ABSTRACT
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.
Subject(s)
N-Ethylmaleimide-Sensitive Proteins/metabolism , Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins/chemistry , Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins/metabolism , Adenosine Triphosphate/metabolism , Animals , Cricetulus , Kinetics , Models, Molecular , N-Ethylmaleimide-Sensitive Proteins/chemistry , N-Ethylmaleimide-Sensitive Proteins/ultrastructure , Protein Conformation , Protein Subunits/chemistry , Protein Subunits/metabolism , Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins/ultrastructure , Substrate SpecificityABSTRACT
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.
Subject(s)
Adaptor Proteins, Vesicular Transport/metabolism , N-Ethylmaleimide-Sensitive Proteins/metabolism , Nerve Tissue Proteins/metabolism , SNARE Proteins/metabolism , Animals , Cricetulus , Fluorescence Resonance Energy Transfer , Kinetics , Mice , Mutant Proteins/metabolism , Mutation/genetics , N-Ethylmaleimide-Sensitive Proteins/ultrastructure , Osmolar Concentration , Protein Binding , Protein Domains , Qa-SNARE Proteins/chemistry , Qa-SNARE Proteins/metabolism , Rats , Single Molecule Imaging , Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins/metabolism , Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins/ultrastructureABSTRACT
Evolutionarily conserved SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptors) proteins form a complex that drives membrane fusion in eukaryotes. The ATPase NSF (N-ethylmaleimide sensitive factor), together with SNAPs (soluble NSF attachment protein), disassembles the SNARE complex into its protein components, making individual SNAREs available for subsequent rounds of fusion. Here we report structures of ATP- and ADP-bound NSF, and the NSF/SNAP/SNARE (20S) supercomplex determined by single-particle electron cryomicroscopy at near-atomic to sub-nanometre resolution without imposing symmetry. Large, potentially force-generating, conformational differences exist between ATP- and ADP-bound NSF. The 20S supercomplex exhibits broken symmetry, transitioning from six-fold symmetry of the NSF ATPase domains to pseudo four-fold symmetry of the SNARE complex. SNAPs interact with the SNARE complex with an opposite structural twist, suggesting an unwinding mechanism. The interfaces between NSF, SNAPs, and SNAREs exhibit characteristic electrostatic patterns, suggesting how one NSF/SNAP species can act on many different SNARE complexes.
Subject(s)
Multiprotein Complexes/chemistry , Multiprotein Complexes/metabolism , SNARE Proteins/chemistry , SNARE Proteins/metabolism , Adenosine Diphosphate/metabolism , Adenosine Triphosphate/metabolism , Animals , Cricetulus , Cryoelectron Microscopy , Models, Molecular , Multiprotein Complexes/ultrastructure , N-Ethylmaleimide-Sensitive Proteins/chemistry , N-Ethylmaleimide-Sensitive Proteins/metabolism , N-Ethylmaleimide-Sensitive Proteins/ultrastructure , Protein Binding , Protein Structure, Tertiary , Rats , SNARE Proteins/ultrastructure , Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins/chemistry , Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins/metabolism , Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins/ultrastructureABSTRACT
The 20S particle, which is composed of the N-ethylmaleimide-sensitive factor (NSF), soluble NSF attachment proteins (SNAPs) and the SNAP receptor (SNARE) complex, has an essential role in intracellular vesicle fusion events. Using single-particle cryo-EM and negative stain EM, we reconstructed four related three-dimensional structures: Chinese hamster NSF hexamer in the ATPγS, ADP-AlFx and ADP states, and the 20S particle. These structures reveal a parallel arrangement between the D1 and D2 domains of the hexameric NSF and characterize the nucleotide-dependent conformational changes in NSF. The structure of the 20S particle shows that it holds the SNARE complex at two interaction interfaces around the C terminus and N-terminal half of the SNARE complex, respectively. These findings provide insight into the molecular mechanism underlying disassembly of the SNARE complex by NSF.
Subject(s)
Adaptor Proteins, Vesicular Transport/chemistry , N-Ethylmaleimide-Sensitive Proteins/chemistry , SNARE Proteins/chemistry , Adaptor Proteins, Vesicular Transport/metabolism , Adaptor Proteins, Vesicular Transport/ultrastructure , Adenosine Diphosphate/chemistry , Adenosine Diphosphate/metabolism , Animals , Cricetinae , Cricetulus , Cryoelectron Microscopy , Models, Molecular , N-Ethylmaleimide-Sensitive Proteins/metabolism , N-Ethylmaleimide-Sensitive Proteins/ultrastructure , Protein Binding , Protein Structure, Quaternary , Protein Structure, Tertiary , SNARE Proteins/metabolism , SNARE Proteins/ultrastructure , Structural Homology, Protein , Substrate SpecificityABSTRACT
The AAA+ATPase p97/VCP, helped by adaptor proteins, exerts its essential role in cellular events such as endoplasmic reticulum-associated protein degradation or the reassembly of Golgi, ER and the nuclear envelope after mitosis. Here, we report the three-dimensional cryo-electron microscopy structures at approximately 20 Angstroms resolution in two nucleotide states of the endogenous hexameric p97 in complex with a recombinant p47 trimer, one of the major p97 adaptor proteins involved in membrane fusion. Depending on the nucleotide state, we observe the p47 trimer to be in two distinct arrangements on top of the p97 hexamer. By combining the EM data with NMR and other biophysical measurements, we propose a model of ATP-dependent p97(N) domain motions that lead to a rearrangement of p47 domains, which could result in the disassembly of target protein complexes.
