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QTY code designed thermostable and water-soluble chimeric chemokine receptors with tunable ligand affinity.

ABSTRACT: Chemokine receptors are of great interest as they play a critical role in many immunological and pathological processes. The ability to study chemokine receptors in aqueous solution without detergent would be significant because natural receptors require detergents to become soluble. We previously reported using the QTY code to design detergent-free chemokine receptors. We here report the design of 2 detergent-free chimeric chemokine receptors that were experimentally unattainable in detergent solution. We designed chimeric receptors by switching the N terminus and 3 extracellular (EC) loops between different receptors. Specifically, we replaced the N terminus and 3 EC loops of CCR5QTY with the N terminus and 3 EC loops of CXCR4. The ligand for CXCR4; namely CXCL12, binds to the chimeric receptor CCR5QTY (7TM)-CXCR4 (N terminus+3 EC loops), but with lower affinity compared to CXCR4; the CCL5 ligand of CCR5 binds the chimeric receptor with ?20× lower affinity. The chimeric design helps to elucidate the mechanism of native receptor-ligand interaction. We also show that all detergent-free QTY-designed chemokine receptors, expressed in Escherichia coli, bind to their respective chemokines with affinities in the nanomolar (nM) range, similar to the affinities of native receptors and SF9-produced QTY variants. These QTY-designed receptors exhibit remarkable thermostability in the presence of arginine and retain ligand-binding activity after heat treatment at 60 °C for 4 h and 24 h, and at 100 °C for 10 min. Our design approach enables affordable scale-up production of detergent-free QTY variant chemokine receptors with tunable functionality for various uses.

SUBMITTER: Qing R

PROVIDER: S-EPMC6926000 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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Project description:Membrane proteins, particularly those that are α-helical, such as transporters and G-protein-coupled receptors (GPCRs), have significant biological relevance. However, their expression and purification pose difficulties because of their poor water solubilities, which impedes progress in this field. The QTY method, a code-based protein-engineering approach, was recently developed to produce soluble transmembrane proteins. Here, we describe a comprehensive Web server built for QTY design and its relevance for in silico analyses. Typically, the simple design model is expected to require only 2 to 4 min of computer time, and the library design model requires 2 to 5 h, depending on the target protein size and the number of transmembrane helices. Detailed protocols for using the server with both the simple design and library design modules are provided. Methods for experiments following the QTY design are also included to facilitate the implementation of this approach. The design pipeline was further evaluated using microbial transmembrane proteins and structural alignment between the designed proteins and their origins by employing AlphaFold2. The results reveal that mutants generated by the developed pipeline were highly identical to their origins in terms of three-dimensional (3D) structures. In summary, the utilization of our Web server and associated protocols will enable QTY-based protein engineering to be implemented in a convenient, fast, accurate, and rational manner. The Protein Solubilizing Server (PSS) is publicly available at http://pss.sjtu.edu.cn. IMPORTANCE Water-soluble expression and purification are of considerable importance for protein identification and characterization. However, there has been a lack of an effective method for water-soluble expression of membrane proteins, which has severely hampered their studies. Here, an enabling comprehensive Web server, PSS, was developed for designing water-soluble mutants of α-helical membrane proteins, based on QTY design, a code-based protein-engineering approach. With microbial transmembrane proteins and GPCRs as examples, we systematically evaluated the server and demonstrated its successful performance. PSS is readily available for worldwide users as a Web-based tool, rendering QTY-based protein engineering convenient, efficient, accurate, and rational.

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QTY code designed thermostable and water-soluble chimeric chemokine receptors with tunable ligand affinity.

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