title: Disulfide Trapping for Modeling and Structure Determination of Receptor: Chemokine Complexes.
abstract: Despite the recent breakthrough advances in GPCR crystallography, structure determination of protein-protein complexes involving chemokine receptors and their endogenous chemokine ligands remains challenging. Here, we describe disulfide trapping, a methodology for generating irreversible covalent binary protein complexes from unbound protein partners by introducing two cysteine residues, one per interaction partner, at selected positions within their interaction interface. Disulfide trapping can serve at least two distinct purposes: (i) stabilization of the complex to assist structural studies and/or (ii) determination of pairwise residue proximities to guide molecular modeling. Methods for characterization of disulfide-trapped complexes are described and evaluated in terms of throughput, sensitivity, and specificity toward the most energetically favorable crosslinks. Due to abundance of native disulfide bonds at receptor:chemokine interfaces, disulfide trapping of their complexes can be associated with intramolecular disulfide shuffling and result in misfolding of the component proteins; because of this, evidence from several experiments is typically needed to firmly establish a positive disulfide crosslink. An optimal pipeline that maximizes throughput and minimizes time and costs by early triage of unsuccessful candidate constructs is proposed.
authors.affiliation: Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, California, USA.
authors.forename: Irina
authors.initials: I
authors.lastname: Kufareva
authors.affiliation: Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, California, USA.
authors.forename: Martin
authors.initials: M
authors.lastname: Gustavsson
authors.affiliation: Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, California, USA.
authors.forename: Lauren G
authors.initials: LG
authors.lastname: Holden
authors.affiliation: Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, California, USA.
authors.forename: Ling
authors.initials: L
authors.lastname: Qin
authors.affiliation: Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, California, USA.
authors.forename: Yi
authors.initials: Y
authors.lastname: Zheng
authors.affiliation: Department of Pharmacology, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, California, USA. Electronic address: thandel@ucsd.edu.
authors.forename: Tracy M
authors.initials: TM
authors.lastname: Handel
mesh_terms: D001671:Biochemistry / Q000379:methods*; D015153:Blotting, Western; D018925:Chemokines / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry*; D004220:Disulfides / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D005434:Flow Cytometry; D008958:Models, Molecular; D046912:Multiprotein Complexes / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D055550:Protein Stability; D019707:Receptors, Chemokine / Q000737:chemistry* / Q000378:metabolism
chemical_list: D018925:Chemokines; D004220:Disulfides; D046912:Multiprotein Complexes; D019707:Receptors, Chemokine; D003545:Cysteine
keywords: ACKR3; CXCR4; Chemokine receptor; Crystallization construct engineering; Cysteine trapping; Disulfide crosslinking; G protein-coupled receptor; Membrane protein complex; Molecular modeling; Residue proximity restraints