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. 2010 Jun 11;285(24):18650-61.
doi: 10.1074/jbc.M109.092072. Epub 2010 Mar 18.

Allosteric drug discrimination is coupled to mechanochemical changes in the kinesin-5 motor core

Elizabeth D Kim  1 , Rebecca Buckley, Sarah Learman, Jessica Richard, Courtney Parke, David K Worthylake, Edward J Wojcik, Richard A Walker, Sunyoung Kim
Affiliations

Allosteric drug discrimination is coupled to mechanochemical changes in the kinesin-5 motor core

Elizabeth D Kim et al. J Biol Chem. .

Abstract

Essential in mitosis, the human Kinesin-5 protein is a target for >80 classes of allosteric compounds that bind to a surface-exposed site formed by the L5 loop. Not established is why there are differing efficacies in drug inhibition. Here we compare the ligand-bound states of two L5-directed inhibitors against 15 Kinesin-5 mutants by ATPase assays and IR spectroscopy. Biochemical kinetics uncovers functional differences between individual residues at the N or C termini of the L5 loop. Infrared evaluation of solution structures and multivariate analysis of the vibrational spectra reveal that mutation and/or ligand binding not only can remodel the allosteric binding surface but also can transmit long range effects. Changes in L5-localized 3(10) helix and disordered content, regardless of substitution or drug potency, are experimentally detected. Principal component analysis couples these local structural events to two types of rearrangements in beta-sheet hydrogen bonding. These transformations in beta-sheet contacts are correlated with inhibitory drug response and are corroborated by wild type Kinesin-5 crystal structures. Despite considerable evolutionary divergence, our data directly support a theorized conserved element for long distance mechanochemical coupling in kinesin, myosin, and F(1)-ATPase. These findings also suggest that these relatively rapid IR approaches can provide structural biomarkers for clinical determination of drug sensitivity and drug efficacy in nucleotide triphosphatases.

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Figures

FIGURE 1.
FIGURE 1.
Substitution of L5 loop residues in Eg5 kinesin, with known sequence and spatial organization, can result in gel shifting in denaturing SDS-PAGE analysis. A, sequence alignment of Eg5 and other members of the Kinesin-5 family was generated using the ClustalW method in MegAlign 6.1.2 (DNASTAR). Conserved residues of interest are colored. Scored in green and aqua are Glu-116/Glu-118 and Asp-130/Ala-133 residues in the insertion loop, respectively. B, PyMOL representation of the crystallographic data from Ref. . Highlighted are the N-terminal Glu-116 and Glu-118 (green) and C-terminal Asp-130 and Ala-133 (aqua) residues in the L5 loop (red) of Eg5 kinesin. Also shown are monastrol (yellow), Mg2+ (gold sphere), and ADP. Substitutions of Glu-116 (C), Glu-118 (D), Asp-130 (E), and Ala-133 (F) were SDS-PAGE-analyzed with a 10% acrylamide gel. The gel migration of the double mutant, E116D/E118D (DD), matched wild type (WT) Eg5 samples (data not shown). Protein samples were expressed, purified, and used in two different laboratories; equivalent results were observed in both settings.
FIGURE 2.
FIGURE 2.
Kinetic effects of N-terminal L5 residue substitutions are residue-dependent, but C-terminal L5 residue substitutions have no clear kinetic correlation. A and B, steady-state, basal ATP hydrolysis rates, for which mean Vmax for wild type (WT) (black) is 0.144 ± 0.003 s−1. For wild type, n = 101, whereas n for all substitutions ranges from 20 to 51. C and D, normalized Eg5 ATP hydrolysis rates were plotted against increasing concentrations of monastrol (left) or STC (right). WT kinetic data (black) are superimposed on Glu-116 (C, top), Glu-118 (C, bottom), Asp-130 (D, top), and Ala-133 mutants (D, bottom). The double mutant E116D/E118D (DD) data are shown in pink hatched boxes (C, top). All reactions contain 3–22.5 mm NaCl. Colors correspond to protein samples in A and B, respectively. ATP hydrolysis rates for each substitution are normalized with respect to the 0 mm monastrol or 0 mm STC measurements of the parent substitution. All data points in the figure reflect the averaged normalized rate from 2–4 separate protein preparations and the S.E. (error bars).
FIGURE 3.
FIGURE 3.
FTIR spectra illustrate quantifiable changes in secondary structure resulting from challenges to L5, either by drug inhibition or residue substitution. A, averaged FTIR spectra of the amide I′ region of wild type (WT), Glu-116 substitutions, and Glu-118 substitutions are plotted. The wild type spectra are superimposed (dotted line) on each trace, and differences in line shape are highlighted, showing changes in secondary structure. FTIR spectra were acquired from two independent purifications and averaged. B, band narrowing of the amide I′ region of the N-terminal L5 mutant proteins resulted in 10 frequency bins. Positive amplitudes indicate a gain of secondary structure at a given frequency from wild type. Negative amplitudes indicate a loss of structure. Wild type was inhibited by both monastrol in 1:1 (black box, dark gray fill) and 1:20 (light gray fill) ratios and STC (striped fill). Other bar colors correspond to those for a given substitution shown in A.
FIGURE 4.
FIGURE 4.
PCA of secondary structural elements in the Eg5 motor domain reveals distinct conformer populations, resulting from L5-initiated effects. The score, or spatial positioning, of the data points is based on changes in percentage area of the secondary structure profiles determined by IR band narrowing. A, PCA clusters the majority of Glu-118 (■) and WT (●) data points together based on secondary structure. The Glu-116 (□) polymorphisms fall into a separate line of degeneracy with a larger disordered component. The 1642 and 1646 cm−1 vectors separate these two populations. B, PCA of C-terminal L5 substitutions, Ala-133 (▴) and Asp-130 (▵), falls predominantly into the same population occupied by the N-terminal Glu-118 mutations and can be differentiated by the 1633 and 1628 cm−1 vectors, respectively. C, a composite of both N- and C-terminal polymorphisms are plotted. Highlighted are the monastrol (blue) and STC (pink) data points.
FIGURE 5.
FIGURE 5.
Comparison of x-ray crystallographic structures of the Eg5 motor domain bound with L5-directed inhibitors shows distortions in β-sheets. Shown is a region of the antiparallel β-sheet of the Eg5 motor domain; other structural motifs that cover the central β-sheet were removed in PyMOL for visual clarity. The wild type Eg5·ADP structure (PDB ID 1II6) in gray is overlaid on the ternary structure in complex with STC (PDB ID 3KEN) in green (A), monastrol (PDB ID 1X88) in blue (B), and a tetrahydroisoquinoline carboxamide in pink (PDB ID 2FME) (C). Appropriate β-strands are labeled.
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