Biology subjects

McCool, R. S.

Publications and source records attributed to McCool, R. S..

5 recordsLinked to original sources

bioRxiv2026

Structure-based design of soluble prefusion-stabilized herpes simplex virus type 2 glycoprotein B antigens

Herpes simplex virus type 2 (HSV-2) causes genital herpes through latent infection and periodic reactivation. Although antivirals alleviate symptoms, no prophylactic or therapeutic vaccines have been licensed. Glycoprotein B (gB) is a class III fusion protein that mediates entry by irreversibly transitioning from a metastable prefusion conformation to a stable postfusion conformation. Leveraging prior structure-based designs for human cytomegalovirus (HCMV) gB, we engineered amino acid substitutions in HSV-2 gB to stabilize its prefusion conformation. Cryo-EM of the engineered construct revealed a prefusion conformation and non-native dimers of gB trimers. Introduction of N-linked glycosylation sites resulted in the gB-G3 variant, which exhibited improved expression and reduced dimerization. Cryo-EM of gB-G3 bound to neutralizing antibodies yielded a 2.8 [A] resolution structure of the stabilized prefusion conformation in a closed state, which differs from the open states observed in recently published HSV gB structures. Both prefusion and postfusion gB variants were evaluated for immunogenicity in mice, delivered either as a protein subunit or mRNA vaccine. Each gB conformation elicited robust humoral and cellular responses. However, prefusion stabilization of gB did not improve neutralizing antibody titers relative to the postfusion construct, consistent with prior observations for HCMV gB. Collectively, these findings reveal insights into prefusion gB conformational dynamics, provide stabilized reagents for studying gB-directed immune responses and inform HSV-2 vaccine design.

microbiology↗

bioRxiv2025

Design, Structure, and Immunogenicity of a Soluble Prefusion-stabilized EBV gB Antigen

Epstein-Barr virus (EBV), the causative agent of mononucleosis, is linked to over 140,000 annual cancer-related deaths globally and increases the risk of multiple sclerosis by up to 32-fold. As a herpesvirus, EBV establishes lifelong infection, and over 90% of U.S. adults are EBV-seropositive. Despite its significant disease burden, no approved EBV vaccines or therapeutics exist. Among EBV envelope glycoproteins, the fusion protein (gB) is strictly required for epithelial and B cell infection. Using a combination of AlphaFold-guided modeling, rational design, and ThermoMPNN-informed optimization, we engineered a stabilized prefusion gB variant, D2C3. This construct incorporates two inter-protomeric disulfide bonds and three cavity-filling substitutions, resulting in a melting temperature of 54 {degrees}C. Cryo-EM analysis of this construct allowed us to determine the prefusion structure of EBV gB, providing insights into the structural transitions required to adopt the postfusion conformation. Murine immunizations and depletion studies with human sera suggested a trend toward improved functional immunogenicity of D2C3 compared to postfusion gB. Collectively, these studies define engineering principles to stabilize class III fusion proteins, provide reagents to interrogate the human antibody response to EBV gB, and lay a foundation for further studies to develop EBV gB-based vaccine candidates.

biochemistry↗

bioRxiv2024

Structure-based design of a soluble human cytomegalovirus glycoprotein B antigen stabilized in a prefusion-like conformation

Human cytomegalovirus (HCMV) glycoprotein B (gB) is a class III membrane fusion protein required for viral entry. HCMV vaccine candidates containing gB have demonstrated moderate clinical efficacy, but no HCMV vaccine has been FDA-approved. Here, we used structure-based design to identify and characterize amino acid substitutions that stabilize gB in its metastable prefusion conformation. One variant containing two engineered interprotomer disulfide bonds and two cavity-filling substitutions (gB-C7), displayed increased expression and thermostability. A 2.8 [A] resolution cryo-electron microscopy structure shows that gB-C7 adopts a prefusion-like conformation, revealing additional structural elements at the membrane-distal apex. Unlike previous observations for several class I viral fusion proteins, mice immunized with postfusion or prefusion-stabilized forms of soluble gB protein displayed similar neutralizing antibody titers, here specifically against an HCMV laboratory strain on fibroblasts. Collectively, these results identify initial strategies to stabilize class III viral fusion proteins and provide tools to probe gB-directed antibody responses. TeaserA structure-based design campaign leads to stabilization of the class III viral fusion protein from HCMV in a prefusion-like conformation.

molecular biology↗

bioRxiv2021

Structural basis for mismatch surveillance by CRISPR/Cas9

The widespread use of CRISPR/Cas9 as a programmable genome editing tool has been hindered by off-target DNA cleavage (Cong et al., 2013; Doudna, 2020; Fu et al., 2013; Jinek et al., 2013). While analysis of such off-target editing events have enabled the development of Cas9 variants with greater discrimination against mismatches (Chen et al., 2017; Kleinstiver et al., 2016; Slaymaker et al., 2016), the underlying molecular mechanisms by which Cas9 rejects or accepts mismatches are poorly understood (Kim et al., 2019; Liu et al., 2020; Slaymaker and Gaudelli, 2021). Here, we used kinetic analysis to guide cryo-EM structure determination of Cas9 at different stages of mismatch surveillance. We observe a distinct, previously undescribed linear conformation of the duplex formed between the guide RNA (gRNA) and DNA target strand (TS), that occurs in the presence of PAM-distal mismatches, preventing Cas9 activation. The canonical kinked gRNA:TS duplex is a prerequisite for Cas9 activation, acting as a structural scaffold to facilitate Cas9 conformational rearrangements necessary for DNA cleavage. We observe that highly tolerated PAM-distal mismatches achieve this kinked conformation through stabilization of a distorted duplex conformation via a flexible loop in the RuvC domain. Our results provide molecular insights into the underlying structural mechanisms that may facilitate off-target cleavage by Cas9 and provides a molecular blueprint for the design of next-generation high fidelity Cas9 variants that selectively reduce off-target DNA cleavage while retaining efficient cleavage of on-target DNA.

biochemistry↗

bioRxiv2021

A glycan gate controls opening of the SARS-CoV-2 spike protein

SARS-CoV-2 infection is controlled by the opening of the spike protein receptor binding domain (RBD), which transitions from a glycan-shielded "down" to an exposed "up" state in order to bind the human ACE2 receptor and infect cells. While snapshots of the "up" and "down" states have been obtained by cryoEM and cryoET, details of the RBD opening transition evade experimental characterization. Here, over 130 s of weighted ensemble (WE) simulations of the fully glycosylated spike ectodomain allow us to characterize more than 300 continuous, kinetically unbiased RBD opening pathways. Together with ManifoldEM analysis of cryo-EM data and biolayer interferometry experiments, we reveal a gating role for the N-glycan at position N343, which facilitates RBD opening. Residues D405, R408, and D427 also participate. The atomic-level characterization of the glycosylated spike activation mechanism provided herein achieves a new high-water mark for ensemble pathway simulations and offers a foundation for understanding the fundamental mechanisms of SARS-CoV-2 viral entry and infection.

biophysics↗