Biology subjects

White, K. I.

Publications and source records attributed to White, K. I..

6 recordsLinked to original sources

bioRxiv2026

Symmetry Breaking under Vectorial Perturbations in Macromolecular X-ray Crystallography

Proteins are molecular machines whose dynamics govern their function. Using time-resolved crystallography, the basis of protein mechanics can be uncovered by physical perturbation on protein crystals. Perturbations are often vector fields that, in crystals, break symmetry. Lacking existing tools for analyzing symmetry breaking, we built open-source software to do so. We applied this method to three time-resolved crystallography experiments and analyzed the effect of vectorial perturbations on macromolecular crystal structure. We demonstrate that our method can handle symmetry reduction in all 65 Sohncke space groups.

biophysics↗

bioRxiv2026

A rational design strategy and validation for protease-resistant fusion-inhibitor antiviral peptides

Peptide-based fusion inhibitors are promising pharmaceuticals in the fight against enveloped viruses relying on membrane fusion for host infection. However, peptide therapeutic applications have long been hindered by their poor stability in vivo. Here, we discovered that peptide inhibitors with the wildtype sequence of the heptad repeat 2 (HR2) domain of the SARS-CoV-2 spike protein are efficiently cleaved by Transmembrane Protease, Serine 2 (TMPRSS2), a key protease involved in the SARS-CoV-2 virus-cell fusion pathway. We then identified the corresponding cleavage sites and designed three protease-resistant peptides using ranking based on deep mutational scanning and natural occurrence. The three candidates all exhibit inhibitory activity in a cell-cell fusion assay. A high-resolution cryo-EM structure of the top candidate, HR2-NHN, bound to its HR1 target reveals the molecular basis for its potent activity. The top candidate of the cell-based screening assay significantly improved efficacy relative to the wildtype peptide when administered 12 h before infection in both an authentic virus-cell infection assay and a mouse assay. More broadly, our results suggest that the design strategies for protease-resistant peptides could be applied to a broad spectrum of other enveloped viruses and pave the way for the development of safe, prophylactic antivirals that can be administered before exposure.

microbiology↗

bioRxiv2026

The Structure of the Picornaviral 2C:RNA holoenzyme: Molecular Basis of RNA binding and specificity by a AAA+ protein

Picornaviruses are one of the leading agents of animal and human infectious disease with at least 8 billion infections a year and cause a range of symptoms including respiratory failure and acute flaccid myelitis1. The most conserved nonstructural protein in picornaviruses is 2C2, a member of the AAA+ family of ATPases that binds RNA, and a broad spectrum antiviral target3-5. Despite its crucial role in the viral life cycle and as a clinical target, no structure of 2C bound to RNA has been structurally determined. Here we present the first structure of 2C as a hexamer bound to single stranded RNA in its central pore; a novel AAA+ protein:substrate interaction. Using the 2C:RNA holoenzyme complex structure, we characterize the mode that this AAA+ protein employs to specifically bind single stranded RNA, and demonstrate that mutations to key residues inhibit both RNA binding and viral replication in Apthovirus and Enterovirus systems, and show that the core residues responsible for binding are broadly conserved in viruses beyond Picornaviridae. Finally, we reveal that the 2C:RNA holoenzyme complex is conformationally more similar to a protein translocase adapted to bind RNA rather than other viral DNA binding SF3 helicases, underscoring how the AAA+ core module can be adapted for a variety of biochemical substrates.

biophysics↗

bioRxiv2024

Pre-fusion AAA+ remodeling of target-SNARE protein complexes enables synaptic transmission

Synaptic vesicles containing neurotransmitters fuse with the plasma membrane upon the arrival of an action potential at the active zone. Multiple proteins organize trans-SNARE complex assembly and priming, leading to fusion. One target membrane SNARE, syntaxin, forms nanodomains at the active zone, and another, SNAP-25, enters non-fusogenic complexes with it. Here, we reveal mechanistic details of AAA+ protein NSF (N-ethylmaleimide sensitive factor) and SNAP (soluble NSF attachment protein) action before fusion. We show that syntaxin clusters are conserved, that NSF colocalizes with them, and characterize SNARE populations that may exist within or near them using cryo-EM. Supercomplexes of NSF, -SNAP, and either a syntaxin tetramer or one of two binary complexes of syntaxin--SNAP-25 reveal atomic details of SNARE processing and show how sequential ATP hydrolysis drives disassembly. These results suggest a functional role for syntaxin clusters as reservoirs and a corresponding role for NSF in syntaxin liberation and SNARE protein quality control preceding fusion.

neuroscience↗

bioRxiv2024

Sec18 side-loading is essential for universal SNARE recycling across cellular contexts

SNARE proteins drive membrane fusion at different cell compartments as their core domains zipper into a parallel four-helix bundle. After fusion, these bundles are disassembled by the AAA+ protein Sec18/NSF and its adaptor Sec17/-SNAP to make them available for subsequent rounds of membrane fusion. SNARE domains are often flanked by C-terminal transmembrane or N-terminal domains. Previous structures of the NSF--SNAP-SNARE complex revealed binding to the D1 ATPase pore, posing a topological constraint as SNARE transmembrane domains would prevent complete substrate threading as suggested for other AAA+ systems. Using mass-spectrometry in yeast cells, we show N-terminal SNARE domain interactions with Sec18, exacerbating this topological issue. We present cryo-EM structures of a yeast SNARE complex, Sec18, and Sec17 in a non-hydrolyzing condition, which show SNARE Sso1 threaded through the D1 and D2 ATPase rings of Sec18, with its folded, N-terminal Habc domain interacting with the D2 ring. This domain does not unfold during Sec18/NSF activity. Cryo-EM structures under hydrolyzing conditions revealed substrate-released and substrate-free states of Sec18 with a coordinated opening in the side of the ATPase rings. Thus, Sec18/NSF operates by substrate side-loading and unloading topologically constrained SNARE substrates.

biophysics↗

bioRxiv2022

Nanomolar inhibition of SARS-CoV-2 infection by an unmodified peptide targeting the pre-hairpin intermediate of the spike protein

Variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) challenge currently available COVID-19 vaccines and monoclonal antibody therapies through epitope change on the receptor binding domain of the viral spike glycoprotein. Hence, there is a specific urgent need for alternative antivirals that target processes less likely to be affected by mutation, such as the membrane fusion step of viral entry into the host cell. One such antiviral class includes peptide inhibitors which block formation of the so-called HR1HR2 six-helix bundle of the SARS-CoV-2 spike (S) protein and thus interfere with viral membrane fusion. Here we performed structural studies of the HR1HR2 bundle, revealing an extended, well-folded N-terminal region of HR2 that interacts with the HR1 triple helix. Based on this structure, we designed an extended HR2 peptide that achieves single-digit nanomolar inhibition of SARS-CoV-2 in cell-based fusion, VSV-SARS-CoV-2 chimera, and authentic SARS-CoV-2 infection assays without the need for modifications such as lipidation or chemical stapling. The peptide also strongly inhibits all major SARS-CoV-2 variants to date. This extended peptide is ~100-fold more potent than all previously published short, unmodified HR2 peptides, and it has a very long inhibition lifetime after washout in virus infection assays, suggesting that it targets a pre-hairpin intermediate of the SARS-CoV-2 S protein. Together, these results suggest that regions outside the HR2 helical region may offer new opportunities for potent peptide-derived therapeutics for SARS-CoV-2 and its variants, and even more distantly related viruses, and provide further support for the pre-hairpin intermediate of the S protein. Significance StatementSARS-CoV-2 infection requires fusion of viral and host membranes, mediated by the viral spike glycoprotein (S). Due to the importance of viral membrane fusion, S has been a popular target for developing vaccines and therapeutics. We discovered a simple peptide that inhibits infection by all major variants of SARS-CoV-2 with nanomolar efficacies. In marked contrast, widely used shorter peptides that lack a key N-terminal extension are about 100 x less potent than this peptide. Our results suggest that a simple peptide with a suitable sequence can be a potent and cost-effective therapeutic against COVID-19 and they provide new insights at the virus entry mechanism.

microbiology↗