Fragment antigen binding domains (Fabs) as tools to study assembly-line polyketide synthases

Abstract

The crystallization of proteins remains a bottleneck in our fundamental understanding of their functions. Therefore, discovering tools that aid crystallization is crucial. In this review, the versatility of fragment-antigen binding domains (F ab s) as protein crystallization chaperones is discussed. F ab s have aided the crystallization of membrane-bound and soluble proteins as well as RNA. The ability to bind three F ab s onto a single protein target has demonstrated their potential for crystallization of challenging proteins. We describe a high-throughput workflow for identifying F ab s to aid the crystallization of a protein of interest (POI) by leveraging phage display technologies and differential scanning fluorimetry (DSF). This workflow has proven to be especially effective in our structural studies of assembly-line polyketide synthases (PKSs), which harbor flexible domains and assume transient conformations. PKSs are of interest to us due to their ability to synthesize an unusually broad range of medicinally relevant compounds. Despite years of research studying these megasynthases, their overall topology has remained elusive. One F ab in particular, 1B2, has successfully enabled X-ray crystallographic and single particle cryo-electron microscopic (cryoEM) analyses of multiple modules from distinct assembly-line PKSs. Its use has not only facilitated multidomain protein crystallization but has also enhanced particle quality via cryoEM, thereby enabling the visualization of intact PKS modules at near-atomic (3–5 Å) resolution. The identification of PKS-binding F ab s can be expected to continue playing a key role in furthering our knowledge of polyketide biosynthesis on assembly-line PKSs.

Discovery and Characterization of Antibody Probes of Module 2 of the 6-Deoxyerythronolide B Synthase

“…Over the past two decades, X-ray crystallography and single-particle cryo-electron microscopy (cryoEM) have been especially powerful structural tools for structure–function analysis of assembly line PKSs. − More recent structural efforts have also relied on the use of fragment antigen-binding domains of antibodies (F ab s) as chaperones for crystallography and cryoEM. ,− For example, the F ab 1B2 binds to the N-terminal helical docking domain of Module 3 of the 6-deoxyerythronolide B synthase (DEBS; Figure ) and has proven invaluable for visualizing entire modules of two unrelated PKSs, the lasalocid PKS and DEBS . We therefore sought to discover additional antibody probes to enhance our understanding of the KS-AT core of a representative assembly line PKS module.…”

Pathway and protein engineering for biosynthesis

“…Corpuz et al reviewed the current progress on protein-protein interface analysis of the non-ribosomal peptide synthetase (NRPS), providing insights for engineering these mega-enzymes [ 2 ]. Similarly, Guzman et al summarized how to use fragment-antigen binding domains as protein crystallization chaperones for structural study of assembly-line polyketide synthases (PKSs), which are of interest to synthesize an unusually broad range of medicinally relevant compounds [ 3 ].…”