Essential in mitosis, the human Kinesin-5 protein is a target for >80 classes of allosteric compounds that bind to a surfaceexposed 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 ␤-sheet hydrogen bonding. These transformations in ␤-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.Allostery is important in controlled catalysis, signal transduction, and apoptosis (1). The classic view of proteins demonstrating this property (2) asserts that binding of a ligand at one site provokes conformational changes at a remote, second site. Recent studies (3) evaluating underlying mechanisms of allostery alternatively suggest that ligand binding results in selection of preexisting conformational substates. Implicit in the latter model is the principle that interactions between the orthosteric and allosteric sites are tightly linked through structure and thermodynamics (4). Active challenges in structural biology, which are central to this work, are deciphering the chemical nature of the ligand-protein interactions as well as how energy is transduced through protein structures to transmit allosteric events.Our experimental model, the human Kinesin-5 motor protein (Eg5 or KSP), plays key roles in bipolar mitotic spindle formation and is a protein target for allosteric compounds (5-7) that alter catalytic ATPase activity of the protein (8, 9). Biochemical studies demonstrate a wide concentration range of inhibition by these compounds (10 -12); there may be differences in the kinetic mechanism of allostery (13-15), and even allosteric activation (16) is possible. The best characterized inhibitors, monastrol (10) and S-trityl-L-cysteine (STC)2 (11), were uncovered from independent chemical screens.Interest in these allosteric compounds has been acute because they are potential anticancer agents. Additionally, these compo...

Kinesin-5 proteins are essential for formation of a bipolar mitotic spindle in most, and perhaps all, eukaryotic cells. Several Kinesin-5 proteins, notably the human version, HsEg5, are targets of a constantly expanding group of small-molecule inhibitors, which hold promise both as tools to probe mechanochemical transduction and as anti-cancer agents. Although most such compounds are selective for HsEg5 and closely related Kinesin-5 proteins, some, such as NSC 622124, exhibit activity against at least one kinesin from outside the Kinesin-5 family. Here we show NSC 622124, despite identification in a screen that yielded inhibitors now known to target the HsEg5 monastrol-binding site, does not compete with 14C-monastrol for binding to HsEg5, and is able to inhibit the basal and microtubule-stimulated ATPase activity of the monastrol-insensitive Kinesin-5, KLP61F. NSC 622124 competes with microtubules, but not ATP, for interaction with HsEg5, and disrupts the microtubule binding of HsEg5, KLP61F and Kinesin-1. Proteolytic degradation of an HsEg5•NSC622124 complex revealed that segments of the α3 and α5 helices map to the inhibitor-binding site. Overall, our results demonstrate that NSC 622124 targets the conserved microtubule-binding site of kinesin proteins. Further, unlike compounds previously reported to target the kinesin microtubule-binding site, NSC 622124 does not produce any enhancement of basal ATPase activity, and thus acts solely as a negative regulator through interaction with a site traditionally viewed as a binding region for positive regulators (i.e., microtubules). Our work emphasizes the concept that microtubule-dependent motor proteins may be controlled at multiple sites by both positive and negative effectors.

No abstract

likely to undergo exocytosis than those without such associations. When endocytosis was inhibited with Dynasore (inhibitor of dynamin 1 and 2), membrane p/s changes following fusion were long-lived (10s of seconds). This is consistent with endocytosis occuring at the site of exocytosis. These experiments are the first to directly visualize plasma membrane changes occuring with secretion. They reveal an association of the granule and plasma membrane for seconds before exocytosis and provide direct evidence for maintained curvature of the plasma membrane after exocytosis.