No: 20), from Oryzias latipes (Accession number XP_004072748.1 - SEQ 21 ), from Danio rerio (Accession Number XP_009302449.1 - SEQ ID No: 22), from Ornithorhynchus anatinus (Accession number XP_007662878.1 - SEQ ID No:

23) , from Python bivittatus (Accession number XP_007440049.1 - SEQ ID No:

24) , and from Aquila chrysaetos canadensis (Accession number XP_01 1573737.1 - SEQ ID No: 25). At least one of the above cited sequence may be aligned within the MSA when the target protein is human NPC1 L1 . In a particular embodiment of the invention, SEQ ID No: 12 to SEQ ID No: 25 may be aligned within the MSA when the target protein is human NPC1 L1 .

[61] In a particular embodiment of the invention, the process is performed, wherein the target protein is the Niemann-Pick C1 -like1 (NPC1 L1 ) and the obtained mutant protein is NPCL1 of sequence SEQ ID No.13.

[62] The invention thus also concerns a mutant protein of Niemann-Pick C1 -like1

(NPC1 L1 ) the amino acid sequence of which is SEQ ID No.13.

[63] According to another aspect, the invention relates to a liposome or a collection of liposomes containing a mutant protein obtained from carrying out the process according to the invention.

[64] The invention also relates to a cell expressing a mutant protein obtained from the process of the invention.

[65] According to a particular embodiment, the invention relates to a cell or a liposome comprising a mutant protein of Excitatory Amino Acid Transporter 1 (EAAT1 ) the amino acid sequence of which is SEQ ID No.1 SEQ ID No.2, SEQ ID No.3 or

SEQ ID No.4.

[66] According to a particular embodiment, the invention relates to a cell or a liposome comprising a mutant protein of Niemann-Pick C1 -Iike1 (NPC1 L1 ) the amino acid sequence of which is SEQ ID No.13.

[67] Each and every embodiment defined in respect to the process to produce a protein with modified thermal stability in a detergent solution according to the invention applies to the process to modify the thermal stability of a target protein and to the process to identify an amino acid sequence which is mutated with respect to the sequence of a target protein.

[68] Other features and advantages of the invention will be apparent from the Examples below and from the figures.

[69] The process of the invention may be followed by additional steps of mutation in the mutant protein such as mutations that enable the improvement of purification or crystallization of the protein.

[70] Table 1 . Data collection and refinement statistics. EAAT1 cryst EAAT1 cryst-l EAAT1 cryst-l EAAT1 cryst

UCPH10I UCPH10I UCPH101 and

TBOATFB

Bound bound

bound

Data collection*

Space group P6₃ P63 P63 P63

Cell dimensions

a=b, c (A) 123.27, 89.87 123.1 1 , 89.62 123.32, 89.57 124.33, 90.8

Alpha=beta(°) 90.0, 120.0 90.0, 120.0 90.0, 120.0 90.0, 120.0

Wavelength 0.979 0.976 1.009 0.977

Resolution (A) 45.89-3.25 45.82-3.1 45.87-3.32 46.31 -3.71

(3.34-3.25) (3.18-3.1) (3.41 -3.32) (3.81 -3.71)

Anisotropy

direction⁸

Resolution where

Overall (A) 3.37 3.1 3.32 3.71 along h, k axis (A) 3.75 3.68 3.85 4.35 along I axis (A) 3.25 3.1 3.32 3.71

Measured 333978 290672 326273 141904 reflections (24261) (21906) (21829) (1 1 176)

Unique reflections 12338 (902) 141 15 (1 032) 11556 (834) 8570 (628)

Completeness (%) 100 (100) 99.9 (99.9) 100 (100) 99.9 (100.0)

Mn (I) half-set 0.99 (0.22) 1 (0.43) 0.99 (0.31) 0.99 (0.373) correlation

l/ (l) 1 1.7 (0.7) 15.8 (0.8) 13 (0.7) 12.1 (0.9)

Emerge 0.20 (6.90) 0.1 (6.71) 0.15 (8.16) 0.14 (3.71)

Redundancy 27.1 (26.9) 20.6 (21.2) 28.2 (26.2) 16.3 (14.3)

Structure

determination

Refinement

Resolution cut-off 45.89-3.25 45.80-3.10 20.00-3.32 25.00-3.71

(A)

No. of Work / Test 9891/475 10725/528 9251/445 6860/684 reflections ftcryst (%) / ftfree (%) 21.9/24.1 21.7/25.9 20.9/25.3 22.7/25.4

No. of protein 3002 2960 2995 3008

atoms

No. of 42 42 10 62

heteroatoms

B factors (A)²

Protein 129.5 1 11.8 137.0 135.5

Heteroatoms 107.3 99.5 125.6 132.7

R.m.s. deviations

from ideal

Bond lengths 0.009 0.01 0.009 0.009

(A)

Bond angles (°) 1.06 1.12 1.05 1.03

One crystal was used to collect diffraction datasets for each structure, except in the EAATl

UCPHioi and TFB-TBOA bound structure, where datasets from three crystals were merged.

5% of reflections were used for calculation of /?f_ree.

*Values in parentheses are for the highest-resolution shell.

$The anisotropy directions where computed with AIMLESS.

[71 ] Legend of the Figures

Some of the figures, to which the present application refers, are in color. The application as filed contains the color print-out of the figures, which can therefore be accessed by inspection of the file of the application at the patent office.

Figure 1 . Size exclusion chromatograms of detergent-solubilized GFP-fusions of EAATI WT and its thermostable variants at different temperatures from clear lysates of HEK293 cells (upper plots). The apparent melting temperature (T_m) was estimated from the GFP-fluorescence value of the peak corresponding to the trimeric protein (lower plots).

Figure 2. Uptake of radioactive L-glutamate by purified ΕΑΑΤ1 τι (left), and ΕΑΑΤ1 τ2 (right) reconstituted in liposomes. Purified EAATI WT did not show significant levels of uptake above the control liposomes without reconstituted transporters (see Fig. 3a). Figure 3. function and architecture of EAAT1 _cryst. a-b, Uptake of radioactive L- glutamate by purified EAAT1 , EAATl cryst (cryst), and EAAT1 _Cryst-n (cryst-ll) reconstituted in liposomes. Transport was abolished when choline (Ch⁺) was used as the main cation in the extra- or intra-liposomal solutions (circles) (a). UCPH101 inhibits glutamate transport EAATl cryst (lower plot), but not in EAAT1 _Cryst-ii (upper plot) in a concentration dependent manner (b). Plots depict an average of three independent experiments performed with duplicate measurements, and error bars represent s.e.m. c-d, Structure of EAATI cryst trimer viewed from the extracellular solution (c) and from the membrane (d), with the ScaD and TranD as surfaces, and U PCH101 bound between them (spheres), e, EAATI cryst monomer viewed parallel to the membrane. The ScaD domain is represented as surface, and several helices and loops in the TranD have been removed for clarity of display, f, Domain organization diagram of EAATI cryst monomer.

Figure 4. Alignment of human SLC1 transporters. Amino acid sequences of EAAT1 -5, ASCT1 -2 and EAATI cryst are compared. The boundaries of the a-helices (cylinders) in the TranD and the ScaD seen in the EAATI cryst structure are shown. In order to confer crystallizability, the region between TM3 and TM4c (arrows) from ASCT2 was transferred to a thermally stabilized EAAT1 . To further improve crystal formation in the absence of U PCH101 , mutations M231 1 and F235I (circles) were introduced to generate EAAT1 cryst-n. These substitutions are found in EAAT2. Other residues involved in U PCH101 coordination are more conserved (triangles). Sequences were aligned with Jalview.

Figure 5. UCPH101 binding site, a, Lateral view of EAATI cryst monomer from the membrane showing UCPH101 bound between the TranD and ScaD. b-c, UCP H101 coordination and Fo-Fc densities contoured at 2.0o in EAATI cryst (b) and EAAT1 _Cryst-ii (c), respectively. Side chains of residues in TM3, TM4c, and TM7 involved in coordination are shown. F369 side chain moves outward in the EAAT1 _Cryst-ii unbound state (c).

Figure 6. UCPHI OI -TBOATFB bound EAATI cryst structure, a, The TranD and ScaD of the EAATI cryst monomer are represented in light grey and grey, respectively. The movement of HP2 partly exposes the substrate-binding pocket to the solvent and shows a molecule of TBOATFB bound to it. UCP H101 is also observed in this structure. b, The tip of HP2 moves as much as 9.5 A in the UCPHI OI -TBOATFB-, compared to the

UCPHioi-substrate bound structures, moving the carbonyl oxygen of A420 away from Na2 (black sphere), c, Omit map Fo-Fc⁽⁶⁸⁾ density for the TBOATFB molecule is contoured at 2.3o (black mesh), and some of the residues at Van deer Waals or H- bond distance from the compound are represented as sticks.

Figure 7. EAAT1 consensus mutants, a-b, Residues exchanged for consensus amino acids in ΕΑΑΤ1 τι (a) and ΕΑΑΤ1 τ2 (b) are mapped into the structure of the EAATI CRYST (PDB 5LLM) trimer viewed from the extracellular medium (left panel), as well as the scaffold (cyan) and the transport (orange) domains viewed from the membrane. These domains are depicted separately for clarity of display, including two views of the transport domain separated ~180° from each other, showing its interface with the scaffold domain (left) and the membrane (right), respectively. Spheres correspond to the alpha carbon atoms of residues that were exchanged by conservative (grey) and non-conservative (black) consensus mutations, c-d, Radioactive L-glutamate uptake in cells expressing transporters (c), including control cells transfected with a vector lacking EAAT1 genes, and in liposomes with purified reconstituted transporters (d). Yellow circles depict the liposomal bilayer separating sodium- (Na⁺), potassium- (K⁺), and choline-based (Ch⁺) solutions, e, Rate of L-glutamate uptake by purified ΕΑΑΤ1 τι (blue) and ΕΑΑΤ1τ2 (red) reconstituted in liposomes, as a function of L-glutamate concentration. Solid lines indicate Michael is-Menten fits to the data with K_m values 30.7 and 18.8 μΜ, and Vmax values 18.3 and 1 2.8 pmol Mg^"1 min^"1 for ΕΑΑΤ1 τι (blue) and ΕΑΑΤ1 τ2 (red), respectively.

Figure 8. Deuterium exchange at 20 °C. a-c, Deuterium uptake kinetics at 20 °C of examples peptides covering both helical and non-helical regions of EAATI WT (grey), ΕΑΑΤ1 -Π (blue), and ΕΑΑΤ1 τ2 (red), respectively. Solid lines represent double- exponential fits to the data, and dotted lines the expected deuterium kinetics of unfolded and solvent exposed peptides, d-f Deuterium incorporation after 1 h at 20 °C in EAAT1 TI (d), EAAT1 Τ2 (e), and EAATI WT (f) mapped into the structure of the EAAT1 cRYTs (PDB 5LLM) trimer viewed from the extracellular medium (upper panel), as well as the scaffold (ScaD) and the transport (TranD) domains viewed from the membrane (lower panel), respectively. These domains are depicted separately for clarity of display. In the trimeric depiction, arrows point to the interface between protomers. Deuterium incorporation was calculated as an average of three experiments, and normalized to the maximal theoretical incorporation based on the number of backbone amide available for exchange in each peptide. A scale bar representing deuterium incorporation is depicted (d).

Figure 9. Melting of trimeric transporters, a-c, Size-exclusion chromatograms of purified ΕΑΑΤ1 τι (a), ΕΑΑΤτ2 (b), and the chimeric transporter EAAT1 -ScaD-n-TranDT2 (c), respectively, pre-heated at different temperatures. Chromatograms show how the trimeric form of the transporters that elutes at ~3.0 ml melts into lower oligomeric state(s), most likely monomers, that elute at ~ 3.5 ml. d-f Melting curves depicting the change in fractional area of the chromatographic peak corresponding to the trimeric transporters (black symbols), as a function of the temperature pre-pulse in ΕΑΑΤ1τι (d), ΕΑΑΪΤ2 (e), and EAAT1 -ScaD-n-TranDT2 (f), respectively. Solid lines indicate fits of a Hill-like equation (see methods) to the data with TSO-FSEC values 49.3, 38.6 and 45.7 °C, and n values -21.5, -9.3, and -18.3 for EAAT1 _T1 , EAAT1 _T2, and EAAT1 - ScaD-n-TranDT2, respectively. The total area under the chromatogram at each temperature, normalized to that at 4 °C, is also shown (empty symbols), and remains relatively constant at all temperatures.

Figure 10. Bimodal m/z envelopes, a-b, m/z envelopes of an example peptide covering residues 174-184 of ΕΑΑΤ1 τι (a), ΕΑΑΤτ2 (b), at different pre-pulse temperatures. Solid symbols represent the average of three experiments and error bars represent s.e.m, and are superimposed on the m/z spectrum of a representative experiment. Solid lines represent fits of a double Gaussian equation to the data, as well as their low- and high-m/z components, c-d, melting plots of the low-m/z component of peptides covering residues 174-184 (c) and 187-194 (d) of ΕΑΑΤ1 τι (solid circles), ΕΑΑΪΤ2 (solid triangles). Solid lines indicate fits of a Hill-like equation to the data with T50-HDX-GAUSS values °C, and n values for peptide 174-184 in ΕΑΑΤ1 τι and ΕΑΑΤ1 τ2, respectively, and TSO-HDX-GAUSS values °C, and n values for peptide 187-194 in ΕΑΑΤ1τι and EAAT1 T2, respectively.

Figure 1 1 . Local thermal unfolding, a-b, Deuterium uptake kinetics at different pre- pulse temperatures of an example peptides containing residues 174-184 in ΕΑΑΤ1 τι (a), and ΕΑΑΤ1 τ2 (b), respectively. Solid lines represent double-exponential fits to the data, and dotted lines the expected deuterium kinetics of the unfolded and solvent exposed peptide. Plots in a-b depict an average of three experiments, and error bars represent s.e.m. The inset represents the temperature protocol used to prepare the protein samples, c-d Apparent melting temperatures of the peptides folded state in EAAT1 TI (C), and EAAT1 _T2 (d) are mapped into the structure of the EAATI CRYST (PDB 5LLM) trimer viewed from the extracellular medium (upper panel), as well as the scaffold (ScaD) and the transport (TranD) domains viewed from the membrane (lower panel), respectively. These domains are depicted separately for clarity of display. The color code representing the apparent melting temperature values is depicted in a scale bar.

Figure 12. Size exclusion chromatograms of detergent-solubilized GFP-fusions of NPC1 L1WT (front, row 1 ) and NPC1 L1 _T1 (back, row 2) from lysates of HEK293 cells. Figure 13. Alignment of ΕΑΑΤ1 τι , ΕΑΑΤ1 τ2 and EAATI WT. Amino acid residues of EAAT1 TI , EAAT1 T2 and EAATI WT are compared. The boundaries of the a-helices (cylinders) in the TranD and the ScaD issued from the structural analysis of the EAAT1 CRYST are shown. ΕΑΑΤ1 τι and EAATI WT share 465 amino acid residues (85 % identity), while ΕΑΑΤ1τ2 and EAATI WT share 513 amino acid residues (95% identity).

Figure 14. Alignment of NPC1 L1WT and NPC1 L1 TL Amino acid residues of NCP1 L1 TI and NCP1 L1WT are compared. NPC1 L1 TI and NPC1 L1WT share 1239 amino acid residues (91 % identity).

Example 1 : THERMAL STABILIZATION OF HUMAN EXCITATORY AMINO ACID TRANSPORTER 1 FOR IN VITRO STRUCTURAL AND FUNCTIONAL STUDIES

This Example describes a process to improve the human Excitatory Amino Acid Transporter 1 (EAAT1 ) thermal stability in detergent solutions, as well as the determination of the X-ray structures of EAAT1 thermostable variants. In particular, amino acid mutations that increase thermal stability have been introduced in EAAT1 that make the transporter amenable for purification in detergent solutions, while preserving the neurotransmitter transport function of the wild type protein. This feature allowed to solve the first three-dimensional structures of thermostable EAAT1 variants and unravel a novel allosteric mechanism of inhibition.

Human members of the solute carrier 1 (SLC1 ) family of transporters take up excitatory neurotransmitters in the brain and amino acids in peripheral organs. Dysregulation of their functions is associated to neurodegenerative disorders and cancer. Here the first crystal structures are presented for a human SLC1 transporter thermostabilized according to the method of the invention, the excitatory amino acid transporter 1 (EAAT1 ), with and without allosteric and competitive inhibitors bound. The structures show novel architectural features of the human transporters, including intra- and extracellular domains with potential roles in transport function, as well as regulation by lipids and post-translational modifications. The coordination of the inhibitor in the structures and the change in the transporter dynamics measured by hydrogen-deuterium exchange mass spectrometry, reveal an allosteric mechanism of inhibition, whereby the transporter is locked in the outward-facing states of the transport cycle. These results provide unprecedented insights into the molecular mechanisms of function and pharmacology of human SLC1 transporters.

SLC1 transporters constitute a large family of ion-coupled amino acid transporters present in all kingdoms of life¹. In humans, there are seven SLC1 transporters that share 40-70% amino acid identity (Fig. 4) and have evolved to serve two specialized functions²: in the central nervous system, SLC1 excitatory amino acid transporters (EAAT1 -5) take up the neurotransmitter glutamate into the cell. In peripheral organs, EAATs take up glutamate and aspartate, while neutral amino acid transporters (ASCT1 -2) exchange small amino acids between the extra- and intracellular compartments, contributing to the cellular solute homeostasis.

Glutamate is the most important excitatory transmitter in the mammalian brain and is involved in most aspects of brain physiology, from development to cognition³. Notably, most of the glutamate in the brain is intracellular, and it has to be continuously pumped into the cytoplasm to allow for rounds of transmission and prevent cytotoxicity. This essential neurological function is done by the EAAT1 -5 isoforms expressed at the plasma membrane of astrocytes and neurons. In particular, astroglial EAAT1 and EAAT2 orthologs are highly expressed in the hind- and forebrain, respectively, and are responsible for most of the glutamate uptake in the rodent brain⁴. EAATs are powerful molecular pumps capable of maintaining up to 10⁴-fold glutamate gradients by using energy stored in sodium, proton and potassium gradients⁵. Remarkably, their dysregulation has been associated with several neurological diseases, including amyotrophic lateral sclerosis⁶, ataxia^{7 8}, stroke⁹, depression¹⁰ and glioma¹¹ , making them important drug targets. Moreover, they are also expressed in intestine and kidney, where mutations in EAAT3 have been associated to dicarboxylic aminoaciduria¹².

ASCTs are structurally similar to EAATs, and function as sodium-dependent neutral amino acid exchangers at the plasma membrane¹³. They are highly expressed in intestine, kidney and testis, where they play a key role in maintaining the amino acid cellular homeostasis. Importantly, ASCT2 is up-regulated in several forms of cancer, including melanoma¹⁴, lung¹⁵, prostate¹⁶ and breast cancer¹⁷, and it is a key drug target for the treatment and diagnosis of these diseases.

Despite the need for small compounds that selectively and allosterically modulate SLC1 human transporters, most of their pharmacology is based on substrate-analogs that inhibit transport with rather low selectivity among EAAT⁶⁰ and ASCT⁶¹ isoforms, respectively. Notably, the only known selective allosteric modulators of SLC1 transporters are a series of non-competitive EAAT1 -selective inhibitors, of which 2-Amino-4-(4-methoxyphenyl)-7-(naphthalen-1 -yl)-5-oxo-5,6,7,8-tetrahydro-4H- chromene-3-carbonitrile (UCPH101 ) is the best studied^{62 63}. However, its mechanism of action is still poorly understood at the molecular level. Despite the expression of wild type EAAT1 at the cell surface of HEK293 cells where the neurotransmitter function is retained (Fig.7c), purification and reconstitution of these transporters in synthetic liposomes lack this function (Fig.7d). The detergent solutions used for solubilization and purification induces irreversible unfolding events in the transporters that render them inactive. Human wild type EAAT1 is extensively unfolded in detergent-lipid micelles loosing several secondary structural elements important for oligomerization and transport function.

The process used and disclosed herein to prepare stable and functional EAAT1 is general, fast, and cost-effective, and can be applied to other human membrane protein families. Moreover, with small variations the method can be applied to membrane proteins from other species, in particular pathogenic bacteria and viruses, or even to globular soluble proteins of different origin.

The following steps have been carried out:

a) The amino acid sequences of SLC1 transporters from selected vertebrate animals used as target sequence that share less than 95% sequence identity with each other have been aligned using JALVIEW software⁵⁹. Other softwares for Multiple Sequence Alignment may be used such as Clustal W algorithm, or others that can be identified on are disclosed in above paragraph [028]. The parent (or query) sequence was the amino acid sequence of the human protein.

b) At each position in the alignment, a consensus residue was defined when it met all the below criteria: i) the residue had the highest frequency of occurrence among the aligned sequences, and this frequency is >20%; ii) the residue occupied a position in the alignment in which gaps constitute < 30% of the entries; iii) the residue's frequency of occurrence differed by more >10% from the corresponding frequency of the residue in the target/parent sequence (human sequence: EAATI WT);

c) In a first aspect of the process to increase the stability residues in the target protein were substituted for consensus residues in regions where secondary structural elements (e.g. alpha helices) are predicted, and in the short unstructured loops connecting them. The EAAT1 homologous transporter EAAT1 thermostable 1 (EAAT1 TI) has been generated using this approach and has the following sequence: Sequence ID No.1

MTKSNGEEPKMGGRMERFQQGVSKRTLLAKKKVQNITKEDVKSFLRRNALLLLTVL AVILGVVLGFLLRPYPLSPREVKYFAFPGELLMRMLKMLILPLIVSSLITGLASLDAKA SGRLGMRAWYYMSTTIIAWLGIILVLIIHPGKGTKENMHREGKIVRVTAADAFLDLIR NMFPENLVEACFQQYKTVYEKRSFKVPIQANETLVGAVINNVSEAMETLTRITEELV PVPGSVDGMNVLGLWFSIVFGIALGKMGEQGQLLVDFFNSLNEATMKLVAIIMWYA PLGILFLIAGKIVEMEDLEVLGGQLGMYMVTVIVGLVIHGLIVLPLIYFLITRKNPFVFIA GILQALITALGTSSSSATLPITFKCLEENNGVDKRITRFVLPVGATINMDGTALYEAVA AIFIAQVNNYELDFGQIITISITATAASIGAAGIPQAGLVTMVIVLTAVGLPTDDITLIIAVD WLLDRFRTMVNVLGDALGAGIVEHLSRKELEKQDAELGNSVIEENEMKKPYQLIAQ DNETEKPIDSETKM

To improve the stability of the transporters in detergent solutions, 44 and 33 amino acid residues have been substituted in the transmembrane helices of the ScaD and TranD respectively, for consensus amino acids among SLC1 animal homologs (Fig.7a where the alpha carbon atoms of the exchanged amino acid residues are illustrated as spheres and Fig. 13 where the sequence of the target protein and the sequence of the mutated protein are aligned). Most of the mutated positions localize to hydrophobic regions of the transporter facing either the trimeric interface of the lipid bilayer, and involve conservative substitutions among hydrophobic amino acids (lie, Leu, Val, Met). EAAT1 TI is 85% identical and 92% similar to EAAT

c') According to an optional aspect of the process, an additional step consisted in calculating pairs of residues that strongly co-evolved, using EVcouplings software⁶⁷ (http://evfold.org/evfold-web/evfold.do) (or others having similar functionality), and substituting these pairs of residues in the target sequence for consensus residues. In a particular embodiment of the process, this additional step is performed so that the substitutions performed in step c) are limited to co-varying residues. The EAAT1 homologous transporter EAAT1 thermostable 2 (ΕΑΑΤ1 τ2) has been generated using this approach and has the following sequence:

Sequence ID No.2

MTKSNGEEPKMGGRMERFQQGVSKRTLLAKKKVQNITKEDVKSYLFRNAFVLLTVT AVILGTILGFTLRPYRMSYREVKYFSFPGELLMRMLKMLVLPLIVSSLITGLAALDSKA SGKMGMRAVVYYMTTTIIAWLGIILVLIIHPGKATKENMHREGKIVRVTAADAFLDLIR NMFPENLVEACFKQFKTNYEKRSFKVPIQANETLVGAVINNVSEAMETLTRITEELVP VPGSVNGVNVLGLWFSIVFGIALGKMGEQGQALREFFDSLNEAIMKLVAVIMWYAP VGILFLIAGKIVEMEDMGVIGGQLAMYMVTVIVGLLIHAVIVLPLIYFLVTRKNPWVFIG GILQALITALGTSSSSATLPITFKCLEENNGVDKRITRFVLPVGATINMDGTALYEAVA AIFIAQVNNFELNFGQIITISITATAASIGAAGIPQAGLVTMVIVLTAVGLPTDDITLIIAVD WFLDRFRTMVNVLGDALGAGIVEHLSRHELKNRDVEMGNSVIEENEMKKPYQLIAQ DNETEKPIDSETKM

In order to decrease the amount of mutagenesis and retain the stability of the purified transporters in detergent solutions, the inventors hypothesized that pairs of co-varying residues predicted to be in close proximity play an important role on the stability of proteins. Accordingly, the consensus mutagenesis was restricted to residues that show strong-covariance within the SLC1 family (Fig.7b where the alpha carbon atoms of the exchanged amino acid residues are illustrated as spheres and Fig. 14 where the sequence of the target protein and the sequence of the mutated protein are aligned). 15 and 14 amino acid residues have been substituted in the transmembrane helices of the ScaD and TranD respectively. ΕΑΑΤ1τ2 is 95% identical and 97% similar to EAAT1 wt.

The homologous transporters ΕΑΑΤ1 τι and ΕΑΑΤ1 τ2 have higher thermostability in detergent solutions and showed an increase in the apparent melting temperature (T_m) of ~13, and ~5 °C, respectively (Fig. 1 ), compared to EAATIWT. The homologous transporters ΕΑΑΤ1 τι and ΕΑΑΤ1 τ2 have conserved transport mechanism (i.e. functional) are when expressed in cells (Fig. 7c). In other words, these mutants are functional. The homologous transporters ΕΑΑΤ1 τι and ΕΑΑΤ1τ2 also have a conserved transport mechanism (i.e. are functional) upon reconstitution of purified transporters in liposomes (Fig. 7d and 7e). ΕΑΑΤ1 τι and ΕΑΑΤ1τ2 also have a good solubility in detergent solutions. As an example, ΕΑΑΤ1 τι shows similar level of neurotransmitter uptake in cells compared to EAATIWT, substantially higher solubilization yields in all detergents tested, and robust glutamate uptake upon purification and reconstitution in synthetic liposomes (Fig. 7d). Moreover, glutamate transport in liposomes was strictly dependent on opposite gradients of sodium and potassium across the bilayer, and the rate of transport was dependent on the concentration of L-glutamate, with a K_m (~30μΜ) similar to the one reported for EAATIWT (Fig. 7e). The transport mechanism is therefore conserved for ΕΑΑΤ1 τι and EAAT1 T2. The neurotransmitter experiment shows that the detergent solutions used for solubilization and purification induce unfolding events that render the EAAT1 WT inactive, while ΕΑΑΤ1 τι and ΕΑΑΤ1 τ2 remain functional. Notably, due to the increase in stability the homologous transporters ΕΑΑΤ1 τι and ΕΑΑΤ1 τ2, but not EAATI WT, could be purified as properly folded transporters and reconstituted in liposomes as functional proteins (Fig. 2). In turn, the stable and functional purified transporters could be used in high-resolution structural studies and would be suitable candidates for high- throughput screening of small compounds using in vitro techniques for drug discovery. Moreover, purified ΕΑΑΤ1 τι and ΕΑΑΤ1 τ2 can be used to develop monoclonal molecules, like antibodies and nanobodies that recognize the transporters in the cell surface using three-dimensional epitopes, as opposed to the linear and intracellular epitopes currently used. Mutations based on consensus amino acids of animal SLC1 transporters kinetically trapped EAAT1 in folded and functional states, and allowed the analysis of intermediate unfolded states along their denaturation pathways.

Example 2: STRUCTURE AND ALLOSTERIC INHIBITION MECHANISM OF EXCITATORY AMINO ACID TRANSPORTER 1

In structural terms, most knowledge of the molecular mechanism of transport and pharmacology of SLC1 transporters comes from the prokaryotic homolog GltPh that has been crystallized in the main conformational states of the transport cycle, outward-²² and inward-facing states^{23 24}, as well as in complex with a non-selective and competitive inhibitor of the EAATs²⁵, DL-threo-p-benzyloxyaspartic acid (TBOA). However, the presence of amino acid insertions and deletions, as well as important differences in the transport function and pharmacology of GltPh, make this homolog a limited structural model to understand the molecular mechanism of the human SLC1 proteins.

Here 3.1 -3.3 A X-ray crystal structures of thermostable EAAT1 variants in complex with a substrate (L-aspartate), and the allosteric inhibitor UCPH101 are presented. The structures, and supporting functional data, show new architectural features of the EAATs and ASCTs, and unravel the allosteric mechanism of UCPH101- like inhibitors in atomic detail. Taken together, these structural data can prove useful for the design of novel allosteric compounds with improved selectivity for both EAATs and ASCTs. EAAT1 engineering and crystallization

Purified wild-type EAAT1 shows a poly-disperse size-exclusion chromatogram in detergent solutions, and lacks transport activity upon reconstitution in synthetic liposomes (Fig. 3a). To confer stability to EAAT1 , consensus mutations²⁶ were introduced in the predicted transmembrane helices, and obtained a biochemically stable and functional transporter (Fig. 4). As the protein was still refractory to crystallization, the hypothesis was made that the extracellular region of the transporter between the predicted transmembrane helix 3 (TM3) and the cytoplasmic half of TM4 (TM4c) could preclude crystal growth due to the presence of a long amino acid insertion compared to GltPh. A chimeric transporter substituting this region with the corresponding amino acid sequence from ASCT2 was therefore engineered, which is the shortest in this region among human SLC1 transporters (Fig. 4). The resulting transporter, called EAATI cryst, shares an overall ~75% sequence identity with wild type EAAT1 , and up to ~90% identity at the C-terminal core of the protein, where the transported substrate and coupled ions are expected to bind^{25 27"31}. Importantly, purified EAATI cryst reconstituted in liposomes showed robust glutamate uptake that depended on opposite gradients of sodium and potassium ions across the bilayer (Fig. 3a), and was inhibited by the EAAT1 -selective compound UCPH101 in a concentration- dependent fashion (IC50 of 4.5±0.3 μΜ, Hill coefficient 0.92±0.07) (Fig. 3b). These data show that the transport mechanism and pharmacological selectivity are conserved in EAAT1 cryst-

Notably, EAATI cryst formed crystals in the presence of UCPH101 that diffract X- rays anisotropically and up to 3.25 A resolution, and its inhibitor-bound structure was solved by molecular replacement (see Methods and Table 1 ). As EAATI cryst was refractory to crystallization in the absence of UCPH101 , it was reasoned that mutations in the inhibitor-binding pocket could aid with the crystallization of the transporter. A construct carrying M231 I and F235I mutations (EAAT1 _Cryst-ii; Fig. 4), crystallized both in the presence and absence of the inhibitor, and diffracted X-rays up to 3.1 and 3.32 A resolution, respectively (Extended Data Table 1 ). Remarkably, purified EAAT1 _Cryst-ii reconstituted in liposomes also showed robust sodium- and potassium-dependent glutamate uptake, while the UCPH101 IC50 increased >30-fold (>131 ±38 μΜ, Hill coefficient 0.92±0.0; Fig. 3a, b), as expected for mutations in the binding pocket of the inhibitor (see below). Domain organization

The structure of EAAT1 _cryst shows a symmetric homotrimer in a substrate- and UCPHi 01 -bound outward-facing conformation (Fig. 3c-e). EAATI cryst adopts an overall GltPh-like fold^{22 23}, in which each monomer is composed of two domains: a trimerization or scaffold domain (ScaD), including TM1 -2 and TM4-5; and a transport domain (TranD), including TM3, TM6-8 and re-entrant helical loops 1 -2 (HP1 -2; Fig. 3f). The ScaD forms all inter-subunit contacts through residues in TM2, TM4 and TM5, that include six salt bridges and bury ~3,000 A² from each subunit. Hence, the three ScaDs form a compact central structure with a propeller-like shape that ensures the trimeric form of the transporter and anchors it to the membrane.

The three TranDs are more peripheral and localize between the blades of the propeller, making protein contacts exclusively with the ScaDs of their own monomer. The TranD-ScaD interface buries ~3,500 A², including a conserved salt bridge between E256 and K364. This interface is mainly formed by cytoplasmic residues in HP1 , TM7, and TM3 (TranD), and TM2, TM4c, and TM5 (ScaD). However, on the extracellular side additional contacts occurred between HP2 and TM4, through residues that are well conserved among human transporters.

Substrate and ion translocation in SLC1 transporters is thought to occur through large rigid-body movements of the TranD, relative to the static ScaD, that move the cargo in an elevator-like fashion across the membrane^{23 32}. Thus, during the isomerization to the inward-facing state the TranD-ScaD interface changes drastically on the TranD side, and the novel features observed at this interface in EAATI cryst might influence the distinct TranD dynamics in human SLC1 proteins.

Transport domain

One of the most remarkable architectural features of the EAAT1 cryst TranD is at the TM8 level, in which deletions and insertions compared to GltPh reshape this helix and its interactions with neighboring structural elements important for transport. In EAATI cryst, TM8 can be divided into extracellular (TM8a), transmembrane (TM8b), and cytoplasmic (TM8c) helices. The loop connecting TM8a and the C-terminal helix of HP2 (HP2b) is six residues shorter in human SLC1 transporters. Consequently, the extracellular ends of TM8a and HP2b are in close proximity and engage in hydrogen bonding and hydrophobic interactions. HP2 is a key component of the gating machinery that controls the access of substrate and ions to their binding sites in the TranD^{25 33-35}, and its interactions with TM8b likely play an important role in determining HP2 movements. Notably, several single-cysteine mutations at positions along TM8a in EAAT1³⁶, and in a rodent EAAT2 ortholog³⁷ impaired glutamate transport, highlighting the significance of this extracellular region for function.

At TM8b level, strong electron density for the substrate (L-aspartate) was found and one of the sodium-binding sites previously observed in the archaeal homologs of SLC1 family (Na2), which was modeled with similar coordination than in their structures^{25 31}. Remarkably, the carboxylate group of D456 (TM8b), which coordinates the alpha-amino group of the substrate, is also at hydrogen bond distance with the hydroxyl group of S343 (HP1 ). Moreover, the guanidinium group of R457 (TM8b) engages in hydrogen bonding with HP1 residue G341 , and possibly L340 and T342 that point their backbone carbonyl oxygen atoms towards TM8b. Residues S343 and R457 are well conserved in human SLC1 transporters, and substitutions at equivalent positions in EAAT1 (S363 and R477)³⁸, and EAAT3 (R445)³⁹ inactivate transport. In addition, the loss-of-function mutation R445W in EAAT3, equivalent to R457 in EAATI cryst, causes human dicarboxylic aminoaciduria due to the lack of aspartate/glutamate reabsorption function in the kidney¹². Overall, the functional studies and our structural data converge to suggest that interactions between conserved human residues at HP1 and TM8b are important to the correct folding and function of the transporters.

On the cytoplasmic side, hydrophilic TM8c extends beyond the membrane plane through a hydrophilic helix (TM8c), and makes contact with residues in TM3 and TM7a. Notably, EAAT2 deletion mutants in this region have a deleterious effect on transport function and membrane trafficking⁴⁰. Accordingly, a deletion of TM8c beyond E501 in EAAT1 (equivalent to E500 in EAAT2 and E481 in EAATI cryst) decreased glutamate uptake rate by ~2-fold. The functional data, and the amino acid conservation in TM8c among EAATs, underscore the pivotal role of this structural motif in protein folding and transport kinetics.

Scaffold domain

The ScaD is less conserved than the TranD in the SLC1 family. In particular,

TM4 is highly divergent (Fig. 4), and shows several unique architectural features in EAATI cryst. On the extracellular side, TM4a forms inter- and intra-monomeric contacts with TM2 and HP2, respectively. Moreover, an amino acid insertion between TM4b and TM4c (TM4b-c loop) that appeared during the evolution of eukaryotic transporters protrudes into the central vestibule of the EAAT1 _cryst trimer. The TM4b-c loop forms the center of the propeller, and makes extensive contacts within and between protomers. Due to the lack of electron density it was not possible to model the outermost residues of the TM4b-c loop (Y200-V210), but they are expected to reach out to the bulk solvent, and expose one of the predicted N-glycosylation sites of the transporter (N204). Notably, despite the lack of sequence identity among human SLC1 transporters, all of them contain predicted N-glycosylation sites in the TM4b-c loop, suggesting a common role of this loop in the posttranslational processing of these proteins.

An additional novel feature of the EAAT1 _cryst ScaD architecture is the N-terminal extension of TM1 by an amphipathic helix (TM1 a). Positioned nearly parallel to the membrane plane, TM1 a forms the tips of the blades in the propeller (Fig. 3c). Remarkably, it does not form inter- or intra-monomeric contacts, and its position and amphipathic nature suggest that TM1 a somehow interacts with the inner leaflet of the membrane. Indeed, there is a hydrophobic crevice between TM1 a and HP1 a from the same monomer, where strong non-protein electron density in EAAT1 _cryst was observed that likely corresponds to bound detergent or lipid molecules. Interestingly, a second hydrophobic crevice is observed between the extracellular part of TM4 and HP2, where there is also strong non-protein electron density. A similar crevice was also noted in a substrate-bound structure of GltPh²².

The lipidic composition of the bilayer regulates the function of SLC1 transporters^{41 -45}. Because TM1 a-HP1 a and TM4-HP2 hydrophobic crevices are at the interface between the TranD and ScaD, where large conformational changes are expected to occur during substrate translocation, they might constitute sites for lipid regulation of transport function.

UCPH₁0₁ binding site

The structure of EAAT1 cryst showed strong electron density for UCPH101 in a hydrophobic pocket facing the inner leaflet of the membrane on the interface between the TranD and ScaD (Fig. 3d,e and Fig. 5a, b). This pocket is formed by residues in TM3, TM7 and TM4c, and extends the TranD-ScaD interface by ~ 500 A².

The chromene skeleton of UCPH101 , the parental group of the UCPH series of compounds, is buried deeply in the domain interface, and coordinated by a direct ring- stacking interaction with F369 (TM7a), as well as hydrophobic interactions with G120 (TM3), V373 (TM7a) and M231 (TM4c) (Fig. 5b). In addition, the amine group of UCPH101 forms a hydrogen bond with the main-chain carbonyl of F369, while its carbonitrile group interacts with Y127 (TM3). The methoxy-phenyl and naphthalene groups are more peripheral and partly facing the hydrocarbon core of the membrane. Yet, the former establishes hydrophobic interactions with V124 (TM3), V373 and M231 , while the latter is mainly coordinated by F235 (TM4c). The majority of the above- mentioned residues have been reported to be important for the inhibition of an EAAT1 rodent ortholog by UCPH101 in cell assays²¹. Furthermore, the EAAT1 _cryst double mutant M231 1-F235I (EAAT1 _Cryst-n) showed >30-fold increase in UCPH101 IC50 compared to EAAT1 _cryst in proteo-liposomes (Fig. 3b). Hence, there is an excellent agreement between the crystallographic and functional data.

Several mechanistically-relevant observations can be made regarding the UCPH101 binding pocket in EAAT1 _cryst: i) it is over 15 A away from the substrate and sodium binding sites, suggesting that UCPH101 does not preclude substrate binding, as expected for a non-competitive allosteric inhibitor; ii) it faces the inner leaflet of the membrane, implying that UCPH101 accesses its binding site from the lipidic, and not the aqueous phase, when applied extracellularly; iii) it is fully contained in a single subunit, in agreement with the lack of cooperativity observed in proteo-liposome (Fig. 3b), and cell assays²¹ ; iv) a comparison of the EAAT1 -5 sequences suggests that the main determinants of UCPH101 selectivity for EAAT1 are in TM4c, where M231 and F235 are the only coordinating residues that differ between EAAT1 and all other EAATs (Fig. 4). Consistently, the equivalent residues in EAAT2 are isoleucine, and the double mutant EAAT1 _Cryst-n, containing M231 I and F235I, shows a >30-fold increase in the UCPH101 IC50 compared to EAAT1 cry st- UCPH ₁₀₁-unbound state

To better understand the conformational changes of the transporter induced by UCPH101 , determination of the structure of the UCPHioi-unbound state was contemplated. As mentioned above, EAAT1 _cryst was refractory to crystallization in the absence of the compound and instead, the structure of EAAT1 _Cryst-ii UCPHioi-unbound state was solved. For comparison, the structure of the EAAT1 _Cryst-ii UCPHioi-bound state was determined, using a large excess of the compound in the crystallization conditions (see Methods). The structure of EAAT1 _Cryst-n in the UCPHioi-bound state is nearly identical to that of the EAATI cryst with the exception of the mutated 1231 and I235 side chains, and a ~2 A movement of UCPH101 methoxy-phenyl and naphthalene groups away from them (Fig. 5c). Interestingly, the EAAT1 _Cryst-ii UCPHioi-unbound structure has an overall similar conformation to the UCPHioi-bound state, but shows notable differences. First, the UCPH101 binding pocket contains no excess electron density, and the side chain of F369 moves outward by as much as 1.9 A, partly occupying the volume for UCPH101 chromene group (Fig. 5c). Second, there is a small rigid-body movement of the entire TranD that is shifted by as much as 0.7 A, compared to the UCPH101 -bound structures. Interestingly, this conformational change shows the EAATI cryst TranD is able to undergo rigid-body movements relative to the ScaD, and highlights the importance of such movements for the function of the human transporters, as it has been shown for the prokaryotic homolog^{23 24}

The structural changes observed in the UCPHioi-unbound structure unambiguously demonstrate that the assigned binding pocket of UCPH101 is correct, and that within the restricted environment of the crystal lattice, UCPH101 induces both local and global conformational changes of the transporter that optimize its coordination in an outward-facing state.

Transport domain dynamics

The coordination of UCPH101 in the crystal structures, wedged between the

TranD (TM3 and TM7a) and the ScaD (TM4c), as well as the effect of the M231 1-F235I mutations on the UCPH101 potency strongly suggest that UCPH101 inhibits transport by trapping the transporter in an outward-facing state. Consistently, the rigid-body movements of the TranD to isomerize into the inward-facing state would separate the coordinating residues in the TranD from those in the ScaD, and disrupt the UCPH101 coordination. Hence, under equilibrium conditions where the transporters are sampling outward- and inward-facing states, the expected effect of UCPH101 binding is to shift the equilibrium in favor of the outward-facing state.

To gain insights into the effects of UCPH101 binding to the transporters at equilibrium, the detergent solubilized EAATI cryst by hydrogen-deuterium exchange mass spectrometry (HDX-MS) was probed. HDX-MS measures the rate of exchange of backbone amide hydrogen atoms that depends on solvent accessibility and hydrogen bonding, and provides valuable information on the dynamics and conformational changes of proteins^{46 47}.

The HDX behavior of the EAAT1 _cryst was compared in the presence and absence of UCPH101. Overall, the deuterium uptake pattern of EAAT1 _cryst shows dynamic structural elements in both the TranD and ScaD, and reveals the unstructured and solvent-exposed nature of several regions that were not resolved in the crystal structures, including the TM3-TM4a (peptide 153-173) and TM4b-c (peptide 200-208) loops, as well as the N- (peptide 1 -28) and C- termini (peptides 490-522).

Binding of UCPH101 significantly decreased deuterium uptake in several areas of the TranD including its binding pocket (residues 1 12-123 and 370-374), and the surrounding area (residues 354-369), while it left the uptake in the ScaD unchanged. It also decreased deuterium uptake in distant residues (336-349 and 420-430) at the tips of HP1 and HP2 involved in substrate coordination and occlusion, suggesting that UCPH101 induces conformational changes in the transporter upon binding. To gain insights into the nature of those conformational changes, the TranD areas in which UCPH101 decreased uptake was first compared with those buried at the interface with ScaD in the EAAT1 _cryst structure, and found that they correlate remarkably well. Second, a model of the EAAT1 _cryst inward-facing state was built, based on a recently solved structure of GltPh²⁴, to assess the changes in solvent accessibility in a possible transition between inward- and outward-facing states. Indeed, the comparison between the structure and the model shows that the UCPHioi-modified areas detected by HDX-MS transit as rigid bodies from being solvent-exposed, in the inward-facing state, to buried at the TranD-ScaD interface, in the outward-facing state. Such conformational change is expected to decrease the dynamics of alpha helices and/or the solvent accessibility of the loops in the UCPHioi-modified areas and thus, is consistent with the observed decrease in deuterium uptake. Overall, the HDX-MS and structural analysis support the stabilization of the outward-facing state, at the expense of the inward-facing state(s), induced by UCPH101.

UCPH₁0₁- and TBOAiFB-bound state

The distant position of UCPH101 from the substrate and the HP2, a structural element that controls extracellular access to the binding site²⁵, suggests that the UCPH101 -bound transporters could undergo the conformational changes required to exchange the substrate with the extracellular solution. To test this, the crystal structure of EAAT1 cryst in complex with both UCPH101 and (2S,3S)-3-[3-[4- (trifluoromethyl)benzoylamino]benzyloxy]aspartate (TBOATFB), a potent and nonselective TBOA derivative⁴⁸, was solved at 3.7 A resolution (Table 1 ).

Overall, the UCPHI OI -TBOATFB bound structure is similar to that of the UCPHioi-bound state, with the exception of HP2 that adopts an "open" conformation and packs against the TM4b-c loop, disrupting the coordination of the Na2 (Fig. 6a, b). These conformational changes resemble those previously observed in the structure of the GltPh-TBOA complex, and are in excellent agreement with the proposed competitive inhibitory mechanism of TBOA-like compounds²⁵.

In the substrate-binding site, excess electron density was observed for the bulky

TBOATFB (Fig. 6c), but due to lack of resolution, it was not possible to unambiguously orient the compound. In order to fit the TBOATFB molecule into the density, the TBOA moiety of TBOATFB was initially positioned using the TBOA-bound GltPh structure as a guide. In this position, it remained stable during several successive cycles of refinement that yielded a reasonable fit into the electron density. The additional benzoylamino and trifluoromethyl groups of TBOATFB localized in a hydrophobic cavity mainly formed by residues in HP1 b and TM7a, and possibly by residues in TM2 and TM4c. Interestingly, the interactions of these groups with the transporter could explain the ~1500-fold increase in inhibitory potency of TBOATFB, compared to TBOA, that has been observed in EAAT1 transport assays⁴⁸.

Indeed, it was also observed that UCPH101 bound in this structure with an identical coordination than in the substrate-bound state. Therefore, despite the lower resolution of the TBOATFB bound structure, it shows that UCPH101 binding at its allosteric site does not preclude the movements of HP2 involved in substrate and sodium binding from the extracellular solution.

Inhibitory mechanisms of EAAT1

The structures of EAAT1 cryst reveal new architectural features of human SLC1 transporters, and the first non-competitive inhibitory mechanism of this family of proteins in molecular detail. UCPH101 is an EAAT1 -selective inhibitor with a bipartite coordination by residues in both the TranD and ScaD, and the downward rigid-body movements of the TranD during transport disrupt such coordination. This implies that upon binding, UCPH101 "glues" the TranD to the ScaD in the outward-facing states, and precludes the translocation reaction of the transport cycle, but not the substrate binding/unbinding reactions from the extracellular solution.

The inhibitory mechanism of UCPH 101 contrasts with that of substrate-analog competitive inhibitors like TBOA. The binding pocket of TBOA-like compounds overlap to some extent with that of the substrate²⁵, and some of these molecules can bind the transporter from both the extra- and intracellular aqueous solutions^{49 50}. Therefore, they inhibit transport by precluding substrate binding on either side of the membrane. Moreover, TBOA-like compounds are not selective among glutamate transporters due to the high amino acid conservation in the substrate-binding sites.

The mechanistic differences observed in UCPH101 over other known inhibitors make it an extremely valuable pharmacological tool, to isolate and study the conformational changes that EAAT1 undergoes upon substrate and ion binding. Remarkably, the UCPH101 allosteric binding site observed in EAAT1 _cryst highlights a cavity that can facilitate the design of selective compounds for other human SLC1 transporters, and possibly the long-sought positive modulators of glutamate uptake.

To sum up the results relative to the EAATCRYST, the structure shows that the transporter is a homo-trimer, in which each protomer contains two structural and functional domains, the scaffold (ScaD) and transport (TranD) domains, previously observed in the structures of a glutamate transporter prokaryotic homologue. The ScaD forms the inter-subunit interface through extensive contacts between transmembrane helices TM2 and TM4 on the extracellular half of the membrane, as well as between TM4 and TM5 on the cytoplasmic side. The remaining alpha helical regions of the protein, including TM3 and TM6-8, as well as two re-entrant helical loops (HP1 -2) fold into the TranD that encages the substrate and the coupled ions and translocates them across the membrane through rigid-body movements in an "elevator-like" fashion.

Example 3: STRUCTURAL COMPARISON BETWEEN EAAT1_WT, EAAT1_Ti AND EAAT1 T2

EAATI WT is inactive in transport assays, while ΕΑΑΤ1 τι and ΕΑΑΤ1 τ2 are active. The detergent solutions used for solubilization and purification may induce unfolding events that render the wild type transporter inactive. The structural differences between the two mutants and the wild type transporter in detergent solutions have therefore been compared. Hydrogen-deuterium exchange behavior of the three proteins using a protease-induced fragmentation approach linked to mass spectrometry (HDX-MS) was used. As previously explained, HDX-MS measures the rate of deuterium exchange of the hydrogen amide in the protein backbone, which depends strongly on the presence of secondary structure due to the engagement of the amide group in hydrogen bonding as well as access to the aqueous solvent. Therefore, HDX-MS provides valuable information on protein folding, stability and dynamics.

EAAT1 TI and ΕΑΑΤ1 τ2 showed similar overall HDX patterns when assayed at 20 °C in detergent solutions, with sequence coverage of ~70 % and unimodal isotopic envelopes across all peptides measured. Analysis of the HDX kinetics (Fig. 8 a-c) revealed that most peptides covering the alpha-helical regions of the TranD and ScaD take up deuterium at rates between 10^~2 to 10^~5 s^~1 , which are several orders of magnitude slower than predicted for unstructured peptides with the same sequence (10² s^"1). Moreover, peptides partly covering at least TM4-5, as well as TM8 and HP2, were completely protected and do not show any deuterium uptake. In contrast, peptides covering predicted unstructured regions like the N- and C-termini, as well as extracellular loops between TM3-TM4a and TM4b-TM4c respectively, which were not resolved in the EAATI CRYST structure, showed saturating levels of deuterium uptake already at 10s, the shortest time measured in the experiments, consistent with a solvent accessible backbone in these regions being fully solvent accessible and lacking secondary structure. To sum up, the wild transporter should have secondary structure but it loses it in detergent and therefore loses its function, while the mutated proteins ΕΑΑΤ1τι and ΕΑΑΤ1 τ2 keep secondary structure. Importantly, the HDX patterns of EAAT1 τι and EAAT1 Τ2 are similar to that of EAAT1 CRYST and EAAT1 CRYST- ii, which are also functional transporters when reconstituted in liposomes.

The HDX behavior of EAATI WT was strikingly different form the above-mentioned mutants and showed an overall dramatic increase in deuterium uptake (Fig. 8 d-f). This increase in backbone amide HDX is most prominent in two regions of the transporter: first, the cytoplasmic half of the TranD, where several peptides covering TM3 (peptide 1 12-123), TM7 (peptides 390-399 and 397-404) and HP1 (peptide 357-369), including the substrate and ion binding sites revealed maximal deuterium uptake at 10s, as observed in the unstructured N- and C-termini (Fig. 8 a-c). The lack of backbone protection implies that the native helical structure of these key regions for transport function is lost upon solubilization and purification of the protein; second, several regions of the ScaD, particularly the extracellular helices of TM4 form extensive inter- subunit contact. Although deuterium uptake in these regions still reveals some level of backbone protection, these results suggest that the ScaD might undergo partial unfolding events that could compromise the oligomeric state of the transporter.

All together the HDX results show that ΕΑΑΤ1 τι and ΕΑΑΤ1 τ2 retain the native fold observed in the structure of EAATI CRYST, while the EAATIWT undergoes partial and extensive unfolding events in both the TranD and ScaD in detergent solutions. Consistently, purified ΕΑΑΤ1 τι and ΕΑΑΤ1 τ2, but not EAATI WT, showed robust sodium- and potassium-dependent neurotransmitter transport function, when reconstituted in synthetic liposomes. The above results prove that the consensus, as well as the consensus-covariance approaches, can be used as semi-rational approaches to produce a stabilized version of the human EAAT1 for biophysical analysis of its molecular mechanisms.

ΕΑΑΤ1 -Π and ΕΑΑΤ1 τ2 showed an overall similar sequence of events during thermal inactivation, despite the difference in amino acid sequence, in which the loss of quaternary structure is an essential early event in the denaturation pathway. Several lines of evidence support this conclusion: the overall kinetic stability of the ScaD is lower than that of the TranD, and mutations in the ScaD determined the stability of trimeric form of the transporters, as shown by the chimera EAAT1 -ScaD-n-TranDT2; The lowest local apparent melting (T50-HDX) observed in both ΕΑΑΤ1τι and ΕΑΑΤ1τ2 map to an extensive area of the trimeric interface formed by TM4a-c and TM2 from neighboring subunits, respectively. This means that the first temperature-induced unfolding events of the transporters occur in TM4a-c and TM2 and therefore, that they constitute important rate-limiting step(s) during thermal inactivation. There is excellent quantitative agreement between the T50s of local unfolding at the trimeric interface (T50-HDX in TM4a-c and TM2), and those that characterize the loss of trimeric state (T50-FSEC). This agreement holds for both EAAT1 _Ti and EAAT1 _T2 despite the ~ 10 °C difference in the T50 values between the transporters. The above results strongly argue that the loss of secondary structure in TM4a-c and TM2 causes the dissociation of the trimers.

Unfolding events occur at higher temperatures in the TranD than in the ScaD. Clear unfolding has been observed for important helical regions for substrate and ion binding (TM3, TM7-8, and HP1 -2) in EAAT1 _Ti at temperatures as high as 65 °C, while the TranD of ΕΑΑΤ1 τ2 showed no major changes in deuterium uptake at 55 °C. This contrasts with the loss of function observed in ΕΑΑΤ1 τ2 pre-heated at 55 °C and suggests that thermal inactivation is due to the loss of trimeric state, and does not require extensive unfolding events of the TranD. This could be explained if dynamic regions of the transporter required for transport (e.g. HP2) were constrained by lipid or detergent molecules in the monomeric form of the transporters, but not in the trimeric one. In fact, decreased in deuterium uptake at the level of HP2 in both ΕΑΑΤ1 τι and EAAT1 T2 at high temperatures, is consistent with this line of thought.

Early unfolding events at the trimeric interface determine the lifetime of the transporters and opens the interesting possibility to target this region by mutagenesis, or even small compounds that could extend the half-life of the transporters and possibly act as the long-sought glutamate transport activators.

Example 4: THERMAL STABILITY OF EAAT1_Ti AND EAAT1_T2

The thermal denaturation of both mutants was assessed. Thermal denaturation is in general an irreversible process in detergent-solubilized membrane proteins. Applying temperature protocols that generate partial denaturation of the proteins offers the possibility to capture kinetic intermediates of the unfolding pathway. Size Exclusion Chromatography has first been used to analyze the effect of twenty-minute temperature pre-pulses on purified ΕΑΑΤ1 τι and ΕΑΑΤ1 τ2 in detergent solutions, at a constant protein concentration, in order to quantify the irreversible effect of temperature on protein solubility, aggregation, and oligomeric state compared to reference transporters that were not pre-heated.

ΕΑΑΤ1 -Π and ΕΑΑΤ1 τ2 reference samples maintained at 4 °C eluted as symmetric and monodisperse peaks that correspond to the trimeric form of the transporters (~3 ml elution volume), and remained stable up to 35 °C and 25 °C respectively (Fig. 9a, 9b and Fig. 9c,9d respectively). At higher temperatures the trimers melt into a lower oligomeric state that elutes at ~3.5 ml, without any signs of aggregation or insolubility, judging by the lack of high molecular-weight peaks and the constant area under the chromatograms at all temperatures respectively. The apparent melting temperatures (TSO-SEC) of ΕΑΑΤ1 τι and ΕΑΑΤ1 τ2 trimeric states were 49.06 ± 0.02 and 38.4 ± 0.3 °C respectively, demonstrating that the quaternary structure of ΕΑΑΤ1 -Π is kinetically more thermo-stable than that of ΕΑΑΤ1 τ2 (Fig.9b and 9d) Interestingly, the appearance of a single low molecular-weight peak in the chromatographic profiles at high temperatures argues that the transporters lose their quaternary structure by melting into monomers, in a cooperative process that does not involve the formation of stable dimers, and that those monomers remain soluble at pre- pulses of up to 65 °C.

In order to probe the transport function of the low molecular-weight species,

EAAT1 T2 samples pre-heated at 55 °C in liposomes were reconstituted and compared to the reference samples maintained at 4 °C. Indeed, the large decrease in neurotransmitter uptake observed in the pre-heated samples shows that the transport function of the "low molecular-weight" species has been largely impaired, and suggests that the monomeric form of the transporter is not functional.

The loss of trimeric state and transport function argues that early events during the thermal inactivation of the transporters involve the formation of partly unfolded monomers, and that the interface between the subunits plays an important role in the thermal stability of the transporters. To gain further insights into the role of the trimeric interface, a chimeric transporter with the ScaD of ΕΑΑΤ1 τι and the TranD of ΕΑΑΤ1 τ2 (EAAT1 -ScaD-n-TranDT2) was built. Notably, the melting curve of EAAT1 -ScaD-n- TranD_T2 approached that of EAAT1_Ti (T50-SEC of 45.2 ± 1 .0 °C) showing that key determinants of the transporter thermal stability localize to the trimeric interface (Fig. 9e,f).

LOCAL THERMAL UNFOLDING OF EAAT1_Ti AND EAAT1_T2

The chromatographic and functional analysis clearly established that the temperature pre-pulse protocols irreversibly generate intermediates of the thermal unfolding pathway. To gain structural insights into the unfolded state of these intermediates, pre-heated transporters were analyzed by HDX-MS using the untreated transporters as reference. Since the sample throughput by HDX-MS is limited, analysis was focused on ΕΑΑΤ1 τι and ΕΑΑΤ1 τ2 to the corresponding apparent melting temperatures observed by SEC (TSO-SEC) as well as selected temperatures below and above it.

The overall HDX pattern of ΕΑΑΤ1 τι and ΕΑΑΤ1 τ2 was similar when the transporters were pre-heated at nearly their TSO-SEC, 40 °C and 50 °C respectively. Both transporters showed a significant increase in deuterium uptake in peptides covering TM4a-b (e.g. peptide 174-184) and the extracellular part of TM2 (e.g. peptide 81 -87) indicating partial loss of secondary structure in these helices. Additional increase in deuterium uptake was observed in the cytoplasmic parts of TM2 and TM3 (e.g. peptides 105-1 1 1 and 1 12-1 15 in EAAT1 _Ti), as well as TM4c (peptide 230-246 in EAAT1 T2), while HDX remained unchanged in the TranD at large. These results map the initial thermal unfolding events to the trimeric interface of the transporters and highlight the key role of this region in thermal stability.

EAAT1 TI and ΕΑΑΤ1 τ2 were also probed at pre-pulse temperatures above the

T50-SEC at which complete loss of trimeric state was observed based on SEC, 65 °C and 55 °C respectively. Under these conditions, both transporters showed a dramatic increase in HDX at the trimeric interface (TM4a-c and external TM2) that went beyond the one observed at their respective TSO-SEC. The HDX profile of the TranD differed greatly between ΕΑΑΤ1 τι and ΕΑΑΤ1 τ2: in the latter, the deuterium uptake remained unchanged compared to the reference condition, with the exception of a slight decrease in uptake observed in HP2; ΕΑΑΤ1 τι showed large HDX changes in most peptides covering the TranD including increased deuterium uptake in HP1 (e.g. peptide 361 -369) and TM7-TM8 (e.g. peptides 390-399 and 483-495 respectively). These results show that the TranD of both ΕΑΑΤ1 τι and EAAT1 i2 are more thermo-stable than their ScaD, and only undergo extensive unfolding events at pre-pulse temperatures overs 55 °C.

CORRELATION BETWEEN LOCAL UNFOLDING AND LOSS OF QUATERNARY STRUCTURE

The thermal denaturation experiments show that increasing temperatures induce on one side loss of the trimeric state, as observed by SEC, and on the other side local unfolding events that initially map to the extracellular part of the trimeric interface between TM4a-b and TM2, as observed by HDX-MS. The important question then raises regarding if there is a quantitative correlation between the two events that could establish a causative link between the local unfolding events and the loss of quaternary structure. To shed light on this problem, the apparent melting temperatures of the individual peptides obtained by HDX (TSO-HDX) were estimated and compared to the corresponding TSO-SEC, the parameter that characterizes melting of the trimeric state.

The peptides covering the extracellular helices TM4a-b (e.g. 174-184 and 187- 194) showed clear bimodal isotopic envelops in the m/z spectra, while all other peptides were unimodal. The bimodal envelopes were most apparent at pre-pulse temperatures close to the TSO-SEC (Fig. 10a and 10b), in which the amplitudes of the high- and low-mass components were similar. In contrast, at temperatures above and below the Tso-sEc the envelopes were nearly unimodal and dominated by the high- and low-mass components respectively. Using Gaussian fitting to calculate fraction of the low-mass components as a function of temperature, and estimated the apparent melting temperature of the peptides at the inter-subunit interface (TSO-HDX-GAUSS), peptides 174-184 and 187-194 in ΕΑΑΤ1 τι showed TSO-HDX-GAUSS values of 49,5 °C and 48,8 °C respectively, while those of EAAT1 _T2 were 36,8 °C and 42,4 °C respectively (Fig. 10c). These values are in excellent agreement with the T50-SEC values observed for EAAT1 TI (49,0 °C) and EAAT1 Τ2 (38,4 °C), strongly suggesting a causative correlation between the unfolding of the TM4a-b helices and the loss of quaternary structure.

In order to estimate the apparent melting temperature of all peptides that showed temperature-dependent HDX changes, and not just those that showed bimodal isotopic envelopes, a kinetic analysis of the deuterium uptake data has been carried out. The deuterium uptake kinetics of all peptides is well described by three components: an initial burst, which corresponds to the uptake measured at 10 s; a slow component with uptake rates < 10^~3 s^~1 , and an intermediate component with rates ~ 10^"1-10^"2 s^"1 (Fig. 1 1 a and 1 1 b). The deuterium uptake of the peptides covering the helical regions of the transporter is dominated by the slow component under reference conditions (e.g. >70% of deuterium uptake in peptide 174-184, Fig. 1 1 a) and therefore, the decrease in the fractional amplitude of this component at increasing temperatures was used as a proxy for the loss of native folded state and to extract the associated apparent melting temperature (TSO-HDX-KINET) (Fig. 1 1 c and 1 1 d).The peptides covering the extracellular part of TM2, as well as TM4a-c showed the lowest TSO-HDX-KINET values that ranged between 50.6-52.8 °C and 40.6-42.1 °C for EAAT1 _T1 and EAAT1 _T2, respectively. These values are in excellent quantitative agreement with the TSO-SEC obtained for the loss of the trimeric state, and strongly support the idea that unfolding of TM4 and TM2 are key initial events that determine the trimeric stability and functional state of the transporters. Interestingly, the T50-HDX-KINET values of peptides covering the TranD of ΕΑΑΤ1 τι ranged between 52.8 °C in the cytoplasmic end of TM3, to 56-58 °C in HP1 , TM7, as well as TM8, demonstrating that the regions involved in substrate and ion coordination unfolds at later stages in the thermal denaturation pathway of the transporters. Conclusion

Mutagenesis based on consensus amino acids may allow the engineering of membrane protein with enhanced stability. The two approaches disclosed therein (substituting one or more amino acid residues in the amino acid sequence of the target protein by its(their) respective consensus amino acid residue(s) or substituting one or more amino acid residues in the amino acid sequence of the target protein by substitution, wherein said amino acid residues are selected among residues which are present in pairs of residues that show co-evolution in the target protein) allow the production of two stabilized human homo-trimeric membrane proteins. The consensus approach is especially dedicated for protein families that lack high selective pressure on their stability determinants. The loss of stability observed in a full-consensus sequence of highly thermostable soluble proteins supports the above. Furthermore, non-specialized ancestral proteins are expected to be more stable than the extant specialized structural homologs; the gain of stability has been observed in several laboratories during resurrection of ancestral soluble proteins.

The consensus approach may be adapted to include the condition of co- variance. Conserved amino acid interactions in the SLC1 family are important to the stability of the fold and therefore, restricting the consensus mutagenesis to conserved interacting positions may improve the protein stability. The determination of pairs of position that show high co-variance in a curated PFAM alignment of the SLC1 family, as a proxy for conserved amino acid interactions, and application of the same consensus amino acid exchanges than in the consensus mutagenesis to those positions only permit to reduce the overall number of mutations. Indeed, the second method decreases the number of consensus mutations from 77 to 29, and yield a transporter that shares ~95% sequence identity with wild type EAAT1 . The proteins produced according to this method remains folded and functional in detergent solutions, and even forms low-diffracting 3D crystals, demonstrating a better stability than the wild type protein. Example 5: THERMAL STABILIZATION OF HUMAN Niemann-Pick C1 -like1 PROTEIN

The consensus approach has been applied to another human integral membrane protein family. Niemann-Pick C1 -Like1 (NPCL1 ) is a cholesterol binding protein and is responsible for cholesterol re-adsorption in the apical plasma membrane of intestinal and hepatic epithelia. Notably, some of the current FDA-approved cholesterol lowering drugs, like ezetimibe, inhibit NPC1 L1 transport function making this protein an important drug target. Wild type human NPC1 L1 (NPC1 L1WT) contains 13 transmembrane helices and three large cytoplasmic domains, and although it expresses well in HEK293 cells, it is barely soluble in detergent solutions. The amino acid sequence of N PC1 L1WT includes 1 ,359 residues (SEQ ID No: 12), and it makes impractical and costly the implementation of methods like amino acid scanning mutagenesis to improve its stability. To overcome this problem, the method of the invention has been applied and a mutant construct called NPC1 L1 TI has been designed.

The following steps have been carried out:

a) The amino acid sequences of wild type human NPC1 L1 from selected vertebrate animals used as target sequence that share less than 95% sequence identity with each other have been aligned using JALVIEW software⁵⁹. Other softwares for Multiple Sequence Alignment may be used such as Clustal W algorithm, or others that can be identified on are disclosed in above paragraph [028]. The parent (or query) sequence was the amino acid sequence of the human protein.

b) At each position in the alignment, a consensus residue was defined when it met all the below criteria: i) the residue had the highest frequency of occurrence among the aligned sequences, and this frequency is >20%; ii) the residue occupied a position in the alignment in which gaps constitute < 30% of the entries; iii) the residue's frequency of occurrence differed by more >10% from the corresponding frequency of the residue in the target/parent sequence (human sequence: NPC1 L1WT);

c) In a first aspect of the process to increase the stability residues in the target protein were substituted for consensus residues in regions where secondary structural elements (e.g. alpha helices) are predicted, and in the short unstructured loops connecting them. The NPC1 L1 homologous transporter NPC1 L1 thermostable T1 (NPC1 L1 TI) has been generated using this approach and has the following sequence:

Sequence ID No.13:

MAEAGLRGWLLWALLLHLAQSEPYTPIHQAGYCAFYDECGKNPELSGSLIPLSNVS CLSNTPARKVTGDHLILLQRICPRLYTGPTTYACCSLKQLVSLELSLSLSKALLTRCP ACAENFANLHCHNTCSPNQSLFINVTRVAQLGAGQLPAWAYEAFYQRSFAEQAYD

SCSRVRIPAAATLAVGTMCGVYGSALCNAQRWLNFQGDTSNGLAPLDITFHLLEPG

QALGSGIQPLNGEVWRCNESQGDGSAACSCQDCAASCPVIAQPPALDSTFRLGQM

PGSLVLIIILCSVFVLLFAFLVGSRVASCRGKDKAKDPKKGTSCSDKLSLSTHTLLGRL

FQSWGTWVASWPLTVLAVSVIVWALAGGLAFIELTTDPVELWSAPNSQARQEKAF

HDQHFGPFFRTNQVILTAPNRPSYRYDSLLLGPKNFSGILSLDLLLELLELQERLRHL

QVWSPEEQRNISLQDICYAPLNPHNASLSDCCVNSLLQYFQNNRTNLLLTANQTLM

GQTSQVDWRDHFLYCANSPLTFKDGTALALSCMADYGAPVFPFLAVGGYKGKDYS

EAEALIMTFSLNNYPAGDPRLAQAKLWEEAFLEEMRAFQRRTAGNFQVTFMAERSL

EDEINRTTAEDLPIFAISYLVIFLYISLALGSYSSWSRVLVDSKATLGLGGVAVVLGAV

LASMGFFSYLGVPSSLVILQVVPFLVLAVGADNIFIFVLEYQRLPRRPGEPREVHIGR

ALGRVAPSMLLCSLSEAICFFLGALTPMPAVRTFALTAGLAVILDFLLQMSAFVALLSL

DSRRQEASRLDVCCCVKAQKLPPPKQGEGLLLRFFRKFYAPFLLHRVTRGVVLLLF

LALFGVSLYFMCHISVGLDQELALPKDSYLLDYFLFLNRYFEVGAPVYFVTTGGYNF

SSEAGMNAICSSAGCDNFSLTQKIQYATEFPEQSYLAIPASSWVDDFIDWLTPSSCC

RLYAFGPNKDEFCPSTVNSLNCLKKCMSITLGPVRPSVEQFHKYLPWFLNDRPNIK

CPKGGLAAYDTSVNLSSDGQILDTVAILSPRLEYSGTISAHCNLYLLDSASRFMAYHK

PLKNSQDYTEALRAARELAANITADLRKVPGTDPAFEVFPYTITNVFYEQYLTIVPEG

LFMLALCLVPTFAVCCLLLGMDLRSGLLNLFSIIMILVDTVGFMALWGISYNAVSLINL

VTAVGISVEFVSHITRSFAISTKPTRLERAKEATISMGSAVFAGVAMTNLPGILVLGLA

KAQLIQIFFFRLNLLITLLGLLHGLVFLPVILSYLGPDVNQALVLEQKRAEEAVAAVME

ASCPNHPSRVSTADNIYVNHSFEHPAKGAGAISSSLPNNGRQF

Alignment between NPC1 L1 TI and NPC1 L1WT is illustrated on Fig. 14. NPC1 L1 TI shares ~ 91 % amino acid sequence identity with NPC1 L1WT (Sequence 12). NPC1 L1 TI expresses at similar levels than NPC1 L1WT in HEK293 cells, but shows a strong improvement in detergent solubility giving rise to a major dominant peak in the size exclusion chromatogram of clear lysates (Fig. 12). These results argue that the consensus approach is a universal method that can be applied to challenging targets with limited phylogenetic information on the protein target.

The consensus-based approaches illustrated by ΕΑΑΤ1 τι , ΕΑΑΤ1 τ2 and NPC1 L1 TI can be generalized to other membrane proteins to obtain stable and functional proteins for structural, as well as biophysical analysis. The methods of the invention allow reduction of the number of constructs to be screened or tested by nearly two orders of magnitude compared to current method like alanine screening and direct evolution, since only a few synthetic genes containing the consensus mutations have to be tested. Furthermore, the methods of the invention allow an easy and eventually automated construct design; based on simple phylogenic analysis of protein sequences and algorithms available online through web-based servers, the calculation and of amino acid consensus sequences is simple.

METHODS

Construct optimization

Fluorescence-detection size-exclusion chromatography (FSEC)⁵¹ was used to screen solubilization conditions and EAAT1 variants fused to enhanced green fluorescent protein (eGFP). EAAT1 N-terminal fusions solubilized in dodecanoyl sucrose (DDS, Anatrace) were found to have good solubility and mono-dispersity by FSEC in clear lysates. However, EAAT1 loses its transport activity and chromatographic monodispersity upon purification. To increase its stability, consensus mutagenesis²⁶ was used according to the method of the invention as described in Example 1 , and EAAT1 variants were screened with different consensus mutations in the predicted transmembrane helices by FSEC. The apparent melting temperature (Tm) of the most stable EAAT1 construct was >20 °C over that of the wild-type EAAT1 , but the mutated transporter was still refractory to crystallization. It The extracellular region between TM3-4c was changed for the shorter TM3-4c sequence from ASCT2 (Fig.4). In addition, the two predicted N-glycosylation sites of the transporter (N155T and N204T mutations) were mutated. The obtained mutant showed improved crystallizability.

The transporter obtained this way, called EAAT1 _cryst, retained the functional and pharmacological properties of the EAATI WT upon purification and has the following sequence:

Sequence-3

MTKSNGEEPKMGGRMERFQQGVSKRTLLAKKKVQNITKEDVKSFLRRNALLLLTVL A VI LG VVLGFLLRPYPLSPREVKYFAFPGELLMRMLKMLILPLI VSSLITGLA SLDAKA SGRLGMRA VVYYMSTTIIA VVL GIIL VLIIHPGAA SAA I TA S VGA A GSA ENAPSKE VLD SFLDLARNIFPSNL VSAAFRS YS TTYEER Tl TG TR VKVP VGQE VEGMNIL GL VVFSMV FGFALGKMGEQGQLL VDFFNSLNEA TMKL VAIIMWYAPLGILFLIA GKI VEMEDLEVL GGQLGMYMVTVIVGLVIHGLIVLPLIYFLITRKNPFVFIAGILQALITALGTSSSSATLPIT FKCLEENNGVDKRITRFVLPVGA TINMDGTALYEA VAAIFIAQVNNYELDFGQIITISIT A TAA SIGAA GIPQA GL VTMVI VLTA VGLPTDDITLIIA VDWLLDRFRTMVNVLGDALGA

Gl VEHLSRKELEKQDAELGNS VIEENEMKKPYQLIA QDNETEKPIDSETKM Expression and purification

All constructs were introduced into pcDNA3.1 (+) (Invitrogen) with N-terminal Strep-tag II affinity tag followed by eGFP and PreScission protease cleavage site, and expressed in HEK293F cells (ATCC, mycoplasma test negative) grown in Ex-Cell®293 medium (Sigma) and supplemented with 4mM L-glutamine (Sigma) and 5 g/ml Phenol red (Sigma-Aldrich) to densities of 2.5 x 10⁶ cells ml^"1. Cells were transiently transfected in Freestyle™293 medium (Invitrogen) using poly-ethylenimine (PEI) (Tebu-bio) at a density of 2.5 x 10⁶ cells ml^"1 , diluted with an equivalent volume of Ex- Cell®293 6 hours post-transfection, and treated with 2.2 mM valproic acid (Sigma) 12 hours after dilution of the cultures. Cells were collected at ~48 h post-transfection.

Initial screens of constructs and detergent solubilization buffers were done in small-scale (5-10 ml), and cells were collected, mechanically disrupted with a douncer and solubilized in 50 mM HEPES/Tris-base, pH 7.4, 200 mM NaCI buffer supplemented with 1 mM L-asp, 1 mM EDTA, 1 mM Phenylmethylsulfonyl fluoride (PMSF), 1 mM Tris(2-carboxyethyl)phosphine (TCEP), 1 :200 (v/v) dilution of mammalian protease inhibitor cocktail (Sigma), 10 % glycerol, 2% detergent and 0.4% cholesterol hemisuccinate (CHS) (Anatrace). After 1 -hour incubation at 4 °C, clear lysates were obtained by ultracentrifugation (247,000 g for 45 min). A high-throughput auto-sampler was used to inject the lysates in a SRT SEC-500 column (Sepax Technologies) equilibrated in 50 mM HEPES/Tris-base, pH 7.4, 200 mM NaCI buffer supplemented with 1 mM L-asp, 1 mM (TCEP), 5 % glycerol, 3 x CMC (Critical Micelle Concentration) detergent, and ~0.01 % CHS, in line with fluorescence detection (Photon technology international) for FSEC analysis.

Large-scale expression was done in 2-4 I cultures with cells collected in 50 mM HEPES/Tris-base, pH 7.4, 50 mM NaCI buffer supplemented with 1 mM L-asp, 1 mM EDTA, 1 mM PMSF, 1 mM TCEP, and 1 :200 (v/v) dilution of mammalian protease inhibitor cocktail (Sigma), and disrupted in a cell homogenizer (EmulsiFlex-C5, Avestin) after 3 runs at 15,000 Psi. The resulting homogenate was clarified by centrifugation (4,500 g, 0.5 h) and the crude membranes were collected by ultracentrifugation (186,000 g for 1 .5 h). Membranes were washed once with the above-mentioned buffer and finally homogenized with a douncer in a buffer containing 50 mM HEPES/Tris-base, pH 7.4, 200 mM NaCI, 1 mM L-asp, 1 mM EDTA, 1 mM TCEP, and 10% Glycerol, snap-frozen in liquid N2 and stored at -80°C at 0.5 g of membranes ml^"1. Membrane solubilization was done by thawing out and supplementing the membrane homogenate with 2% DDS, 0.4% CHS, and 25 μΜ UCPHioi (Abeam). After 1 -hour incubation, the insoluble material was removed by ultracentrifugation (186,000g for 1 h), and Strep-Tactin® sepharose® resin (GE Healthcare) was added to the supernatant and rotated for 2 h. Resin was washed with 25 column volumes of 50 mM HEPES/Tris-base, pH 7.4, 200 mM NaCI, 1 mM L-asp, 1 mM TCEP, 5% Glycerol, 0.05% DDS, 0.01 % CHS and 25 μΜ UCPHioi , and the protein was eluted with the same buffer supplemented with 2.5 mM D-desthiobiotin.

The eluted eGFP-transporter fusion was concentrated to 1 -2 mg ml^"1 using 100- kDa cutoff membranes (Millipore), and digested with His-tagged PreScission protease overnight at 4 °C. The protease was removed by reverse Ni-NTA (Qiagen) affinity chromatography, and the flow through containing the transporter was concentrated to 500 μΙ, ultra-centrifuged (86,900 g, 20 min), and applied to a Superose 6 10/300 gel filtration column (GE Healthcare) equilibrated with 50 mM HEPES/Tris-base, pH 7.4, 200 mM NaCI, 1 mM L-asp, 1 mM TCEP, 5% Glycerol, 0.25% decanoyl sucrose (DS, Sigma), 0.05% CHS and 100 μΜ UCPHioi . To obtain the UCPH101 unbound structure, the protocol was identical, but the allosteric inhibitor was omitted from all buffers. To obtain the UCP HI OI -TBOATFB bound structure, the protein sample was supplemented with 3 mM TBOATFB (Tocris) before the injection in the gel filtration column equilibrated with 50 mM HEPES/Tris-base, pH 7.4, 200 mM NaCI, 1 mM TCEP, 5% Glycerol, 0.25% decanoyl sucrose (DS, Sigma), 0.05% CHS, 300 μΜ TBOATFB and 100 μΜ UCPHioi . Protein samples after the solubilization step were kept on ice or at 4 °C at all times.

Crystallization and structure determination

Purified protein was concentrated to 3.5-4.0 mg ml^"1 and 1 mM UCP H 101 was added in experiments with the inhibitor-bound transporters. Initial vapor diffusion crystallization screens were done by mixing 300 nl of protein and reservoir solution in sitting drops, dispensed by a Mosquito® robot (TTP labtech) in 96-well Greiner plates. The purified transporters form three-dimensional crystals in several conditions containing low molecular weight polyethylene glycols. The best-diffracting crystals were obtained after manual optimization using 1.6 μΙ hanging drops at 4 °C, obtained by mixing equal volumes of protein supplemented with 0.2% n-Octyl-b-D- glucopyranoside (BOG, Anatrace) and 0.04% CHS, and reservoir solutions containing 100 mM Tris, pH 8.2, 50 mM CaCI₂, 50 mM BaCI₂, and 28-30% PEG 400. Crystals appeared after 24-48 h and reached their maximum size after a week. Crystals were flash-frozen in liquid nitrogen before X-ray diffraction data collection without any further cryo protection.

X-ray diffraction data were collected at beamlines PROXIMA-1 at the SOLEIL synchrotron (St Aubin, France) and at beamlines at the European Synchrotron Radiation Facility (Grenoble, France). In general, 2-3 data sets from single crystals were collected, and indexed, integrated, scaled and merged using XDS package⁵². Due to the anisotropic nature of the diffraction data, the DEBYE and STARANISO programs were applied to scale it using the STARANISO server (http://staraniso.globalphasing.org/). The software performs an anisotropic cut-off of merged intensity data with a Bayesian estimation of the structure amplitudes, and applies an anisotropic correction to the data. Table 1 shows the refinement statistics for the full sets of reflections truncated at the best high-resolution along h, k or I axis, values given by AIMLESS⁵³, before the anisotropic corrections computed by the STARANISO software. The corrected anisotropic amplitudes were then used for molecular replacement in PHASER⁵⁴, using the scaffold and transport domains of GltPh (PDB code 2NWL) as independent search models. The initial electron density maps were clearly interpretable, and the final model was obtained through rounds of manual building in COOT⁵⁵ and refinement in Buster⁵⁶, until reaching good crystallographic statistics and stereochemistry. The model contains one EAAT1 _cryst monomer per asymmetric unit and most of the EAAT1 _cryst polypeptide (residues 37- 487), with the exception of some residues in the extracellular loops between TM3-4a, TM4b-4c, TM5-6 and TM7b-HP2a. Sequence assignment was aided by anomalous difference Fourier maps from diffraction data collected with low energy X-rays (1.77 A) to highlight the sulfur atoms of methionine and cysteine residues. EAAT1 _Cryst-n, as well as the EAATI cryst UCPHIOI -TBOATFB bound structures were solved by the same approach above mentioned, but using the EAAT1 _cryst TranD and ScaD as independent search models for molecular replacement.

The stereochemical properties of the final models were analyzed with the Molprobity server (http://molprobity.biochem.duke.edu/). At least 95% of the residues in all models are in the Ramachandran favored region. Protein interfaces were analyzed with the PISA server (http://www.ebi.ac.uk/msd-srv/prot_int/pistart.html). Structural alignments were done with Superpose in the CCP4 suite. All structural figures were prepared with PyMOL Molecular Graphics System, Schrodinger, LLC. Radioactive substrate transport assays

Unilamellar liposomes were made at 9: 1 molar ratio of 1 -palmitoyl-2-oleoyl-sn- glycero-3-phosphocholine (Avanti Polar Lipids) and CHS, in a buffer containing 50 mM HEPES/Tris-base, pH 7.4, 200 mM NaCI and 1 mM L-asp. The transporters were purified as described above, but excluding the reverse chromatography step after protease cleavage, and using a Superose 6 10/300 column equilibrated with 50 mM HEPES/Tris-base, pH 7.4, 200 mM NaCI, 1 mM L-asp, 0.5 mM TCEP, 0.0632% DDS, 0.01264% CHS, and 5% glycerol.

To reconstitute the protein, liposomes were first mixed with DDS at a 1 :2 (w/w) lipid-to-detergent ratio for 1 h, and then the purified transporters were added at a 1 :40 (w/w) protein-to-lipid ratio. Detergent removal was done at 4° C using SM-2 biobeads (BioRad) at 100 mg ml^"1. The internal solution of the liposomes was exchanged using 10 freeze-thaw cycles in the appropriate buffer. After extrusion through 400-nm polycarbonate membranes (Avanti Polar Lipids), the proteoliposomes were concentrated by ultracentrifugation (150,000 g for 30 min at 4°C) and resuspended at 20 mg of lipids ml^"1 , for immediate use.

Substrate transport was assayed at 37°C. The uptake reaction was initiated by diluting the proteo-liposomes 10-fold into a buffer containing 50 mM HEPES/Tris-base, pH 7.4, 200 mM NaCI, 50 μΜ L-glutamate, and 5 μΜ [¹⁴C]-L-glutamate (PerkinElmer), and 2.5% glycerol. After 30 min, 200-μΙ aliquots were diluted 5-fold into ice-cold quench buffer (50 mM HEPES/Tris-base, pH 7.4, 200 mM ChCI, and 2.5% glycerol), followed by immediate filtration and wash on nitrocellulose 0.22-μηΊ filters (Millipore). Radioactivity was quantified by liquid scintillation using a Tri-Carb 31 10TR counter (PerkinElmer). For the UCPH101 titrations, proteo-liposomes were both pre-incubated for 20 min at room temperature, and assayed in the presence of UCPH101. Background radioactivity was estimated from protein-free liposomes, and subtracted from the uptake data. Data was fitted to a Hill equation of the form:

F=F- + deltaF-/(1 +(IC₅o/[UCPHioi])ⁿ)

Where F~ is the final level of inhibition, deltaF~ is the final amplitude of the UCPH101 effect, and n is the Hill coefficient.

To titrate the rate of L-glutamate transport by EAAT1 _cryst, proteo-liposomes were assayed in the presence of 0, 5, 50 or 200 μΜ L-glutamate supplemented with 1 , 5, 5, or 5 μΜ [¹⁴C]-L-glutamate, respectively. At each substrate concentration, the initial rate of transport was calculated by a linear fit to 120 s and 180 s uptake measurements with origin fixed at zero. Background radioactivity was estimated from protein-free liposomes, and subtracted from the uptake data.

For the cell-based transport uptake, cells were collected 36 h post-transfection, and washed three times and resuspended at a density of 50 x 10⁶ cells ml^"1 in 1 1 mM HEPES/Tris-base, pH 7.4, 140 mM ChCI, 4.7 mM KCI, 2.5 mM CaCI₂, 1.2 mM MgCI₂, and 10 mM D-glucose, for immediate use. The uptake assay was performed similarly to the one described for the proteo-liposomes, but using a reaction buffer containing 1 1 mM HEPES/Tris-base, pH 7.4, 140 mM NaCI, 4.7 mM KCI, 2.5 mM CaCI₂, 1 .2 mM MgC , 10 mM D-glucose, 50 μΜ L-glutamate, and 5 μΜ [¹⁴C]-L-glutamate, and 0.8- m nitrocellulose filters. Background radioactivity was estimated from cells transfected with empty vector, and subtracted from the uptake data.

Hydrogen-deuterium exchange mass spectrometry

HDX-MS experiments were performed with transporters purified as described in the proteo-liposome section, and using a Superose 6 5/150 gel filtration column equilibrated with 50 mM HEPES/Tris-base, 200 mM NaCI, pH7.4, 1 mM L-asp, 0.5 mM TCEP, 0.0632% DDS, 0.01264% CHS, and 5% glycerol.

The purified EAAT1 _cryst was incubated in ice for 30 min with 2.2% DMSO at a monomer concentration of 5.2 μΜ, in the presence and absence of 102 μΜ UCPHioi , respectively. Prior to labeling, 10 μΙ_ of the unbound and UCPHioi-bound EAAT1 _cryst solution was equilibrated for 10 min at room temperature. Deuterium exchange was initiated by adding 40 μΙ_ of D₂0 buffer (50 mM HEPES, pH 7.3, 200 mM NaCI, 1 mM L-asp, 5% glycerol, 0.0632% DDS, 0.01264% CHS, 0.5 mM TCEP) supplemented or not with 101 .2 μΜ UCPHioi . Assuming that the K_D and IC50 values of UCPH101 are similar, ~96% of the transporters would remain bound during deuterium labeling. Aliquots of 10.4 pmols of protein were removed at defined deuterium exchange time points (from 10 sec to 60 min) and quenched upon mixing with an ice-cold acidic solution (0.75% formic acid, 5% glycerol) to decrease the pH to 2.6. Quenched samples were immediately snap-frozen in liquid N₂ and stored at -80°C until analysis.

Prior to mass analysis, quenched samples were rapidly thawed and immediately injected into a cooled nanoACQUITY UPLC® HDX system (Waters corp.) maintained at 0°C. 8.6-pmol protein samples were on-line digested for 2 min at 20 °C using an in- house packed immobilized pepsin cartridge (2.0 x 20 mm, 66 μΙ_ bed volume). The resulting peptides were trapped and desalted onto a C18 Trap column (VanGuard BEH 1 .7 μίτι, 2.1 x 5 mm, Waters corp.) at a flow rate of 100 μΙ_Ληίη of 0.15% formic acid, and then separated in 10 min by a linear gradient of acetonitrile from 5 to 40% at 40 ML/min using an ACQUITY UPLC^® BEH C18 analytical column (1 .7 Mm, 1 x 100 mm, Waters corp.). After each run, the pepsin cartridge was manually cleaned with two consecutive washes of 1 % formic acid, 5% acetonitrile, 1.5 M guanidinium chloride, pH 2.5. Blank injections were performed between each run to confirm the absence of carry-over.

Mass spectra were acquired in resolution and positive mode on a Synapt G2-Si HDMS mass spectrometer (Waters corp.) equipped with a standard electrospray ionization source, as described previously⁵⁷. Peptides were identified from undeuterated protein samples acquired in MS^E mode by database searching in ProteinLynX Global Server 3.0 (Waters corp.). Each fragmentation spectrum was manually inspected for assignment validation. Deuterium uptake values were calculated for each peptide using DynamX 3.0 (Waters corp.). Only one unique charge state was considered per peptide and no adjustment was made for back-exchange. HDX-MS results are reported as relative deuterium uptake values expressed in mass unit or fractional exchange⁵⁷. A statistical analysis was performed with MEMHDX⁵⁸ using a False Discovery Rate of 1 %. References

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Claims

A process to produce a protein with modified thermal stability in a detergent solution with respect to the thermal stability of a target protein comprising: a. providing a multiple sequence alignment (MSA) wherein at least a portion comprising at least 50 % of the full-length amino acid sequence of a target protein is aligned, in particular the full-length amino acid sequence of the target protein is aligned, with the amino acid sequence of homologous sequences of said protein present (i) in other species and/or (ii) in protein variants of the same species, using a determined alignment tool; wherein the homologous sequences are each different from the amino acid sequence of the target protein by at least one amino acid residue in their amino acid sequence, in particular wherein each homologous sequence shares at least 50% identity, more preferably at least 70% identity, with the amino acid sequence of the target protein or with the aligned portion thereof;

c. providing a mutated amino acid sequence defining a mutant protein of the target sequence by substituting one or more amino acid residues in the amino acid sequence of the target protein by its(their) respective consensus amino acid residue(s) defined for said each amino acid residue according to step b.; d. obtaining a polynucleotide encoding the mutant protein of step c. for expression in a cellular expression system;

e. producing the mutant protein from the mutated amino acid sequence provided in step c in a cellular expression system;

A process to design a protein with modified thermal stability in a detergent solution with respect to the thermal stability of a target protein comprising: a. providing a multiple sequence alignment (MSA) wherein at least a portion comprising at least 50 % of the full-length amino acid sequence of a target protein is aligned, in particular the full-length amino acid sequence of the target protein is aligned, with the amino acid sequence of homologous sequences of said protein present (i) in other species and/or (ii) in protein variants of the same species, using a determined alignment tool; wherein the homologous sequences are each different from the amino acid sequence of the target protein by at least one amino acid residue in their amino acid sequence, in particular wherein each homologous sequence shares at least 50% identity, more preferably at least 70% identity, with the amino acid sequence of the target protein or with the aligned portion thereof;

iii. a frequency of occurrence which is higher than 10% with respect to the frequency of occurrence of the residue at the same position in the target amino acid sequence when said frequency is evaluated among the aligned amino acid sequences; c. providing a mutated amino acid sequence defining a consensus mutant protein of the target sequence by substituting one or more amino acid residues in the amino acid sequence of the target protein by its(their) respective consensus amino acid residue(s) defined for said each amino acid residue according to step b.

3. A process according to claim 1 or 2, wherein the amino acid residues targeted for substitution are selected among residues located in predicted or characterized secondary structural element(s) of the target protein and/or in loops structures.

4. A process according to any one of claims 1 to 3, wherein the amino acid residues targeted for substitution are selected among residues which are present in pairs of residues that show co-evolution in the target protein.

5. A process according to any one of claims 1 to 4, wherein the amino acid residues targeted for substitution are contained in a characterized or a predicted alpha-helice structure of the target protein.

6. A process according to any one of claims 1 to 5, wherein the protein is selected from integral membrane proteins, membrane protein complexes, water-soluble proteins.

7. A process according to any one of claims 1 to 6, wherein the target protein is selected in the group of membrane proteins, integral membrane proteins, soluble proteins, globular soluble proteins.

8. A process according to any one of claims 1 to 7, wherein the target protein is selected in the group of membrane proteins, integral membrane proteins, soluble proteins, globular soluble proteins, wherein said protein originates from a pathogenic bacterium, virus or parasite.

9. A process according to any one of claims 1 to 8, wherein the target protein is selected in the group of membrane proteins and is a eukaryotic membrane protein, in particular a mammalian membrane protein, especially a human membrane protein, and in particular a human integral membrane protein.

10. A process according to any one of claims 1 to 9, wherein the membrane protein is selected among channels, enzymes, and primary or secondary active transporters.

11. A process according to any one of claims 1 to 10, wherein the thermal stability in detergent solution of the mutant protein is evaluated by size-exclusion chromatography of detergent-solubilized protein expressed in step e. and optionally compared to the thermal stability of the target protein in the same conditions.

12. A process according to any one of claims 1 to 1 1 , wherein the mutant protein is fused to a detectable marker, such as a fluorescent marker, for the evaluation of the melting temperature of the mutant protein.

13. A process to improve thermal stability of a target protein in detergent solution comprising carrying out the process of any one of claims 1 to 12 and selecting the mutant protein(s) having improved thermal stability with respect to the target protein.

14. A process according to any one of claims 1 to 13, wherein the mutant protein has a sequence identity with the target protein which is at least 80%, more preferably at least 85%, in particular the mutant protein has a sequence similarity with SEQ ID No: 1 1 or SEQ ID No: 12 which is at least 85%.

15. A process according to any one of claims 1 to 13, wherein the number of homologous sequences used in the MSA is at least 7, and more preferably at least 10.

16. A process for the preparation of a protein with modified crystallization capability comprising preparing a protein having modified thermal stability in a detergent solution according to any one of claims 1 to 15, wherein an optional step of mutation is performed after step c. and in particular before step d. to further improve crystallization capability, such optional step of mutation being in particular a step of mutation in the extracellular region of the target protein.

17. A process according to any one of claims 1 to 16, wherein the target protein is the Excitatory Amino Acid Transporter 1 (EAAT1 ) and the obtained mutant protein is EAAT1 of sequence SEQ ID No.1 SEQ ID No.2, SEQ ID No.3 or SEQ ID No.4.

18. A process according to any one of claims 1 to 16, wherein the target protein is the Niemann-Pick C1 -Likel (NPCL1 ) and the obtained mutant is NPCL1 of SEQ ID No.13.

19. A process according to any one of claims 1 to 18, wherein the determined alignment tool is JALVIEW software.

20. A mutant protein of Excitatory Amino Acid Transporter 1 (EAAT1 ) the amino acid sequence of which is SEQ ID No.1 SEQ ID No.2, SEQ ID No.3 or SEQ ID No.4.

21. A mutant protein of Niemann-Pick C1 -Likel (NPCL1 ) the amino acid sequence of which is SEQ ID No. 13.

22. A liposome or a collection of liposomes containing a mutant protein obtained from the process according to any one of claims 1 to 19.

23. A cell expressing a mutant protein obtained from the process according to any one of claims 1 to 19.

24. A cell or a liposome comprising a mutant protein of Excitatory Amino Acid Transporter 1 (EAAT1 ) the amino acid sequence of which is SEQ ID No.1 SEQ ID No.2, SEQ ID No.3 or SEQ ID No.4.

25. A cell or a liposome comprising a mutant protein of Niemann-Pick C1-like1 (NPCL1 ) the amino acid sequence of which is SEQ ID No. 13.