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. 2014 Nov 4;107(9):2204-13.
doi: 10.1016/j.bpj.2014.09.019.

Kinesin-5 Allosteric Inhibitors Uncouple the Dynamics of Nucleotide, Microtubule, and Neck-Linker Binding Sites

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Free PMC article

Kinesin-5 Allosteric Inhibitors Uncouple the Dynamics of Nucleotide, Microtubule, and Neck-Linker Binding Sites

Guido Scarabelli et al. Biophys J. .
Free PMC article

Abstract

Kinesin motor domains couple cycles of ATP hydrolysis to cycles of microtubule binding and conformational changes that result in directional force and movement on microtubules. The general principles of this mechanochemical coupling have been established; however, fundamental atomistic details of the underlying allosteric mechanisms remain unknown. This lack of knowledge hampers the development of new inhibitors and limits our understanding of how disease-associated mutations in distal sites can interfere with the fidelity of motor domain function. Here, we combine unbiased molecular-dynamics simulations, bioinformatics analysis, and mutational studies to elucidate the structural dynamic effects of nucleotide turnover and allosteric inhibition of the kinesin-5 motor. Multiple replica simulations of ATP-, ADP-, and inhibitor-bound states together with network analysis of correlated motions were used to create a dynamic protein structure network depicting the internal dynamic coordination of functional regions in each state. This analysis revealed the intervening residues involved in the dynamic coupling of nucleotide, microtubule, neck-linker, and inhibitor binding sites. The regions identified include the nucleotide binding switch regions, loop 5, loop 7, ?4-?5-loop 13, ?1, and ?4-?6-?7. Also evident were nucleotide- and inhibitor-dependent shifts in the dynamic coupling paths linking functional sites. In particular, inhibitor binding to the loop 5 region affected ?-sheet residues and ?1, leading to a dynamic decoupling of nucleotide, microtubule, and neck-linker binding sites. Additional analyses of point mutations, including P131 (loop 5), Q78/I79 (?1), E166 (loop 7), and K272/I273 (?7) G325/G326 (loop 13), support their predicted role in mediating the dynamic coupling of distal functional surfaces. Collectively, our results and approach, which we make freely available to the community, provide a framework for explaining how binding events and point mutations can alter dynamic couplings that are critical for kinesin motor domain function.

Figures

Figure 1
Figure 1
The distinct flexibilities of nucleotide- (switch I and switch II), inhibitor- (loop 5), and NL-binding regions in ATP- (red line), ADP- (green line), and inhibitor- (blue line) bound states. Regions that show statistically significant differences between states (with p < 0.01) and are composed of at least three consecutive residues are highlighted with a light-blue-shaded background. The consensus secondary structure is reported schematically with β strands in gray and α helices in black (top and bottom).
Figure 2
Figure 2
Community analysis reveals nucleotide- and inhibitor-dependent differences in the dynamic coupling of nucleotide-, inhibitor-, and NL-binding regions. (A) Community partitioning from correlation network analysis. (B) Front and back views of the motor domain structure for each state along with a zoomed-in view of the nucleotide- and microtubule-binding regions. The colors match the community partitioning shown in A.
Figure 3
Figure 3
Differences in residue-wise network centralities for ATP (red), ADP (green), and inhibitor (blue) networks. Regions of significant difference are highlighted with a light-blue-shaded background. Note the reduced inhibitor centralities for the central β-sheet (β3, β4, β6, and β7), as well as the α4-loop 12-α5-loop 13 and NL regions.
Figure 4
Figure 4
The extent of unique dynamic couplings per state defines the regions affected by nucleotide binding. (A and B) Front (A) and rear (B) views of the motor domain, colored by the proportion of unique couplings per residue. Yellow spheres correspond to residues with three or more unique couplings in both ATP and ADP states (see Table 1 for details).
Figure 5
Figure 5
Optimal and suboptimal path analysis reveals nucleotide- and inhibitor-dependent coupling routes between functional sites. (A–C) Paths linking nucleotide and the NL (with source and sink nodes represented by the β-phosphate of the nucleotide and residue 368 of the NL). (D–F) Paths from the nucleotide to α5 (β-phosphate and residue 318) and (G–I) paths from loop5 to the NL (residues 131 and 368). The ATP state is represented in A, D, and G; ADP is represented in B, E, and H; and inhibitor is represented in C, F, and I. Source and sink nodes are highlighted with spheres with related paths (passing through the same structural elements) between these nodes shown in the same color. See main text for details. To see this figure in color, go online.

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