Fig. 8: Principal component analysis of NSF D1 protomer coordinates reveals quantitative mechanistic details of sequential hydrolysis.

A 132 D1 domain models were aligned by large subdomain, subjected to Cɑ coordinate PCA, and clustered with HDBSCAN in 11-dimensional space (left). Protomers cluster into discrete groups based on conformation, visible upon projection into a plane defined by the first and fifth modes. A proposed hydrolysis cycle is indicated. Superimposed models corresponding to these transitions are shown (right). The cluster corresponding to pre-hydrolysis protomers E is circled. B Protomer nucleotide state, mapped to the 2D PCA coordinate from (A). The ATP + Mg²⁺ state is unique to protomers E (circled). C The angle (°) between helices ɑ₃ and ɑ₈ (a proxy for the relative angle between the D1 large and small subdomains). Protomers E have the largest angle (circled). D A mutant form of NSF, NSF(Δlatch), in which part of the latch loop (residues 460–466) was replaced with a short glycine-serine linker (SGS), was assayed for its ability to disassemble the ternary SNARE complex composed of syntaxin, SNAP-25, and synaptobrevin⁶. Traces show the mean ± SEM of eight replicates for each form of NSF, with and without adding MgCl₂. Disassembly rates were calculated by linear regression to each trace in the linear region of the time course, from ~95–350 s. NSF(Δlatch) disassembled SNARE complex at 28 ± 1% the rate of wild type. E Three conformational features distinguish the pre-hydrolysis protomer E cluster (circled). These features are in contact and structurally coupled. The position of nucleotide in the active site is modulated by the position of the Walker A helix (ɑ₃), which occupies three discrete states; pre-hydrolysis protomers E occupy the “down” state (left). The position of the ɑ₃ is allosterically modulated by at least two inputs. The N-D1 linker conformation (center) is connected to the state of N-domain binding to ɑ-SNAP, and it interacts with the latch loop of the up-ring protomer upon rebinding. The latch loop of the up-ring protomer contacts this region in trans and is also structurally coupled (right). These two elements push against the C-terminal end of ɑ₃ and ultimately modulate the position of the ATP γ-phosphate relative to key catalytic residues.