Discovery and Characterization of a Thioesterase-Specific Monoclonal Antibody That Recognizes the 6-Deoxyerythronolide B Synthase
Abstract
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 Fab (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. Fab 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 Bmore »
- Authors:
-
[3];
[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)
- Publication Date:
- Research Org.:
- SLAC National Accelerator Lab., Menlo Park, CA (United States)
- Sponsoring Org.:
- USDOE Office of Science (SC), Biological and Environmental Research (BER); USDOE Office of Science (SC), Basic Energy Sciences (BES)
- OSTI Identifier:
- 1490870
- Grant/Contract Number:
- P41CA196276; AC02-76SF00515; GM087934, GM104659, P41GM103393
- Resource Type:
- Accepted Manuscript
- Journal Name:
- Biochemistry
- Additional Journal Information:
- Journal Volume: 57; Journal Issue: 43; Journal ID: ISSN 0006-2960
- Publisher:
- American Chemical Society (ACS)
- Country of Publication:
- United States
- Language:
- English
- Subject:
- 37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; 59 BASIC BIOLOGICAL SCIENCES
Citation Formats
Li, Xiuyuan, Sevillano, Natalia, La Greca, Florencia, Hsu, Jake, Mathews, Irimpan I., Matsui, Tsutomu, Craik, Charles S., and Khosla, Chaitan. Discovery and Characterization of a Thioesterase-Specific Monoclonal Antibody That Recognizes the 6-Deoxyerythronolide B Synthase. United States: N. p., 2018.
Web. doi:10.1021/acs.biochem.8b00886.
Li, Xiuyuan, Sevillano, Natalia, La Greca, Florencia, Hsu, Jake, Mathews, Irimpan I., Matsui, Tsutomu, Craik, Charles S., & Khosla, Chaitan. Discovery and Characterization of a Thioesterase-Specific Monoclonal Antibody That Recognizes the 6-Deoxyerythronolide B Synthase. United States. https://doi.org/10.1021/acs.biochem.8b00886
Li, Xiuyuan, Sevillano, Natalia, La Greca, Florencia, Hsu, Jake, Mathews, Irimpan I., Matsui, Tsutomu, Craik, Charles S., and Khosla, Chaitan. Fri .
"Discovery and Characterization of a Thioesterase-Specific Monoclonal Antibody That Recognizes the 6-Deoxyerythronolide B Synthase". United States. https://doi.org/10.1021/acs.biochem.8b00886. https://www.osti.gov/servlets/purl/1490870.
@article{osti_1490870,
title = {Discovery and Characterization of a Thioesterase-Specific Monoclonal Antibody That Recognizes the 6-Deoxyerythronolide B Synthase},
author = {Li, Xiuyuan and Sevillano, Natalia and La Greca, Florencia and Hsu, Jake and Mathews, Irimpan I. and Matsui, Tsutomu and Craik, Charles S. and Khosla, Chaitan},
abstractNote = {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 Fab (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. Fab 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.},
doi = {10.1021/acs.biochem.8b00886},
journal = {Biochemistry},
number = 43,
volume = 57,
place = {United States},
year = {Fri Oct 05 00:00:00 EDT 2018},
month = {Fri Oct 05 00:00:00 EDT 2018}
}
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Figures / Tables:
Figure 1: (A) Synthesis of 6-deoxyerythronolide B by DEBS. Abbreviations: ACP, acyl carrier protein; AT, acyltransferase; DH, dehydratase; KR, ketoreductase; KR0, inactive ketoreductase; KS, ketosynthase. Whereas the loading didomain (LDD) and the first two modules of DEBS occur within a single protein (DEBS!) in nature, our in vitro reconstituted systemmore »
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Figures/Tables have been extracted from DOE-funded journal article accepted manuscripts.