Design of Diverse Asymmetric Pockets in De Novo Homo-oligomeric Proteins

Journal Article · Tue Jan 10 04:00:00 EST 2023 · Biochemistry

DOI:https://doi.org/10.1021/acs.biochem.2c00497· OSTI ID:2471018

Gerben, Stacey R. ^[1]; Borst, Andrew J. ^[1]; Hicks, Derrick R. ^[1]; Moczygemba, Isabelle ^[1]; Feldman, David ^[1]; Coventry, Brian ^[1]; Yang, Wei ^[1]; Bera, Asim K. ^[1]; Miranda, Marcos ^[1]; Kang, Alex ^[1]; Nguyen, Hannah ^[1]; Baker, David ^[1]

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1. Univ. of Washington, Seattle, WA (United States)

A challenge for design of protein–small-molecule recognition is that incorporation of cavities with size, shape, and composition suitable for specific recognition can considerably destabilize protein monomers. This challenge can be overcome through binding pockets formed at homo-oligomeric interfaces between folded monomers. Interfaces surrounding the central homo-oligomer symmetry axes necessarily have the same symmetry and so may not be well suited to binding asymmetric molecules. To enable general recognition of arbitrary asymmetric substrates and small molecules, we developed an approach to designing asymmetric interfaces at off-axis sites on homo-oligomers, analogous to those found in native homo-oligomeric proteins such as glutamine synthetase. We symmetrically dock curved helical repeat proteins such that they form pockets at the asymmetric interface of the oligomer with sizes ranging from several angstroms, appropriate for binding a single ion, to up to more than 20 Å across. Of the 133 proteins tested, 84 had soluble expression in E. coli, 47 had correct oligomeric states in solution, 35 had small-angle X-ray scattering (SAXS) data largely consistent with design models, and 8 had negative-stain electron microscopy (nsEM) 2D class averages showing the structures coming together as designed. Both an X-ray crystal structure and a cryogenic electron microscopy (cryoEM) structure are close to the computational design models. The nature of these proteins as homo-oligomers allows them to be readily built into higher-order structures such as nanocages, and the asymmetric pockets of these structures open rich possibilities for small-molecule binder design free from the constraints associated with monomer destabilization.

• Research Organization: Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States). Advanced Light Source (ALS); Argonne National Laboratory (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
• Sponsoring Organization: USDOE Office of Science (SC), Biological and Environmental Research (BER); National Institute of General Medical Sciences (NIGMS); National Institutes of Health (NIH); USDOE Office of Science (SC), Basic Energy Sciences (BES). Scientific User Facilities (SUF)
• Grant/Contract Number: AC02-05CH11231; AC02-06CH11357
• OSTI ID: 2471018
• Journal Information: Biochemistry, Journal Name: Biochemistry Journal Issue: 2 Vol. 62; ISSN 0006-2960
• Publisher: American Chemical Society (ACS)
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES