Discovery and Characterization of a Thioesterase-Specific Monoclonal Antibody That Recognizes the 6-Deoxyerythronolide B Synthase

Li, Xiuyuan; Sevillano, Natalia; La Greca, Florencia; Hsu, Jake; Mathews, Irimpan I.; Matsui, Tsutomu; Craik, Charles S.; Khosla, Chaitan

doi:10.1021/acs.biochem.8b00886

Title: Discovery and Characterization of a Thioesterase-Specific Monoclonal Antibody That Recognizes the 6-Deoxyerythronolide B Synthase

Journal Article · Fri Oct 05 00:00:00 EDT 2018 · Biochemistry

DOI:https://doi.org/10.1021/acs.biochem.8b00886· OSTI ID:1490870

Li, Xiuyuan ^[1]; Sevillano, Natalia ^[2]; La Greca, Florencia ^[2]; Hsu, Jake ^[1];

^[3]; Matsui, Tsutomu ^[2];

^[2];

^[1]

Stanford Univ., Stanford, CA (United States)
Univ. of California San Francisco, San Francisco, CA (United States)
Stanford Univ., Menlo Park, CA (United States)

Assembly line polyketide synthases (PKSs) are large multimodular enzymes responsible for the biosynthesis of diverse antibiotics in bacteria. Structural and mechanistic analysis of these megasynthases can benefit from the discovery of reagents that recognize individual domains or linkers in a site-specific manner. Monoclonal antibodies not only have proven themselves as premier tools in analogous applications but also have the added benefit of constraining the conformational flexibility of their targets in unpredictable but often useful ways. Here we have exploited a library based on the naïve human antibody repertoire to discover a F_ab (3A6) that recognizes the terminal thioesterase (TE) domain of the 6-deoxyerythronolide B synthase with high specificity. Biochemical assays were used to verify that 3A6 binding does not inhibit enzyme turnover. The co-crystal structure of the TE–3A6 complex was determined at 2.45 Å resolution, resulting in atomic characterization of this protein–protein recognition mechanism. F_ab binding had minimal effects on the structural integrity of the TE. In turn, these insights were used to interrogate via small-angle X-ray scattering the solution-phase conformation of 3A6 complexed to a catalytically competent PKS module and bimodule. Furthermore, we have developed a high-affinity monoclonal antibody tool that recognizes the TE domain of the 6-deoxyerythronolide B synthase while maintaining its native function.

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Research Organization:: SLAC National Accelerator Lab., Menlo Park, CA (United States)

Sponsoring Organization:: USDOE Office of Science (SC), Biological and Environmental Research (BER); USDOE Office of Science (SC), Basic Energy Sciences (BES)

Grant/Contract Number:: P41CA196276; AC02-76SF00515; GM087934, GM104659, P41GM103393

OSTI ID:: 1490870

Journal Information:: Biochemistry, Vol. 57, Issue 43; ISSN 0006-2960

Publisher:: American Chemical Society (ACS)Copyright Statement

Country of Publication:: United States

Language:: English

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Cited by: 5 works

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