Biology subjects

Gonzalez, K. J.

Publications and source records attributed to Gonzalez, K. J..

3 recordsLinked to original sources

bioRxiv2024

Systematic computer-aided disulfide design as a general strategy to stabilize prefusion class I fusion proteins

Numerous enveloped viruses, such as coronaviruses, influenza, and respiratory syncytial virus (RSV), utilize class I fusion proteins for cell entry. During this process, the proteins transition from a prefusion to a postfusion state, undergoing substantial and irreversible conformational changes. The prefusion conformation has repeatedly shown significant potential in vaccine development. However, the instability of this state poses challenges for its practical application in vaccines. While non-native disulfides have been effective in maintaining the prefusion structure, identifying stabilizing disulfide bonds remains an intricated task. Here, we present a general computational approach to systematically identify prefusion-stabilizing disulfides. Our method assesses the geometric constraints of disulfide bonds and introduces a ranking system to estimate their potential in stabilizing the prefusion conformation. We found, for the RSV F protein, that disulfides restricting the initial stages of the conformational switch can offer higher stability to the prefusion state than those preventing unfolding at a later stage. The implementation of our algorithm on the RSV F protein led to the discovery of prefusion-stabilizing disulfides, providing evidence that supports our hypothesis. Furthermore, the evaluation of our top design as a vaccine candidate in a cotton rat model demonstrated robust protection against RSV infection, highlighting the potential of our approach for vaccine development. Significance statementClass I fusion proteins, pivotal for cellular entry, constitute primary targets for neutralizing antibodies across a wide range of highly pathogenic viruses. Notably, the prefusion conformation of these proteins has demonstrated to be a robust immunogen, presenting a promising avenue for vaccine development. Despite of this, the inherent structural instability of this conformation poses a substantial hurdle for vaccine formulations. To overcome this limitation and to optimize these proteins as vaccines candidates, we developed an automated computational approach to enhance the structural stability of the prefusion conformation without compromising its immunogenic properties. Given the clinical significance of class I fusion proteins, our computational tool offers a mechanism to accelerate the development of preventive measures against a multitude of viral infections.

bioengineering↗

bioRxiv2023

A general computational design strategy for stabilizing viral class I fusion proteins

Many pathogenic viruses, including influenza virus, Ebola virus, coronaviruses, and Pneumoviruses, rely on class I fusion proteins to fuse viral and cellular membranes. To drive the fusion process, class I fusion proteins undergo an irreversible conformational change from a metastable prefusion state to an energetically more favorable and stable postfusion state. An increasing amount of evidence exists highlighting that antibodies targeting the prefusion conformation are the most potent. However, many mutations have to be evaluated before identifying prefusion-stabilizing substitutions. We therefore established a computational design protocol that stabilizes the prefusion state while destabilizing the postfusion conformation. As a proof of concept, we applied this principle to the fusion protein of the RSV, hMPV, and SARS-CoV-2 viruses. For each protein, we tested less than a handful of designs to identify stable versions. Solved structures of designed proteins from the three different viruses evidenced the atomic accuracy of our approach. Furthermore, the immunological response of the RSV F design compared to a current clinical candidate in a mouse model. While the parallel design of two conformations allows identifying and selectively modifying energetically less optimized positions for one conformation, our protocol also reveals diverse molecular strategies for stabilization. We recaptured many approaches previously introduced manually for the stabilization of viral surface proteins, such as cavity-filling, optimization of polar interactions, as well as postfusion-disruptive strategies. Using our approach, it is possible to focus on the most impacting mutations and potentially preserve the immunogen as closely as possible to its native version. The latter is important as sequence re-design can cause perturbations to B and T cell epitopes. Given the clinical significance of viruses using class I fusion proteins, our algorithm can substantially contribute to vaccine development by reducing the time and resources needed to optimize these immunogens.

microbiology↗

bioRxiv2019

Skp1 Dimerization Conceals its F-box Protein Binding Site

Skp1 is an adapter that links F-box proteins to cullin-1 in the Skp1/cullin-1/F-box (SCF) protein family of E3 ubiquitin ligases that targets specific proteins for polyubiquitination and subsequent protein degradation. Skp1 from the amoebozoan Dictyostelium forms a stable homodimer in vitro with a Kd of 2.5 {micro}M as determined by sedimentation velocity studies, yet is monomeric in crystal complexes with F-box proteins. To investigate the molecular basis for the difference, we determined the solution NMR structure of a doubly truncated Skp1 homodimer (Skp1{Delta}{Delta}). The solution structure of Skp1{Delta}{Delta} dimer reveals a 2-fold symmetry with an interface that buries [~]750 [A]2 of predominantly hydrophobic surface. The dimer interface overlaps with subsite-1 of the F-box interaction area, explaining why only the Skp1 monomer binds F-box proteins (FBPs). To confirm the model, Rosetta was used to predict amino acid substitutions that might disrupt the dimer interface, and the F97E substitution was chosen to potentially minimize interference with F-box interactions. A nearly full-length version of Skp1 with this substitution (Skp1{Delta}F97E) behaved as a stable monomer at concentrations up to 500 {micro}M and actively bound a model FBP, mammalian Fbs1, which suggests that the dimeric state is not required for Skp1 to carry out a basic biochemical function. Finally, Skp1{Delta}F97E is expected to serve as a monomer model for high-resolution NMR studies previously hindered by dimerization.

biochemistry↗