Uncoupling the ATPase activity of the N-ethylmaleimide sensitive factor (NSF) from 20S complex disassembly.

The N-ethylmaleimide sensitive factor (NSF) plays a critical role in intracellular trafficking by disassembling soluble NSF attachment protein receptor (SNARE ) complexes. The NSF protomer consists of three domains (NSF-N, NSF-D1, and NSF-D2). Two domains (NSF-D1 and NSF-D2) contain a conserved approximately 230 amino acid cassette, which includes a distinctive motif termed the second region of homology (SRH) common to all ATPases associated with various cellular activities (AAA). In hexameric NSF, several SRH residues become trans elements of the ATP binding pocket. Mutation of two conserved arginine residues in the NSF-D1 SRH (R385A and R388A) did not effect basal or soluble NSF attachment protein (SNAP)-stimulated ATPase activity; however, neither mutant underwent ATP-dependent release from SNAP-SNARE complexes. A trans element of the NSF-D2 ATP binding site (K631) has been proposed to limit the ATPase activity of NSF-D2, but a K631D mutant retained wild-type activity. A mutation of the equivalent residue in NSF-D1 (D359K) also did not affect nucleotide hydrolysis activity but did limit NSF release from SNAP-SNARE complexes. These trans elements of the NSF-D1 ATP binding site (R385, R388, and D359) are not required for nucleotide hydrolysis but are important as nucleotide-state sensors. NSF-N mediates binding to the SNAP-SNARE complex. To identify the structural features required for binding, three conserved residues (R67, S73, and Q76) on the surface of NSF-N were mutated. R67E completely eliminated binding, while S73R and Q76E showed limited effect. This suggests that the surface important for SNAP binding site lies in the cleft between the NSF-N subdomains adjacent to a conserved, positively charged surface.     

Multiple binding proteins suggest diverse functions for the N-ethylmaleimide sensitive factor

The hexameric ATPase, N-ethylmaleimide sensitive factor (NSF), is essential to vesicular transport and membrane fusion because it affects the conformations and associations of the soluble NSF attachment protein receptor (SNARE) proteins. NSF binds SNAREs through adaptors called soluble NSF attachment proteins (alpha- or beta-SNAP) and disassembles SNARE complexes to recycle the monomers. NSF contains three domains, two nucleotide-binding domains (NSF-D1 and -D2) and an amino terminal domain (NSF-N) that is required for SNAP-SNARE complex binding. Mutagenesis studies indicate that a cleft between the two sub-domains of NSF-N is critical for binding. The structural conservation of N domains in NSF, p97/VCP, and VAT suggests that a similar type of binding site could mediate substrate recognition by other AAA proteins. In addition to SNAP-SNARE complexes, NSF also binds other proteins and protein complexes such as AMPA receptor subunits (GluR2), beta2-adrenergic receptor, beta-Arrestin1, GATE-16, LMA1, rabs, and rab-containing complexes. The potential for these interactions indicates a broader role for NSF in the assembly/disassembly cycles of several cellular complexes and suggests that NSF may have specific regulatory effects on the functions of the proteins involved in these complexes. The structural requirements for these interactions and their physiological significance will be discussed.     

Nucleotide-dependent conformational changes in the N-Ethylmaleimide Sensitive Factor (NSF) and their potential role in SNARE complex disassembly

Homohexameric, N-Ethylmaleimide Sensitive Factor (NSF) disassembles Soluble NSF Attachment Protein Receptor (SNARE) complexes after membrane fusion, an essential step in vesicular trafficking. NSF contains three domains (NSF-N, NSF-D1, and NSF-D2), each contributing to activity. We combined electron microscopic (EM) analysis, analytical ultracentrifugation (AU) and functional mutagenesis to visualize NSF's ATPase cycle. 3D density maps show that NSF-D2 remains stable, whereas NSF-N undergoes large conformational changes. NSF-Ns splay out perpendicular to the ADP-bound hexamer and twist upwards upon ATP binding, producing a more compact structure. These conformations were confirmed by hydrodynamic, AU measurements: NSF-ATP sediments faster with a lower frictional ratio (f/f(0)). Hydrodynamic analyses of NSF mutants, with specific functional defects, define the structures underlying these conformational changes. Mapping mutations onto our 3D models allows interpretation of the domain movement and suggests a mechanism for NSF binding to and disassembly of SNARE complexes.     

Dissecting the N-Ethylmaleimide-sensitive Factor: REQUIRED ELEMENTS OF THE N AND D1 DOMAINS*

N-Ethylmaleimide-sensitive factor (NSF) is a homo-hexameric member of the AAA⁺ (ATPases associated with various cellular activities plus) family. It plays an essential role in most intracellular membrane trafficking through its binding to and disassembly of soluble NSF attachment protein (SNAP) receptor (SNARE) complexes. Each NSF protomer contains an N-terminal domain (NSF-N) and two AAA domains, a catalytic NSF-D1 and a structural NSF-D2. This study presents detailed mutagenesis analyses of NSF-N and NSF-D1, dissecting their roles in ATP hydrolysis, SNAP·SNARE binding, and complex disassembly. Our results show that a positively charged surface on NSF-N, bounded by Arg⁶⁷ and Lys¹⁰⁵, and the conserved residues in the central pore of NSF-D1 (Tyr²⁹⁶ and Gly²⁹⁸) are involved in SNAP·SNARE binding but not basal ATP hydrolysis. Mutagenesis of Sensor 1 (Thr³⁷³–Arg³⁷⁵), Sensor 2 (Glu⁴⁴⁰–Glu⁴⁴²), and Arginine Fingers (Arg³⁸⁵ and Arg³⁸⁸) in NSF-D1 shows that each region plays a discrete role. Sensor 1 is important for basal ATPase activity and nucleotide binding. Sensor 2 plays a role in ATP- and SNAP-dependent SNARE complex binding and disassembly but does so in cis and not through inter-protomer interactions. Arginine Fingers are important for SNAP·SNARE complex-stimulated ATPase activity and complex disassembly. Mutants at these residues have a dominant-negative phenotype in cells, suggesting that Arginine Fingers function in trans via inter-protomer interactions. Taken together, these data establish functional roles for many of the structural elements of the N domain and of the D1 ATP-binding site of NSF.     

Electron cryomicroscopy structure of N-ethyl maleimide sensitive factor at 11 A resolution

N-ethyl maleimide sensitive factor (NSF) belongs to the AAA family of ATPases and is involved in a number of cellular functions, including vesicle fusion and trafficking of membrane proteins. We present the three-dimensional structure of the hydrolysis mutant E329Q of NSF complexed with an ATP-ADP mixture at 11 A resolution by electron cryomicroscopy and single-particle averaging of NSF.alpha-SNAP.SNARE complexes. The NSF domains D1 and D2 form hexameric rings that are arranged in a double-layered barrel. Our structure is more consistent with an antiparallel orientation of the two rings rather than a parallel one. The crystal structure of the D2 domain of NSF was docked into the EM density map and shows good agreement, including details at the secondary structural level. Six protrusions corresponding to the N domain of NSF (NSF-N) emerge from the sides of the D1 domain ring. The density corresponding to alpha-SNAP and SNAREs is located on the 6-fold axis of the structure, near the NSF-N domains. The density of the N domain is weak, suggesting conformational variability in this part of NSF.     

Crystal structure of the amino-terminal domain of N-ethylmaleimide-sensitive fusion protein

The cytosolic ATPase N-ethylmaleimide-sensitive fusion protein (NSF) disassembles complexes of membrane-bound proteins known as SNAREs, an activity essential for vesicular trafficking. The amino-terminal domain of NSF (NSF-N) is required for the interaction of NSF with the SNARE complex through the adaptor protein alpha-SNAP. The crystal structure of NSF-N reveals two subdomains linked by a single stretch of polypeptide. A polar interface between the two subdomains indicates that they can move with respect to one another during the catalytic cycle of NSF. Structure-based sequence alignments indicate that in addition to NSF orthologues, the p97 family of ATPases contain an amino-terminal domain of similar structure.     

Bcs1, a AAA protein of the mitochondria with a role in the biogenesis of the respiratory chain

The family of AAA+ proteins in eukaryotes has many members in various cellular compartments with a broad spectrum of functions in protein unfolding and degradation. The mitochondrial AAA protein Bcs1 plays an unusual role in protein translocation. It is involved in the topogenesis of the Rieske protein, Rip1, and thereby in the biogenesis of the cytochrome bc(1) complex of the mitochondrial respiratory chain. Bcs1 mediates the export of the folded FeS domain of Rip1 across the mitochondrial inner membrane and the insertion of its transmembrane segment into an assembly intermediate of the cytochrome bc(1) complex. We discuss structural elements of the Bcs1 protein compared to other AAA proteins in an attempt to understand the mechanism of its function. In this context, we discuss human diseases caused by mutations in Bcs1 that lead to different properties of the protein and subsequently to different symptoms.     

Nucleotide-dependent conformational changes in a protease-associated ATPase HsIU.

BACKGROUND: The bacterial heat shock locus ATPase HslU is an AAA(+) protein that has structures known in many nucleotide-free and -bound states. Nucleotide is required for the formation of the biologically active HslU hexameric assembly. The hexameric HslU ATPase binds the dodecameric HslV peptidase and forms an ATP-dependent HslVU protease. RESULTS: We have characterized four distinct HslU conformational states, going sequentially from open to closed: the empty, SO(4), ATP, and ADP states. The nucleotide binds at a cleft formed by an alpha/beta domain and an alpha-helical domain in HslU. The four HslU states differ by a rotation of the alpha-helical domain. This classification leads to a correction of nucleotide identity in one structure and reveals the ATP hydrolysis-dependent structural changes in the HslVU complex, including a ring rotation and a conformational change of the HslU C terminus. This leads to an amended protein unfolding-coupled translocation mechanism. CONCLUSIONS: The observed nucleotide-dependent conformational changes in HslU and their governing principles provide a framework for the mechanistic understanding of other AAA(+) proteins.     

Demonstration of differential quantitative requirements for NSF among multiple vesicle fusion pathways of GLUT4 using a dominant-negative ATPase-deficient NSF

In this study, we investigated the relative participation of N-ethylmaleimide-sensitive factor (NSF) in vivo in a complex multistep vesicle trafficking system, the translocation response of GLUT4 to insulin in rat adipose cells. Transfections of rat adipose cells demonstrate that over-expression of wild-type NSF has no effect on total, or basal and insulin-stimulated cell-surface expression of HA-tagged GLUT4. In contrast, a dominant-negative NSF (NSF-D1EQ) can be expressed at a low enough level that it has little effect on total HA-GLUT4, but does reduce both basal and insulin-stimulated cell-surface HA-GLUT4 by approximately 50% without affecting the GLUT4 fold-translocation response to insulin. However, high expression levels of NSF-D1EQ decrease total HA-GLUT4. The inhibitory effect of NSF-D1EQ on cell-surface HA-GLUT4 is reversed when endocytosis is inhibited by co-expression of a dominant-negative dynamin (dynamin-K44A). Moreover, NSF-D1EQ does not affect cell-surface levels of constitutively recycling GLUT1 and TfR, suggesting a predominant effect of low-level NSF-D1EQ on the trafficking of GLUT4 from the endocytic recycling compared to the intracellular GLUT4-specific compartment. Thus, our data demonstrate that the multiple fusion steps in GLUT4 trafficking have differential quantitative requirements for NSF activity. This indicates that the rates of plasma and intracellular membrane fusion reactions vary, leading to differential needs for the turnover of the SNARE proteins.     

Crystal structure of the SF3 helicase from adeno-associated virus type 2

We report here the crystal structure of an SF3 DNA helicase, Rep40, from adeno-associated virus 2 (AAV2). We show that AAV2 Rep40 is structurally more similar to the AAA(+) class of cellular proteins than to DNA helicases from other superfamilies. The structure delineates the expected Walker A and B motifs, but also reveals an unexpected "arginine finger" that directly implies the requirement of Rep40 oligomerization for ATP hydrolysis and helicase activity. Further, the Rep40 AAA(+) domain is novel in that it is unimodular as opposed to bimodular. Altogether, the structural connection to AAA(+) proteins defines the general architecture of SF3 DNA helicases, a family that includes simian virus 40 (SV40) T antigen, as well as provides a conceptual framework for understanding the role of Rep proteins during AAV DNA replication, packaging, and site-specific integration.     

NSF and p97/VCP: similar at first, different at last

N-Ethylmaleimide sensitive factor (NSF) and p97/valosin-containing protein (VCP) are distantly related members of the ATPases associated with a variety of cellular activities (AAA) family of proteins. While both proteins have been implied in cellular morphology changes involving membrane compartments or vesicles, more recent evidence seems to imply that NSF is primarily involved in the soluble NSF attachment receptor (SNARE)-mediated vesicle fusion by disassembling the SNARE complex whereas p97/VCP is primarily involved in the extraction of membrane proteins. These functional differences are now corroborated by major structural differences based on recent crystallographic and cryo-electron microscopy studies. This review discusses these recent findings.     

Structure of the AAA ATPase p97

p97, an abundant hexameric ATPase of the AAA family, is involved in homotypic membrane fusion. It is thought to disassemble SNARE complexes formed during the process of membrane fusion. Here, we report two structures: a crystal structure of the N-terminal and D1 ATPase domains of murine p97 at 2.9 A resolution, and a cryoelectron microscopy structure of full-length rat p97 at 18 A resolution. Together, these structures show that the D1 and D2 hexamers pack in a tail-to-tail arrangement, and that the N domain is flexible. A comparison with NSF D2 (ATP complex) reveals possible conformational changes induced by ATP hydrolysis. Given the D1 and D2 packing arrangement, we propose a ratchet mechanism for p97 during its ATP hydrolysis cycle.     

Crystal structure of E. coli Hsp100 ClpB nucleotide-binding domain 1 (NBD1) and mechanistic studies on ClpB ATPase activity

E. coli Hsp100 ClpB was recently identified as a critical part in a multi-chaperone system to play important roles in protein folding, protein transport and degradation in cell physiology. ClpB contains two nucleotide-binding domains (NBD1 and NBD2) within their primary sequences. NBD1 and NBD2 of ClpB can be classified as members of the large ATPase family known as ATPases associated with various cellular activities (AAA). To investigate how ClpB performs its ATPase activities for its chaperone activity, we have determined the crystal structure of ClpB nucleotide-binding domain 1 (NBD1) by MAD method to 1.80 A resolution. The NBD1 monomer structure contains one domain that comprises 11 alpha-helices and six beta-strands. When compared with the typical AAA structures, the crystal structure of ClpB NBD1 reveals a novel AAA topology with six-stranded beta-sheet as its core. The N-terminal portion of NBD1 structure has an extra beta-strand flanked by two extra alpha-helices that are not present in other AAA structures. Moreover, the NBD1 structure does not have a C-terminal helical domain as other AAA proteins do. No nucleotide molecule is bound with ClpB NBD1 in the crystal structure probably due to lack of the C-terminal helix domain in the structure. Isothermal titration calorimetry (ITC) studies of ClpB NBD1 and other ClpB deletion mutations showed that either ClpB NBD1 or NBD2 alone does not bind to nucleotides. However, ClpB NBD2 combined with ClpB C-terminal fragment can interact with one ADP or ATP molecule. ITC data also indicated that full-length ClpB could bind two ADP molecules or one ATP analogue ATPgammaS molecule. Further ATPase activity studies of ClpB and ClpB deletion mutants showed that only wild-type ClpB have ATPase activity. None of ClpB NBD1 domain, NBD2 domain and NBD2 with C-terminal fragment has detectable ATPase activities. On the basis of our structural and mutagenesis data, we proposed a "see-saw" model to illustrate the mechanisms by which ClpB performs its ATPase activities for chaperone functions.     

Hierarchical binding of cofactors to the AAA ATPase p97

The hexameric AAA ATPase p97 is involved in several human proteinopathies and mediates ubiquitin-dependent protein degradation among other essential cellular processes. Via its N-terminal domain (N domain), p97 interacts with multiple regulatory cofactors including the UFD1/NPL4 heterodimer and members of the "ubiquitin regulatory X" (UBX) domain protein family; however, the principles governing cofactor selectivity remain to be deciphered. Our crystal structure of the FAS-associated factor 1 (FAF1)UBX domain in complex with the p97N domain reveals that the signature Phe-Pro-Arg motif known to be crucial for interactions of UBX domains with p97 adopts a cis-proline configuration, in contrast to a cis-trans mixture we derive for the isolated FAF1UBX domain. Biochemical studies confirm that binding critically depends on a proline at this position. Furthermore, we observe that the UBX proteins FAF1 and UBXD7 only bind to p97-UFD1/NPL4, but not free p97, thus demonstrating for the first time a hierarchy in p97-cofactor interactions.     

Conformational transitions regulate the exposure of a DNA-binding domain in the RuvBL1–RuvBL2 complex

RuvBL1 and RuvBL2, also known as Pontin and Reptin, are AAA+ proteins essential in small nucleolar ribonucloprotein biogenesis, chromatin remodelling, nonsense-mediated messenger RNA decay and telomerase assembly, among other functions. They are homologous to prokaryotic RuvB, forming single- and double-hexameric rings; however, a DNA binding domain II (DII) is inserted within the AAA+ core. Despite their biological significance, questions remain regarding their structure. Here, we report cryo-electron microscopy structures of human double-ring RuvBL1–RuvBL2 complexes at ∼15 Å resolution. Significantly, we resolve two coexisting conformations, compact and stretched, by image classification techniques. Movements in DII domains drive these conformational transitions, extending the complex and regulating the exposure of DNA binding regions. DII domains connect with the AAA+ core and bind nucleic acids, suggesting that these conformational changes could impact the regulation of RuvBL1–RuvBL2 containing complexes. These findings resolve some of the controversies in the structure of RuvBL1–RuvBL2 by revealing a mechanism that extends the complex by adjustments in DII.     

A screen for dominant negative mutants of SEC18 reveals a role for the AAA protein consensus sequence in ATP hydrolysis

An evolutionarily ancient mechanism is used for intracellular membrane fusion events ranging from endoplasmic reticulum-Golgi traffic in yeast to synaptic vesicle exocytosis in the human brain. At the heart of this mechanism is the core complex of N-ethylmaleimide-sensitive fusion protein (NSF), soluble NSF attachment proteins (SNAPs), and SNAP receptors (SNAREs). Although these proteins are accepted as key players in vesicular traffic, their molecular mechanisms of action remain unclear. To illuminate important structure-function relationships in NSF, a screen for dominant negative mutants of yeast NSF (Sec18p) was undertaken. This involved random mutagenesis of a GAL1-regulated SEC18 yeast expression plasmid. Several dominant negative alleles were identified on the basis of galactose-inducible growth arrest, of which one, sec18-109, was characterized in detail. The sec18-109 phenotype (abnormal membrane trafficking through the biosynthetic pathway, accumulation of a membranous tubular network, growth suppression, increased cell density) is due to a single A-G substitution in SEC18 resulting in a missense mutation in Sec18p (Thr(394)-->Pro). Thr(394) is conserved in most AAA proteins and indeed forms part of the minimal AAA consensus sequence that serves as a signature of this large protein family. Analysis of recombinant Sec18-109p indicates that the mutation does not prevent hexamerization or interaction with yeast alpha-SNAP (Sec17p), but instead results in undetectable ATPase activity that cannot be stimulated by Sec17p. This suggests a role for the AAA protein consensus sequence in regulating ATP hydrolysis. Furthermore, this approach of screening for dominant negative mutants in yeast can be applied to other conserved proteins so as to highlight important functional domains in their mammalian counterparts.     

A multisubunit particle implicated in membrane fusion

The N-ethylmaleimide sensitive fusion protein (NSF) is required for fusion of lipid bilayers at many locations within eukaryotic cells. Binding of NSF to Golgi membranes is known to require an integral membrane receptor and one or more members of a family of related soluble NSF attachment proteins (alpha-, beta-, and gamma-SNAPs). Here we demonstrate the direct interaction of NSF, SNAPs and an integral membrane component in a detergent solubilized system. We show that NSF only binds to SNAPs in the presence of the integral receptor, resulting in the formation of a multisubunit protein complex with a sedimentation coefficient of 20S. Particle assembly reveals striking differences between members of the SNAP protein family; gamma-SNAP associates with the complex via a binding site distinct from that used by alpha- and beta-SNAPs, which are themselves equivalent, alternative subunits of the particle. Once formed, the 20S particle is subsequently able to disassemble in a process coupled to the hydrolysis of ATP. We suggest how cycles of complex assembly and disassembly could help confer specificity to the generalized NSF-dependent fusion apparatus.