Chaitan Khosla, Postdoctoral Faculty Sponsor
Discovery and Characterization of Antibody Probes of Module 2 of the 6-Deoxyerythronolide B Synthase.
Fragment antigen-binding domains of antibodies (Fabs) are powerful probes of structure-function relationships of assembly line polyketide synthases (PKSs). We report the discovery and characterization of Fabs interrogating the structure and function of the ketosynthase-acyltransferase (KS-AT) core of Module 2 of the 6-deoxyerythronolide B synthase (DEBS). Two Fabs (AC2 and BB1) were identified to potently inhibit the catalytic activity of Module 2. Both AC2 and BB1 were found to modulate ACP-mediated reactions catalyzed by this module, albeit by distinct mechanisms. AC2 primarily affects the rate (kcat), whereas BB1 increases the KM of an ACP-mediated reaction. A third Fab, AA5, binds to the KS-AT fragment of DEBS Module 2 without altering either parameter; it is phenotypically reminiscent of a previously characterized Fab, 1B2, shown to principally recognize the N-terminal helical docking domain of DEBS Module 3. Crystal structures of AA5 and 1B2 bound to the KS-AT fragment of Module 2 were solved to 2.70 and 2.65 A resolution, respectively, and revealed entirely distinct recognition features of the two antibodies. The new tools and insights reported here pave the way toward advancing our understanding of the structure-function relationships of DEBS Module 2, arguably the most well-studied module of an assembly line PKS.
View details for DOI 10.1021/acs.biochem.3c00156
View details for PubMedID 37184546
In vivo visualization and molecular targeting of the cardiac conduction system.
The Journal of clinical investigation
Accidental injury to the cardiac conduction system (CCS), a network of specialized cells embedded within the heart and indistinguishable from the surrounding heart muscle tissue, is a major complication in cardiac surgeries. Here, we addressed this unmet need by engineering targeted antibody-dye conjugates directed against CCS, allowing for the visualization of the CCS in vivo following a single intravenous injection in mice. These optical imaging tools showed high sensitivity, specificity, and resolution, with no adverse effects to CCS function. Further, with the goal of creating a viable prototype for human use, we generated a fully human monoclonal Fab, that similarly targets the CCS with high specificity. We demonstrate that, when conjugated to an alternative cargo, this Fab can also be used to modulate CCS biology in vivo providing a proof-of-principle for targeted cardiac therapeutics. Finally, in performing differential gene expression analyses of the entire murine CCS at single-cell resolution, we uncovered and validated a suite of additional cell surface markers that can be used to molecularly target the distinct subcomponents of the CCS, each prone to distinct life-threatening arrhythmias. These findings lay the foundation for translational approaches targeting the CCS for visualization and therapy in cardiothoracic surgery, cardiac imaging and arrhythmia management.
View details for DOI 10.1172/JCI156955
View details for PubMedID 35951416
Mapping the catalytic conformations of an assembly-line polyketide synthase module.
Science (New York, N.Y.)
2021; 374 (6568): 729-734
[Figure: see text].
View details for DOI 10.1126/science.abi8358
View details for PubMedID 34735239
Antibody Probes of Module 1 of the 6-Deoxyerythronolide B Synthase Reveal an Extended Conformation During Ketoreduction.
Journal of the American Chemical Society
The 6-deoxyerythronolide B synthase (DEBS) is a prototypical assembly line polyketide synthase (PKS) that synthesizes the macrocyclic core of the antibiotic erythromycin. Each of its six multidomain modules presumably sample distinct conformations, as biosynthetic intermediates tethered to their acyl carrier proteins interact with multiple active sites during the courses of their catalytic cycles. The spatiotemporal details underlying these protein dynamics remain elusive. Here, we investigate one aspect of this conformational flexibility using two domain-specific monoclonal antibody fragments (Fabs) isolated from a very large naive human antibody library. Both Fabs, designated 1D10 and 2G10, were bound specifically and with high affinity to the ketoreductase domain of DEBS module 1 (KR1). Comparative kinetic analysis of stand-alone KR1 as well as a truncated bimodular derivative of DEBS revealed that 1D10 inhibited KR1 activity whereas 2G10 did not. Co-crystal structures of each KR1-Fab complex provided a mechanistic rationale for this difference. A hybrid PKS module harboring KR1 was engineered, whose individual catalytic domains have been crystallographically characterized at high resolution. Size exclusion chromatography coupled to small-angle X-ray scattering (SEC-SAXS) of this hybrid module bound to 1D10 provided further support for the catalytic relevance of the "extended" model of a PKS module. Our findings reinforce the power of monoclonal antibodies as tools to interrogate structure-function relationships of assembly line PKSs.
View details for DOI 10.1021/jacs.0c05133
View details for PubMedID 32786753