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...

To understand the mechanism for assembly of Lys-linked polyubiquitin degradation signals, we previously demonstrated that the E6AP/UBE3A ligase harbors two functionally distinct E2∼ubiquitin-binding sites: a high-affinity Site 1 required for E6AP Cys∼ubiquitin thioester formation and a canonical Site 2 responsible for subsequent chain elongation. Ordered binding to Sites 1 and 2 is here revealed by observation of UbcH7∼ubiquitin-dependent substrate inhibition of chain formation at micromolar concentrations. To understand substrate inhibition, we exploited the PatchDock algorithm to model UbcH7∼ubiquitin bound to Site 1, validated by chain assembly kinetics of selected point mutants. The predicted structure buries an extensive solvent-excluded surface bringing the UbcH7∼ubiquitin thioester bond within 6 Å of the Cys nucleophile. Modeling onto the active E6AP trimer suggests that substrate inhibition arises from steric hindrance between Sites 1 and 2 of adjacent subunits. Confirmation that Sites 1 and 2 function in was demonstrated by examining the effect of E6APC820A on wild-type activity and single-turnover pulse-chase kinetics. A cyclic proximal indexation model proposes that Sites 1 and 2 function in tandem to assemble thioester-linked polyubiquitin chains from the proximal end attached to Cys before stochastic transfer to the target protein. Non-reducing SDS-PAGE confirms assembly of the predicted Cys-linked I-polyubiquitin thioester intermediate. Other studies suggest that Glu serves as a general base to generate the Cys thiolate within the low dielectric binding interface and Arg functions to orient Glu and to stabilize the incipient anionic transition state during thioester exchange.

How signals between the kinesin active and cytoskeletal binding sites are transmitted is an open question and an allosteric question. By extracting correlated evolutionary changes within 700+ sequences, we built a model of residues that are energetically coupled and that define molecular routes for signal transmission. Typically, these coupled residues are located at multiple distal sites and thus are predicted to form a complex, non-linear network that wires together different functional sites in the protein. Of note, our model connected the site for ATP hydrolysis with sites that ultimately utilize its free energy, such as the microtubule-binding site, drug-binding loop 5, and necklinker. To confirm the calculated energetic connectivity between non-adjacent residues, double-mutant cycle analysis was conducted with 22 kinesin mutants. There was a direct correlation between thermodynamic coupling in experiment and evolutionarily derived energetic coupling. We conclude that energy transduction is coordinated by multiple distal sites in the protein rather than only being relayed through adjacent residues. Moreover, this allosteric map forecasts how energetic orchestration gives rise to different nanomotor behaviors within the superfamily.

Human Kinesin-5 (Eg5) has a large number of known allosteric inhibitors that disrupt its mitotic function. Small-molecule inhibitors of Eg5 are candidate anti-cancer agents and important probes for understanding the cellular function. Here we show that Eg5 is capable of more than one type of microtubule interaction, and these activities can be controlled by allosteric agents. While both monastrol and S-trityl-L-cysteine inhibit Eg5 motility, our data reveal an unexpected ability of these loop5 targeting inhibitors to differentially control a novel Eg5 microtubule depolymerizing activity. Remarkably, small molecule loop5 effectors are able to independently modulate discrete functional interactions between the motor and microtubule track. We establish that motility can be uncoupled from the microtubule depolymerase activity and argue that loop5-targeting inhibitors of Kinesin-5 should not all be considered functionally synonymous. Also, the depolymerizing activity of the motor does not contribute to the genesis of monopolar spindles during allosteric inhibition of motility, but instead reveals a new function. We propose that, in addition to its canonical role in participating in the construction of the three-dimensional mitotic spindle structure, Eg5 also plays a distinct role in regulating the dynamics of individual microtubules, and thereby impacts the density of the mitotic spindle.

How signals between the kinesin active and cytoskeletal binding sites are transmitted is an open question and an allosteric question. By extracting correlated evolutionary changes within 700؉ sequences, we built a model of residues that are energetically coupled and that define molecular routes for signal transmission. Typically, these coupled residues are located at multiple distal sites and thus are predicted to form a complex, non-linear network that wires together different functional sites in the protein. Of note, our model connected the site for ATP hydrolysis with sites that ultimately utilize its free energy, such as the microtubule-binding site, drug-binding loop 5, and necklinker. To confirm the calculated energetic connectivity between non-adjacent residues, double-mutant cycle analysis was conducted with 22 kinesin mutants. There was a direct correlation between thermodynamic coupling in experiment and evolutionarily derived energetic coupling. We conclude that energy transduction is coordinated by multiple distal sites in the protein rather than only being relayed through adjacent residues. Moreover, this allosteric map forecasts how energetic orchestration gives rise to different nanomotor behaviors within the superfamily.Biological motors function by converting the chemical energy of ATP hydrolysis into mechanical work in the cell. Thus, molecular motors are free energy transducers, i.e. free energy from the active site is redistributed through the motor protein and ultimately produces a new protein conformational state. Diverse microtubule (MT)-based 2 functions arise in part from differences in their mechanotransduction cycle. For example, members of certain kinesin families are capable of transporting cargo, whereas others modify the MT track (reviewed in Ref. 1).Our goal here is identification of key residues that choreograph transduction between the active site and the microtubule-binding site (see Fig. 1A). It is anticipated that this set of residues couples components that catalyze the free energy-donating reaction with the free energy-accepting ones that result in directed motion. Furthermore, this wiring network should be shared among kinesins and predict adjustment of mechanotransduction between motor families. Such knowledge would reveal mutations that can be used to systematically tune motor protein function.By definition, mechanotransduction is one form of allostery, given that its quintessential property is long range communication. Long range effects in kinesin have been reported. In the first type of study, allosteric mechanisms are inferred from comparisons of well populated conformational states (2, 3) and are primarily descriptive. In the second, molecular dynamics calculations describe dynamic properties of motor proteins as thermally stochastic and yet asymmetric (see Fig. 1B; reviewed in Refs. 4 -6). Theoretical treatments of allostery in other systems also show promise in uncovering fundamental principles of energy conversion, such as the idea that energy storage and transmi...

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Kar3Cik1 is a S. cerevisae Kinesin-14 motor protein that functions to shorten cytoplasmic microtubule (MT) during yeast mating for nuclear fusion, yet crosslinks interpolar MTs (ipMTs) during anaphase. The Kar3 head contains both an ATP and MT binding site, yet the Cik1 head lacks an ATP catalytic site. Presteady-state and steady state experiments were conducted to define the mechanochemical pathway by which Kar3Cik1 stabilizes anti-parallel ipMTs for its mitotic function. To initiate the cycle, we used a high ADP strategy to promote MT-binding by the Cik1 head at 4.9 ˛1/4M-1s-1. The initial association is then followed by a 4-5 s-1 conformational change to induce Kar3 head binding to the MT with rapid ADP release from the active site at 109 s-1. MantATP binding to the nucleotide free MTKar3Cik1 is fast at 2.1 ˛1/4M-1s-1 with koff ¼ 16.6 s-1. Pulse-chase methodology further reveals that MgATP binding to MTKar3Cik1 follows a two step process, formation of a collision complex followed by a 64 s-1 isomerization step. ATP hydrolysis occurs at 26 s-1 followed by motor detachment from microtubule at 11.5 s-1. The rate-limiting step for steady-state ATP turnover at 5 s-1 is hypothesized to be the conformational change leading to Kar3 head binding to MT. These initial results suggest a model in which Kar3-Cik1 interacts with the MT through an alternating cycle of Cik1 binding followed by Kar3 binding. Because Cik1 does not have a nucleotide binding site, we propose that head-head communication is mediated by a strain-dependent mechanism. Supported by NIH GM54141.

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