Biology subjects

Brunger, A. T.

Publications and source records attributed to Brunger, A. T..

5 recordsLinked to original sources

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↗

bioRxiv2022

Screening of hydrocarbon-stapled peptides for inhibition of calcium-triggered exocytosis

The so-called primary interface between the SNARE complex and synaptotagmin-1 (Syt1) is essential for Ca2+-triggered neurotransmitter release in neuronal synapses. The interacting residues of the primary interface are conserved across different species for synaptotagmins (Syt1, Syt2, Syt9), SNAP-25, and syntaxin-1A homologs involved in fast synchronous release. This Ca2+-independent interface forms prior to Ca2+-triggering and plays a role in synaptic vesicle priming. This primary interface is also conserved in the fusion machinery that is responsible for mucin granule membrane fusion. Ca2+-stimulated mucin secretion is mediated by the SNAREs syntaxin-3, SNAP-23, VAMP8, synaptotagmin-2, and other proteins. Here, we designed and screened a series of hydrocarbon-stapled peptides consisting of SNAP-25 fragments that included some of the key residues involved in the primary interface as observed in high-resolution crystal structures. We selected a subset of four stapled peptides that were highly -helical as assessed by circular dichroism and that inhibited both Ca2+-independent and Ca2+-triggered ensemble lipid-mixing with neuronal SNAREs and Syt1. In a single-vesicle contentmixing assay with reconstituted neuronal SNAREs and synaptotagmin-1 or with reconstituted airway SNAREs and synaptotagmin-2, the selected peptides also suppressed Ca2+-triggered fusion. Taken together, hydrocarbon-stapled peptides that interfere with the primary interface consequently inhibit Ca2+-triggered exocytosis. Our inhibitor screen suggests that these compounds may be useful to combat mucus hypersecretion that is a major cause of airway obstruction in the pathophysiology of COPD, asthma and cystic fibrosis.

pharmacology and toxicology↗

bioRxiv2021

Molecular characterization of AMPA receptor trafficking vesicles

Regulated delivery of AMPA receptors (AMPARs) to the postsynaptic membrane is an essential step in synaptic strength modification, and in particular, long-term potentiation (LTP). While LTP has been extensively studied using electrophysiology and light microscopy, several questions regarding the molecular mechanisms of AMPAR delivery via trafficking vesicles remain outstanding, including the gross molecular make up of AMPAR trafficking organelles and identification and location of calcium sensors required for SNARE complex-dependent membrane fusion of such trafficking vesicles with the plasma membrane. Here, we isolated AMPAR trafficking vesicles (ATVs) from whole mouse brains via immunoprecipitation and characterized them using immunoelectron microscopy, immunoblotting, and liquid chromatography tandem mass spectrometry (LC-MS/MS). We identified several proteins on ATVs that were previously found to play a role in AMPAR trafficking, including SNARES (including synaptobrevin 2), Rabs, the SM protein Munc18-1, a calcium-sensor (synaptotagmin-1), as well as several new markers, including synaptophysin and synaptogyrin on ATV membranes. Additionally, we identified two populations of ATVs based on size and molecular composition: small-diameter, synaptobrevin-2- and GluA1-containing ATVs and larger transferrin-receptor-, GluA1-, GluA2-, GluA3-containing ATVs. The smaller population of ATVs likely represents a trafficking vesicle whose fusion is essential for LTP. These findings reveal the important role of AMPAR sorting into fusion-competent trafficking vesicles that are implicated in synaptic strength modification and reveal candidates of putative effectors and regulators of AMPAR trafficking.

neuroscience↗

bioRxiv2021

Sec17/Sec18 can support membrane fusion without help from completion of SNARE zippering

Membrane fusion requires R-, Qa-, Qb-, and Qc-family SNAREs that zipper into RQaQbQc coiled coils, driven by the sequestration of apolar amino acids. Zippering has been thought to provide all the force driving fusion. Sec17/SNAP can form an oligomeric assembly with SNAREs with the Sec17 C-terminus bound to Sec18/NSF, the central region bound to SNAREs, and a crucial apolar loop near the N-terminus poised to insert into membranes. Though Sec17 aids zippering, we now report that Sec17 and Sec18 will drive robust fusion without requiring zippering completion. Zippering-driven fusion is blocked by deleting the C-terminal quarter of any Q-SNARE domain or by replacing the apolar amino acids of the Qa-SNARE which face the center of the 4-SNARE coiled coils with polar residues. These blocks to fusion, singly or combined, are bypassed by Sec17 and Sec18, and SNARE-dependent fusion is restored without help from completing zippering.

biochemistry↗

bioRxiv2020

Conformational dynamics of auto-inhibition in the ER calcium sensor STIM1

The dimeric ER Ca2+ sensor STIM1 controls store-operated Ca2+ entry (SOCE) through the regulated binding of its CRAC activation domain (CAD) to Orai channels in the plasma membrane. In resting cells, the STIM1 CC1 domain interacts with CAD to suppress SOCE, but the structural basis of this interaction is unclear. Using single-molecule Forster resonance energy transfer (smFRET) and protein crosslinking approaches, we show that CC1 interacts dynamically with CAD in a domain-swapped configuration with an orientation predicted to sequester its Orai- binding region adjacent to the ER membrane. Following ER Ca2+ depletion and release from CAD, cysteine crosslinking indicates that the two CC1 domains become closely paired along their entire length in the active Orai-bound state. These findings provide a structural basis for the dual roles of CC1: sequestering CAD to suppress SOCE in resting cells and propelling it towards the plasma membrane to activate Orai and SOCE after store depletion.

biophysics↗