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

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

Arne Moeller et al. J Struct Biol. 2012 Feb.

Abstract

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.

Copyright © 2012 Elsevier Inc. All rights reserved.

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Conflict of interest statement

Competing financial interests

The authors declare no competing financial interests.

Figures

Fig. 1
Fig. 1

Surface representations of NSF in three different nucleotide-bound states and the N domain deletion mutant D1D2-ADP. Surface representations of the reconstructed density maps at mass correlating thresholds filtered to 18 Å (~510 and ~390 kDa). Rows a–c show NSF-AMP-PNP, -ADP and NSF-D1D2-ADP, respectively. The maps are shown as top, bottom, oblique and side views (from left to right). All maps have a diameter of ~115 Å and a height of ~80 Å for the D1 and D2 domains together. In NSF-AMP-PNP map arm densities protrude upwards, in NSF-ADP they splay outwards, and no arm densities are apparent in the D1D2-ADP map.

Fig. 2
Fig. 2

Docking of the individual domains into the surface representations. Results of the automated docking procedure are depicted for the ATP- (a and b) and ADP-states (c and d) of full-length NSF as side views with (a and c) and without the 3D-densities (b and d). Only one out of the six monomers is shown for clarity. Alternative views of the docking of the full hexamer are presented in lower panel (e and f) and in Movies 1 and 2. The automated positioning of the individual PDBs is in good agreement with the individual density maps. No significant overlap between individual domains is observed. N domains (PDB entry 1QDN) are colored in blue, D1 in orange and D2 (PDB entry 1D2N) in. Note the stability of D2, relative to D1, which tilts and rotates: the N arms pivoting by almost 180° around the α-helical part of D1 (Movie 3). Residues of special interest are labeled: yellow (R10, K104, K105, D142, K143) and red (R67) residues: important for SNAP/SNARE binding; purple: Sensor 2 (E440, L441, E442); blue: Arg-fingers (R385, R388); green: pore residues (Y296, G298).

Fig. 3
Fig. 3

Hydrodynamic bead models. Hydrodynamic bead models of full-length NSF in AMP-PNP and ADP-bound states and of D1D2-ADP and D2-ANP-PNP were constructed based on the models in Fig. 2 and the crystal structure of NSF-D2 (PDB entry 1D2N). The models were built using the Solution Modeler (SOMO) routine (Brookes et al., 2010a,b) and presented as top and side views. Bead colors reflect degree of surface exposure as assigned by SOMO.

Fig. 4
Fig. 4

Sedimentation velocity characterization of NSF mutant conformation in ADP or AMP-PNP state. Panel a: sedimentation velocity analysis of NSF-R67A in ADP- and AMP-PNP- bound states. His6-NSF-R67A in ADP (closed circles) or AMP-PNP (open circles) was labeled with (Ni2+-NTA)2-Cy3 and sedimentation velocity was performed as in the Methods. Radial intensity scan data were collected, converted to 2-pseudoabsorbance data and fitted to the c(s) model by SEDFIT program, returning the spectrum of s-values shown. Panel b: differential scan of sedimentation velocity of the AMP-PNP and ADP forms of (Ni2+-NTA)2-Cy3-His6-NSF-R67 (see Section 2). Solutions placed in the sample and reference sectors of the rotor were matched for OD550nm and volume. The downward deflection in the scan, taken after 80 min of sedimentation, indicates that NSF-R67A in its AMP-PNP-bound form sediments more rapidly. Panel c: the c(s) spectrum of (Ni2+-NTA)2-Cy3-His6-NSF-L441A-ADP (closed circles) or L441A-AMP-PNP (open circles) derived from the sedimentation velocity data as in panel A. Panel d: differential sedimentation of the AMP-PNP and ADP forms of (Ni2+-NTA)2-Cy3-His6-NSF-L441A. The lack of deflection in the scan indicates that NSF-L441A in its AMP-PNP-bound form sediments in the same speed as that in the ADP-bound form.

Fig. 5
Fig. 5

Potential mechanism of NSF. NSF-ATP binds to the C-terminus of α-SNAP (red wedge), which is bound to the coiled coil of the SNARE complex (a and b). Upon ATP hydrolysis the N-domains (blue block) pivot around the D1-domain, away from the center pulling, the α-SNAP and SNARE complex (c and d). This radial force serves to separate the SNAREs and thus disassemble the complex (e and f). The positively charged surface of NSF-N, which is important for SNAP–SNARE binding, is depicted as a yellow bar. The pore residues in NSF-D1 are depicted as green spheres. NSF-D1 and NSF-D2 are depicted as orange and green blocks, respectively.

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