Rohweder, P. J. | bioRxiv Science

bioRxiv2025

Antibody Reveals Conformational Latch Controlling Herpesvirus Proteases

Human herpesviruses (HHVs) are widespread pathogens that cause severe disease. Their replication depends on the HHV protease (HHV Pr), an enzyme essential for capsid maturation. Because HHV Pr must dimerize to become catalytically active, disrupting dimer formation is a promising strategy for antiviral therapeutic development. We isolated a conformationally selective antibody inhibitor, Fab5, from a fully human naive Fab-phage library that recognizes monomeric human cytomegalovirus protease (HCMV Pr). A 2.6 [A] cryoelectron microscopy (cryo-EM) structure of the Fab5-HCMV Pr complex revealed that Fab5 binds a flexible loop distal from the active site and dimer interface which we call the latch loop. In HCMV Pr dimers, this loop secures the C-terminal tail to the protein core. Structure-guided mutagenesis confirmed that the latch loop is essential for HCMV Pr dimerization and activity. This loop is structurally conserved across all HHV Prs, and we show its functional role in Kaposis Sarcoma-associated herpesvirus (KSHV) Pr as well. The latch loop plays a mechanistic role in the conformational transition required for HHV Pr activity, and it forms a cryptic site that presents a new avenue for future allosteric inhibitor development.

biochemistry↗

bioRxiv2017

Extending Chemical Perturbations Of The Ubiquitin Fitness Landscape In A Classroom Setting

Although the primary protein sequence of ubiquitin (Ub) is extremely stable over evolutionary time, it is highly tolerant to mutation during selection experiments performed in the laboratory. We have proposed that this discrepancy results from the difference between fitness under laboratory culture conditions and the selective pressures in changing environments over evolutionary time scales. Building on our previous work (Mavor et al 2016), we used deep mutational scanning to determine how twelve new chemicals (3-Amino-1,2,4-triazole, 5-fluorocytosine, Amphotericin B, CaCl2, Cerulenin, Cobalt Acetate, Menadione, Nickel Chloride, p-fluorophenylalanine, Rapamycin, Tamoxifen, and Tunicamycin) reveal novel mutational sensitivities of ubiquitin residues. We found sensitization of Lys63 in eight new conditions. In total, our experiments have uncovered a sensitizing condition for every position in Ub except Ser57 and Gln62. By determining the Ubiquitin fitness landscape under different chemical constraints, our work helps to resolve the inconsistencies between deep mutational scanning experiments and sequence conservation over evolutionary timescales.\n\nBuilds onMavor D, Barlow KA, Thompson S, Barad BA, Bonny AR, Cario CL, Gaskins G, Liu Z, Deming L, Axen SD, Caceres E, Chen W, Cuesta A, Gate R, Green EM, Hulce KR, Ji W, Kenner LR, Mensa B, Morinishi LS, Moss SM, Mravic M, Muir RK, Niekamp S, Nnadi CI, Palovcak E, Poss EM, Ross TD, Salcedo E, See S, Subramaniam M, Wong AW, Li J, Thorn KS, Conchuir SO, Roscoe BP, Chow ED, DeRisi JL, Kortemme T, Bolon DN, Fraser JS. Determination of Ubiquitin Fitness Landscapes Under Different Chemical Stresses in a Classroom Setting. eLife. 2016.\n\nImpact StatementWe organized a project-based course that used deep mutational scanning in multiple chemical conditions to resolve the inconsistencies between tolerance to mutations in laboratory conditions and sequence conservation over evolutionary timescales.

bioinformatics↗