Use of the substituted cysteine accessibility method to study the structure and function of G protein-coupled receptors

doi:10.1016/S0076-6879(02)43131-X

Methods in Enzymology

Volume 343, 2002, Pages 137-156

https://doi.org/10.1016/S0076-6879(02)43131-X Get rights and content

Publisher Summary

Cysteine substitution and covalent modification have been used to study structure–function relationships and the dynamics of protein function in a variety of membrane proteins. Charged, hydrophilic, sulfhydryl reagents have been used to probe systematically the accessibility of substituted cysteines in putative transmembrane segments of a number of proteins. This approach, the substituted cysteine accessibility method (SCAM), has been used to map channel-lining residues in the nicotinic acetylcholine receptor, the GABA receptor, the cystic fibrosis transmembrane conductance regulator, the UhpT transporter, and potassium channels, among others. This chapter uses this approach to map the surface of the binding-site crevice in the dopamine D2 receptor, a member of the G protein-coupled receptor (GPCR) superfamily. SCAM provides an approach to map systematically residues on the water-accessible surface of a protein. These residues are identified by substituting them with cysteine and assessing them for the reaction of charged, hydrophilic, sulfhydryl reagents with the engineered cysteines. Consecutive residues in putative transmembrane segments are mutated to cysteine, one at a time, and the mutant proteins are expressed in heterologous cells.

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Mapping Protein Binding Sites and Conformational Epitopes Using Cysteine Labeling and Yeast Surface Display
2017, Structure
Citation Excerpt :
A significant disadvantage of this technique is that one needs to express and purify all of the cysteine mutants individually. Other approaches have involved labeling of single Cys mutants in membrane proteins, followed by functional assays to determine residues in ligand binding sites (Frillingos et al., 1998; Javitch et al., 2002). However, these require mutants to be individually transfected and assayed.
We describe a facile method for mapping protein:ligand binding sites and conformational epitopes. The method uses a combination of Cys scanning mutagenesis, chemical labeling, and yeast surface display. While Ala scanning is widely used for similar purposes, often mutation to Ala (or other amino acids) has little effect on binding, except at hotspot residues. Many residues in physical contact with a binding partner are insensitive to substitution with Ala. In contrast, we show that labeling of Cys residues in a binding site consistently abrogates binding. We couple this methodology to yeast surface display and deep sequencing to map conformational epitopes targeted by both monoclonal antibodies and polyclonal sera as well as a protein:ligand binding site. The method does not require purified protein, can distinguish buried and exposed residues, and can be extended to other display formats, including mammalian cells and viruses, emphasizing its wide applicability.
Probing the Structure and Function Relationships of Presenilin by Substituted-Cysteine Accessibility Method
2017, Methods in Enzymology
Presenilin is a catalytic subunit of γ-secretase, which hydrolyzes several transmembrane proteins within the lipid bilayer, together with binding cofactors such as nicastrin, Aph-1, and Pen-2. However, the structural basis as well as molecular mechanism of this unusual proteolytic process remains unknown. We have analyzed the structure and function relationships of presenilin using the substituted-cysteine accessibility method (SCAM), which enables identification of the hydrophilic environment by the accessibility of sulfhydryl reagents to cysteine residues introduced at a desired position. In combination with small molecule inhibitors/modulators and cross-linking experiments, we were able to identify certain residues and regions of presenilin that contribute to its intramembrane-cleaving activity. In addition, we revealed the structural dynamics of the transmembrane domains of presenilin during the formation of the complex and its proteolytic process. The SCAM provides new insights into the relationship between the structure and activity of presenilin, and is useful for probing the protein dynamics of the membrane-embedded enzymes.
Identification of transmembrane domain 1 & 2 residues that contribute to the formation of the ligand-binding pocket of the urotensin-II receptor
2014, Biochemical Pharmacology
Citation Excerpt :
Using this approach, we had previously identified residues in TM3, TM4, TM5, TM6 and TM7 that participated in the formation of the rUT receptor binding pocket [34,36]. The SCAM method is based on the reactivity of engineered cysteines to MTSEA, a reagent that reacts 109 times faster with ionized thiol compared to un-ionized thiol [28] and covalently alkylates any cysteine located in a hydrophilic environment. Indeed lipid-exposed, buried, or disulfide-bonded cysteines are unlikely to ionize to a significant extent and hence are assumed to be unaffected by such modification induced by MTSEA.
The vasoactive urotensin-II (UII), a cyclic undecapeptide widely distributed in cardiovascular, renal and endocrine systems, specifically binds the UII receptor (UT receptor), a G protein-coupled receptor (GPCR). The involvement of this receptor in numerous pathophysiological conditions including atherosclerosis, heart failure, hypertension, renal impairment and diabetes potentially makes it an interesting therapeutic target. To elucidate how UII binds the UT receptor through the identification of specific residues in transmembrane domains (TM) one (TM1) and two (TM2) that are involved in the formation of the receptor's binding pocket, we used the substituted-cysteine accessibility method (SCAM). Each residue of TM1 (V49^(1.30) to M76^(1.57)) and TM2 (V88^(2.41) to H117^(2.70)) was mutated, one by one, to a cysteine. The resulting mutants were then expressed in COS-7 cells and subsequently treated with the sulfhydryl-specific alkylating agent methanethiosulfonate-ethylammonium (MTSEA). MTSEA treatment resulted in a significant binding inhibition of ¹²⁵I-UII to mutant I54C^(1.35) in TM1 and mutants Y100C^(2.53), S103C^(2.56), F106C^(2.59), I107C^(2.60), T110C^(2.63) and Y111C^(2.64) in TM2. These results identify key structural residues in TM1 and TM2 that participate in the formation of the UT receptor binding pocket. Together with previous SCAM analysis of TM3, TM4, TM5, TM6 and TM7, these results have led us to identify residues within all 7 TMs that participate in UT's binding pocket and have enabled us to propose a model of this receptor's orthosteric binding site.
Structural-functional analysis of the third transmembrane domain of the corticotropin-releasing factor type 1 receptor role in activation and allosteric antagonism
2014, Journal of Biological Chemistry
The corticotropin-releasing factor (CRF) type 1 receptor (CRF₁R) for the 41-amino acid peptide CRF is a class B G protein-coupled receptor, which plays a key role in the response of our body to stressful stimuli and the maintenance of homeostasis by regulating neural and endocrine functions. CRF and related peptides, such as sauvagine, bind to the extracellular regions of CRF₁R and activate the receptor. In contrast, small nonpeptide antagonists, which are effective against stress-related disorders, such as depression and anxiety, have been proposed to interact with the helical transmembrane domains (TMs) of CRF₁R and allosterically antagonize peptide binding and receptor activation. Here, we aimed to elucidate the role of the third TM (TM3) in the molecular mechanisms underlying activation of CRF₁R. TM3 was selected because its tilted orientation, relative to the membrane, allows its residues to establish key interactions with ligands, other TM helices, and the G protein. Using a combination of pharmacological, biochemical, and computational approaches, we found that Phe-203^3.40 and Gly-210^3.47 in TM3 play an important role in receptor activation. Our experimental findings also suggest that Phe-203^3.40 interacts with nonpeptide antagonists.
Analysis by substituted cysteine scanning mutagenesis of the fourth transmembrane domain of the CXCR4 receptor in its inactive and active state
2013, Biochemical Pharmacology
The chemokine SDF-1 (CXCL12) selectively binds to CXCR4, a member of the G protein-coupled receptor (GPCR) superfamily. In this study, we used the substituted-cysteine accessibility method (SCAM) to identify specific residues of the fourth transmembrane domain (TM4) that contribute to the formation of the binding pocket of CXCR4 in its inactive and active state. We successively substituted each residue from E179^(4.68) to K154^(4.43) with cysteine and expressed the mutants in COS-7 cells. Mutant receptors were then alkylated with methanethiosulfonate-ethylammonium (MTSEA), and binding inhibition was monitored using the CXCR4 antagonist FC131 [cyclo(-D-Tyr¹-Arg²-Arg³-Nal⁴-Gly⁵-)], which displays anti-HIV activity. MTSEA treatment resulted in a significant reduction of FC131 binding to D171C^(4.60) and P170C^(4.59). To assess TM4 accessibility in an active state of CXCR4, TM4 cysteine mutants were transposed within the constitutively active mutant N119S^(3.35). MTSEA treatment of TM4 mutants N119S-S178C^(4.67), N119S-V177C^(4.66) and N119S-I173C^(4.62) resulted in a significant reduction in FC131 binding. Protection assays using FC131 prior to MTSEA treatment significantly reduced the alkylation of all MTSEA-sensitive mutants. The accessibility of the D171C^(4.60) and P170C^(4.59) residues suggests that they are oriented towards a water-accessible area of the binding pocket of CXCR4. S178C^(4.67), V177C^(4.66) and I173C^(4.62) showed binding inhibition only in an N119S^(3.35) background. Taken together our results suggest that TM4 and ECL2 undergo conformational changes during CXCR4 activation and also demonstrate how TM4 is an important feature for the binding of anti-HIV compounds.
Identification of transmembrane domain 3, 4 & 5 residues that contribute to the formation of the ligand-binding pocket of the urotensin-II receptor
2013, Biochemical Pharmacology
Urotensin-II (UII), a cyclic undecapeptide, selectively binds the urotensin-II receptor (UT receptor), a G protein-coupled receptor (GPCR) involved in cardiovascular effects and associated with numerous pathophysiological conditions including hypertension, atherosclerosis, heart failure, pulmonary hypertension and others. In order to identify specific residues in transmembrane domains (TM) three (TM3), four (TM4) and five (TM5) that are involved in the formation of the UT receptor binding pocket, we used the substituted-cysteine accessibility method (SCAM). Each residue in the F118^(3.20) to S146^(3.48) fragment of TM3, the L168^(4.44) to G194^(4.70) fragment of TM4 and the W203^(5.30) to V232^(5.59) fragment of TM5, was mutated, individually, to a cysteine. The resulting mutants were then expressed in COS-7 cells and subsequently treated with the positively charged sulfhydryl-specific alkylating agent methanethiosulfonate-ethylammonium (MTSEA). MTSEA treatment resulted in a significant reduction in the binding of ¹²⁵I-UII to TM3 mutants L126C^(3.28), F127C^(3.29), F131C^(3.33) and M134C^(3.36) and TM4 mutants M184C^(4.60) and I188C^(4.64). No loss of binding was detected following treatment by MTSEA for all TM5 mutants tested. In absence of a crystal structure of UT receptor, these results identify key determinants in TM3, TM4 and TM5 that participate in the formation of the UT receptor binding pocket and has led us to propose a homology model of the UT receptor.

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Use of the substituted cysteine accessibility method to study the structure and function of G protein-coupled receptors

Publisher Summary

J. Biol. Chem.

Biophys. J.

Methods Enzymol.

Neuron

Biophys. J.

J. Biol. Chem.

J. Biol. Chem.

Biophys. J.

Neuron

J. Biol. Chem.

J. Biol. Chem.

Anal. Biochem.

J. Biol. Chem.

Eur. J. Pharmacol.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Proteins

Science

Biochemistry

Science

Biochemistry

Biochemistry

Biochemistry

Biochemistry