Synthetic antibodies against BRIL as universal fiducial marks for single\u2212particle cryoEM structure determination of membrane proteins

Somnath Mukherjee,Seungil Han,Michael D Purdy,Frances J D Alvarez,Yanyong Kang,Kimberly F Fennell,H Eric Xu,Przemysław Dutka,Anthony A Kossiakoff,Mark Ammirati,Mark Yeager,Satchal K Erramilli,Katarzyna Radziwon,Blazej M Skrobek,Xiang Gao,John Coukos,Xiayang Qiu

doi:10.1038/s41467-020-15363-0

Abstract

We propose the concept of universal fiducials based on a set of pre-made semi-synthetic antibodies (sABs) generated by customized phage display selections against the fusion protein BRIL, an engineered variant of apocytochrome b562a. These sABs can bind to BRIL fused either into the loops or termini of different GPCRs, ion channels, receptors and transporters without disrupting their structure. A crystal structure of BRIL in complex with an affinity-matured sAB (BAG2) that bound to all systems tested delineates the footprint of interaction. Negative stain and cryoEM data of several examples of BRIL-membrane protein chimera highlight the effectiveness of the sABs as universal fiducial marks. Taken together with a cryoEM structure of sAB bound human nicotinic acetylcholine receptor, this work demonstrates that these anti-BRIL sABs can greatly enhance the particle properties leading to improved cryoEM outcomes, especially for challenging membrane proteins.

Highlights

We propose the concept of universal fiducials based on a set of pre-made semi-synthetic antibodies generated by customized phage display selections against the fusion protein BRIL, an engineered variant of apocytochrome b562a
We focused on identifying and characterizing a module that could be seamlessly engineered into transmembrane α-helices (TMH), since TMHs represent the most prevalent type of transmembrane structural organization in many types of membrane proteins
The binding kinetics of the affinity-matured variants show an increase in affinity between 50–150-fold compared to the wt sAB24 (Supplementary Table 2, Fig. 3d) that can be attributed both to the enhanced on-rate and slower off-rate for all the three variants. While these results indicated that all the variants should be more potent as fiducial marks than the wild-type synthetic antibodies (sABs), we selected the best expressing variant to determine which mutations in the CDR-H1 loop conferred high affinity to this variant

Summary

Introduction

We propose the concept of universal fiducials based on a set of pre-made semi-synthetic antibodies (sABs) generated by customized phage display selections against the fusion protein BRIL, an engineered variant of apocytochrome b562a. These sABs can bind to BRIL fused either into the loops or termini of different GPCRs, ion channels, receptors and transporters without disrupting their structure. While the display methods are in common practice, they are not yet turnkey for generation of high-affinity binders of the type needed for structural analyses, especially for membrane proteins To overcome these shortcomings, we endeavored to develop an approach that eliminates the need to generate Fabs for each system being studied. The attributes of the Fab for orientation are directly related to its order with respect to the target; if the Fab is connected to a less ordered part of the molecule, this will greatly diminish its contributions to structure determination

Methods

Results

Conclusion