Sensory deficit screen identifies nsf mutation that differentially affects SNARE recycling and quality control.

The AAA+ NSF complex is responsible for SNARE complex disassembly both before and after membrane fusion. Loss of NSF function results in pronounced developmental and degenerative defects. In a genetic screen for sensory deficits in zebrafish, we identified a mutation in nsf, I209N, that impairs hearing and balance in a dosage-dependent manner without accompanying defects in motility, myelination, and innervation. In vitro experiments demonstrate that while the I209N NSF protein recognizes SNARE complexes, the effects on disassembly are dependent upon the type of SNARE complex and I209N concentration. Higher levels of I209N protein produce a modest decrease in binary (syntaxin-SNAP-25) SNARE complex disassembly and residual ternary (syntaxin-1A-SNAP-25-synaptobrevin-2) disassembly, whereas at lower concentrations binary disassembly activity is strongly reduced and ternary disassembly activity is absent. Our study suggests that the differential effect on disassembly of SNARE complexes leads to selective effects on NSF-mediated membrane trafficking and auditory/vestibular function.

Structure-based design of a SARS-CoV-2 Omicron-specific inhibitor.

The Omicron variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) introduced a relatively large number of mutations, including three mutations in the highly conserved heptad repeat 1 (HR1) region of the spike glycoprotein (S) critical for its membrane fusion activity. We show that one of these mutations, N969K induces a substantial displacement in the structure of the heptad repeat 2 (HR2) backbone in the HR1HR2 postfusion bundle. Due to this mutation, fusion-entry peptide inhibitors based on the Wuhan strain sequence are less efficacious. Here, we report an Omicron-specific peptide inhibitor designed based on the structure of the Omicron HR1HR2 postfusion bundle. Specifically, we inserted an additional residue in HR2 near the Omicron HR1 K969 residue to better accommodate the N969K mutation and relieve the distortion in the structure of the HR1HR2 postfusion bundle it introduced. The designed inhibitor recovers the loss of inhibition activity of the original longHR2_42 peptide with the Wuhan strain sequence against the Omicron variant in both a cell-cell fusion assay and a vesicular stomatitis virus (VSV)-SARS-CoV-2 chimera infection assay, suggesting that a similar approach could be used to combat future variants. From a mechanistic perspective, our work suggests the interactions in the extended region of HR2 may mediate the initial landing of HR2 onto HR1 during the transition of the S protein from the prehairpin intermediate to the postfusion state.

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

Variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) challenge currently available coronavirus disease 2019 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 heptad repeat 1 and 2 (HR1HR2) six-helix bundle of the SARS-CoV-2 spike (S) protein and thus interfere with viral membrane fusion. 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 and virus-based 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 prehairpin 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 prehairpin intermediate of the S protein.

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 Statement: SARS-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.

Structural conservation among variants of the SARS-CoV-2 spike postfusion bundle.

Variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) challenge currently available COVID-19 vaccines and monoclonal antibody therapies due to structural and dynamic changes of the viral spike glycoprotein (S). The heptad repeat 1 (HR1) and heptad repeat 2 (HR2) domains of S drive virus­host membrane fusion by assembly into a six-helix bundle, resulting in delivery of viral RNA into the host cell. We surveyed mutations of currently reported SARS-CoV-2 variants and selected eight mutations, including Q954H, N969K, and L981F from the Omicron variant, in the postfusion HR1HR2 bundle for functional and structural studies. We designed a molecular scaffold to determine cryogenic electron microscopy (cryo-EM) structures of HR1HR2 at 2.2­3.8 Å resolution by linking the trimeric N termini of four HR1 fragments to four trimeric C termini of the Dps4 dodecamer from Nostoc punctiforme. This molecular scaffold enables efficient sample preparation and structure determination of the HR1HR2 bundle and its mutants by single-particle cryo-EM. Our structure of the wild-type HR1HR2 bundle resolves uncertainties in previously determined structures. The mutant structures reveal side-chain positions of the mutations and their primarily local effects on the interactions between HR1 and HR2. These mutations do not alter the global architecture of the postfusion HR1HR2 bundle, suggesting that the interfaces between HR1 and HR2 are good targets for developing antiviral inhibitors that should be efficacious against all known variants of SARS-CoV-2 to date. We also note that this work paves the way for similar studies in more distantly related viruses.

The AAA+ superfamily: a review of the structural and mechanistic principles of these molecular machines.

ATPases associated with diverse cellular activities (AAA+ proteins) are a superfamily of proteins found throughout all domains of life. The hallmark of this family is a conserved AAA+ domain responsible for a diverse range of cellular activities. Typically, AAA+ proteins transduce chemical energy from the hydrolysis of ATP into mechanical energy through conformational change, which can drive a variety of biological processes. AAA+ proteins operate in a variety of cellular contexts with diverse functions including disassembly of SNARE proteins, protein quality control, DNA replication, ribosome assembly, and viral replication. This breadth of function illustrates both the importance of AAA+ proteins in health and disease and emphasizes the importance of understanding conserved mechanisms of chemo-mechanical energy transduction. This review is divided into three major portions. First, the core AAA+ fold is presented. Next, the seven different clades of AAA+ proteins and structural details and reclassification pertaining to proteins in each clade are described. Finally, two well-known AAA+ proteins, NSF and its close relative p97, are reviewed in detail.

Role of Aberrant Spontaneous Neurotransmission in SNAP25-Associated Encephalopathies.

SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptor) complex, composed of synaptobrevin, syntaxin, and SNAP25, forms the essential fusion machinery for neurotransmitter release. Recent studies have reported several mutations in the gene encoding SNAP25 as a causative factor for developmental and epileptic encephalopathies of infancy and childhood with diverse clinical manifestations. However, it remains unclear how SNAP25 mutations give rise to these disorders. Here, we show that although structurally clustered mutations in SNAP25 give rise to related synaptic transmission phenotypes, specific alterations in spontaneous neurotransmitter release are a key factor to account for disease heterogeneity. Importantly, we identified a single mutation that augments spontaneous release without altering evoked release, suggesting that aberrant spontaneous release is sufficient to cause disease in humans.

Structures of neurexophilin-neurexin complexes reveal a regulatory mechanism of alternative splicing.

Neurexins are presynaptic, cell-adhesion molecules that specify the functional properties of synapses via interactions with trans-synaptic ligands. Neurexins are extensively alternatively spliced at six canonical sites that regulate multifarious ligand interactions, but the structural mechanisms underlying alternative splicing-dependent neurexin regulation are largely unknown. Here, we determined high-resolution structures of the complex of neurexophilin-1 and the second laminin/neurexin/sex-hormone-binding globulin domain (LNS2) of neurexin-1 and examined how alternative splicing at splice site #2 (SS2) regulates the complex. Our data reveal a unique, extensive, neurexophilin-neurexin binding interface that extends the jelly-roll ß-sandwich of LNS2 of neurexin-1 into neurexophilin-1. The SS2A insert of LNS2 augments this interface, increasing the binding affinity of LNS2 for neurexophilin-1. Taken together, our data reveal an unexpected architecture of neurexophilin-neurexin complexes that accounts for the modulation of binding by alternative splicing, which in turn regulates the competition of neurexophilin for neurexin binding with other ligands.

Calibration of rotation axes for multi-axis goniometers in macromolecular crystallography.

The installation of multi-axis goniometers such as the ESRF/EMBL miniKappa goniometer system has allowed the increased use of sample reorientation in macromolecular crystallography. Old and newly appearing data collection methods require precision and accuracy in crystal reorientation. The proper use of such multi-axis systems has necessitated the development of rapid and easy to perform methods for establishing and evaluating device calibration. A new diffraction-based method meeting these criteria has been developed for the calibration of the motors responsible for rotational motion. This method takes advantage of crystal symmetry by comparing the orientations of a sample rotated about a given axis and checking that the magnitude of the real rotation fits the calculated angle between these two orientations. Hence, the accuracy and precision of rotational motion can be assessed. This rotation calibration procedure has been performed on several beamlines at the ESRF and other synchrotrons. Some resulting data are presented here for reference.

Structural principles of SNARE complex recognition by the AAA+ protein NSF.

The recycling of SNARE proteins following complex formation and membrane fusion is an essential process in eukaryotic trafficking. A highly conserved AAA+ protein, NSF (N-ethylmaleimide sensitive factor) and an adaptor protein, SNAP (soluble NSF attachment protein), disassemble the SNARE complex. We report electron-cryomicroscopy structures of the complex of NSF, αSNAP, and the full-length soluble neuronal SNARE complex (composed of syntaxin-1A, synaptobrevin-2, SNAP-25A) in the presence of ATP under non-hydrolyzing conditions at ~3.9 Å resolution. These structures reveal electrostatic interactions by which two αSNAP molecules interface with a specific surface of the SNARE complex. This interaction positions the SNAREs such that the 15 N-terminal residues of SNAP-25A are loaded into the D1 ring pore of NSF via a spiral pattern of interactions between a conserved tyrosine NSF residue and SNAP-25A backbone atoms. This loading process likely precedes ATP hydrolysis. Subsequent ATP hydrolysis then drives complete disassembly.

NSF-mediated disassembly of on- and off-pathway SNARE complexes and inhibition by complexin.

SNARE complex disassembly by the ATPase NSF is essential for neurotransmitter release and other membrane trafficking processes. We developed a single-molecule FRET assay to monitor repeated rounds of NSF-mediated disassembly and reassembly of individual SNARE complexes. For ternary neuronal SNARE complexes, disassembly proceeds in a single step within 100 msec. We observed short- (<0.32 s) and long-lived (≥0.32 s) disassembled states. The long-lived states represent fully disassembled SNARE complex, while the short-lived states correspond to failed disassembly or immediate reassembly. Either high ionic strength or decreased αSNAP concentration reduces the disassembly rate while increasing the frequency of short-lived states. NSF is also capable of disassembling anti-parallel ternary SNARE complexes, implicating it in quality control. Finally, complexin-1 competes with αSNAP binding to the SNARE complex; addition of complexin-1 has an effect similar to that of decreasing the αSNAP concentration, possibly differentially regulating cis and trans SNARE complexes disassembly.

Electric-field-stimulated protein mechanics.

The internal mechanics of proteins-the coordinated motions of amino acids and the pattern of forces constraining these motions-connects protein structure to function. Here we describe a new method combining the application of strong electric field pulses to protein crystals with time-resolved X-ray crystallography to observe conformational changes in spatial and temporal detail. Using a human PDZ domain (LNX2PDZ2) as a model system, we show that protein crystals tolerate electric field pulses strong enough to drive concerted motions on the sub-microsecond timescale. The induced motions are subtle, involve diverse physical mechanisms, and occur throughout the protein structure. The global pattern of electric-field-induced motions is consistent with both local and allosteric conformational changes naturally induced by ligand binding, including at conserved functional sites in the PDZ domain family. This work lays the foundation for comprehensive experimental study of the mechanical basis of protein function.

Origins of Allostery and Evolvability in Proteins: A Case Study.

Proteins display the capacity for adaptation to new functions, a property critical for evolvability. But what structural principles underlie the capacity for adaptation? Here, we show that adaptation to a physiologically distinct class of ligand specificity in a PSD95, DLG1, ZO-1 (PDZ) domain preferentially occurs through class-bridging intermediate mutations located distant from the ligand-binding site. These mutations provide a functional link between ligand classes and demonstrate the principle of "conditional neutrality" in mediating evolutionary adaptation. Structures show that class-bridging mutations work allosterically to open up conformational plasticity at the active site, permitting novel functions while retaining existing function. More generally, the class-bridging phenotype arises from mutations in an evolutionarily conserved network of coevolving amino acids in the PDZ family (the sector) that connects the active site to distant surface sites. These findings introduce the concept that allostery in proteins could have its origins not in protein function but in the capacity to adapt.