The atomic-level mechanisms by which G protein-coupled receptors (GPCRs) transmit extracellular ligand binding events through their transmembrane helices to activate intracellular G proteins remain unclear. Using a comprehensive library of mutations covering all 352 residues of the GPCR CXC chemokine receptor 4 (CXCR4), we identified 41 amino acids that are required for signaling induced by the chemokine ligand CXCL12 (stromal cell-derived factor 1). CXCR4 variants with each of these mutations do not signal properly but remain folded, based on receptor surface trafficking, reactivity to conformationally sensitive monoclonal antibodies, and ligand binding. When visualized on the structure of CXCR4, the majority of these residues form a continuous intramolecular signaling chain through the transmembrane helices; this chain connects chemokine binding residues on the extracellular side of CXCR4 to G proteincoupling residues on its intracellular side. Integrated into a cohesive model of signal transmission, these CXCR4 residues cluster into five functional groups that mediate (i) chemokine engagement, (ii) signal initiation, (iii) signal propagation, (iv) microswitch activation, and (v) G protein coupling. Propagation of the signal passes through a "hydrophobic bridge" on helix VI that coordinates with nearly every known GPCR signaling motif. Our results agree with known conserved mechanisms of GPCR activation and significantly expand on understanding the structural principles of CXCR4 signaling.T he CXC chemokine receptor 4 (CXCR4) belongs to the G protein-coupled receptor (GPCR) superfamily of proteins, the largest class of integral membrane proteins encoded in the human genome, comprising greater than 30% of current drug targets. Deregulation of CXCR4 expression in multiple human cancers, its role in hematopoietic stem cell migration, and the utilization of CXCR4 by HIV-1 for T-cell entry, make this receptor an increasingly important therapeutic target (1). One FDA-approved drug against CXCR4 is currently on the market (Mozobil, for hematopoietic stem cell mobilization), and multiple additional drugs against this target are in development for oncology and other indications (2).The crystal structures of class A GPCR superfamily members in their active and inactive conformations (reviewed in refs. 3 and 4) provide unprecedented insight into the structural basis of ligand binding, G protein coupling, and activation of GPCRs via rearrangements of transmembrane (TM) helices. GPCR helices V and VI in particular, and in some cases III and VII, are known to undergo significant conformational changes upon activation (5-7). However, static images alone have not been able to explain the residue-level mechanisms underlying the dynamic helical shifts that mediate GPCR signal transduction. Additionally, only inactive state structures have been solved for CXCR4 and most other GPCRs (8,9). Over the last two decades, extensive mutagenesis studies of GPCRs in general [collectively describing >8,000 mutations (gpcrdb.org)] and of CX...
Alanine (Ala) scanning is a widely used mutagenesis approach for systematic evaluation of protein function, which for large targets can be laborious and time-consuming. We have developed a platform technology, Shotgun Mutagenesis, for rapid and comprehensive alanine scanning mutagenesis for protein mapping and engineering applications. Using this technology, every residue in a target protein is individually mutated, expressed in human cells, and assayed for desired functions. Critical mutations are rapidly identified using high-throughput 384-well assays including flow cytometry. Here we used Shotgun Mutagenesis to create comprehensive mutation libraries for different classes of targets including the GPCR CXCR4, bitter taste receptor TAS2R16 and the therapeutic antibody Palivizumab (Synagis). For the cancer target CXCR4, we identified critical residues for antibody binding (epitope mapping) and ligand-dependent activation (functional domain mapping). We also discovered TAS2R16 residues that can be altered for increased expression (protein optimization) and Palivizumab residues which tolerate germline substitutions permitting engineering of a maximally humanized antibody variant (antibody humanization). This platform allows comprehensive protein mapping and engineering of challenging targets previously considered prohibitively time-consuming or expensive, enabling discovery pathways for many untapped therapeutic targets. Citation Format: Cheryl Paes, Jason Goodman, Melanie Wescott, Yana Thaker, Anu Thomas, Joseph Couto, Joseph Rucker, Benjamin J. Doranz. Engineering and mapping difficult proteins using comprehensive mutagenesis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2441. doi:10.1158/1538-7445.AM2015-2441
Identifying antibody epitopes on membrane proteins can help differentiate antibody binding mechanisms, identify immunodominant structures, distinguish and classify antibodies, and identify cancer state-specific antigens, providing valuable information for therapeutic and diagnostic applications. However, membrane proteins represent challenging targets for epitope mapping studies using conventional methodologies such as X-ray crystallography or peptide scanning. We have developed a technology, ‘Shotgun Mutagenesis Epitope Mapping’ for rapidly mapping antibody epitopes on structurally-intact proteins expressed in mammalian cells. Here, we applied Shotgun Mutagenesis to create a comprehensive mutation library for CXCR4, a GPCR associated with tumor metastasis. The CXCR4 mutation library, which comprised >700 clones with 2.7x coverage of each of the 352 residues, was screened in triplicate with 5 different anti-CXCR4 MAbs to identify their epitopes. Our analysis revealed that all CXCR4 MAb epitopes were conformational and showed a requirement for charged residues located on the second extracellular loop. CXCR4 MAb epitopes were mapped onto the crystal structure to visualize and identify common structural features. Using epitope information, we generated point mutations in CXCR4 at positions of critical contact residues, incorporated them into virus-like particles (‘Lipoparticles’) and measured their binding kinetics. These approaches helped define the electrostatic contribution of individual amino acid residues on CXCR4 to the association rate, dissociation rate, and overall binding interaction with CXCR4 MAbs. Taking the same approach, we generated a comprehensive mutation library for the biomarker Claudin-4, a tight junction protein upregulated in epithelial cancers. The Claudin-4 mutation library, which comprised >400 clones with complete coverage of each of the 209 residues, was screened with a panel of 7 different anti-Claudin-4 antibodies to map their epitopes. We identified several distinct conformational epitopes that all map exclusively to the large first extracellular loop, identifying this domain as an immunodominant region of claudin-4. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4742. doi:1538-7445.AM2012-4742
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