Inter-organelle interactions between the ER and mitotic spindle facilitates Zika protease cleavage of human Kinesin-5 and results in mitotic defects.

Here we identify human Kinesin-5, Kif11/HsEg5, as a cellular target of Zika protease. We show that Zika NS2B-NS3 protease targets several sites within the motor domain of HsEg5 irrespective of motor binding to microtubules. The native integral ER-membrane protease triggers mitotic spindle positioning defects and a prolonged metaphase delay in cultured cells. Our data support a model whereby loss of function of HsEg5 is mediated by Zika protease and is spatially restricted to the ER-mitotic spindle interface during mitosis. The resulting phenotype is distinct from the monopolar phenotype that typically results from uniform inhibition of HsEg5 by RNAi or drugs. In addition, our data reveal novel inter-organelle interactions between the mitotic apparatus and the surrounding reticulate ER network. Given that Kif11 is haplo-insufficient in humans, and reduced dosage results in microcephaly, we propose that Zika protease targeting of HsEg5 may be a key event in the etiology of Zika syndrome microcephaly.

Small molecule allosteric uncoupling of microtubule depolymerase activity from motility in human Kinesin-5 during mitotic spindle assembly.

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.

Disruption of the Putative Ribosome-Binding Motif of a Scaffold Protein Impairs Cytochrome c Oxidase Subunit Expression in Leishmania major.

During their parasitic life cycle, through sandflies and vertebrate hosts, Leishmania parasites confront strikingly different environments, including abrupt changes in pH and temperature, to which they must rapidly adapt. These adaptations include alterations in Leishmania gene expression, metabolism, and morphology, allowing them to thrive as promastigotes in the sandfly and as intracellular amastigotes in the vertebrate host. A critical aspect of Leishmania metabolic adaptation to these changes is maintenance of efficient mitochondrial function in the hostile vertebrate environment. Such functions, including generation of ATP, depend upon the expression of many mitochondrial proteins, including subunits of cytochrome c oxidase (COX). Significantly, under mammalian temperature conditions, expression of Leishmania major COX subunit IV (LmCOX4) and virulence are dependent upon two copies of LACK, a gene that encodes the ribosome-associated scaffold protein, LACK (Leishmania ortholog of RACK1 [receptor for activated C kinase]). Targeted replacement of an endogenous LACK copy with a putative ribosome-binding motif-disrupted variant (LACKR34D35G36→LACKD34D35E36) resulted in thermosensitive parasites that showed diminished LmCOX4 expression, mitochondrial fitness, and replication in macrophages. Surprisingly, despite these phenotypes, LACKD34D35E36 associated with monosomes and polysomes and showed no major impairment of global protein synthesis. Collectively, these data suggest that wild-type (WT) LACK orchestrates robust LmCOX4 expression and mitochondrial fitness to ensure parasite virulence, via optimized functional interactions with the ribosome.IMPORTANCELeishmania parasites are trypanosomatid protozoans that persist in infected human hosts to cause a spectrum of pathologies, from cutaneous and mucocutaneous manifestations to visceral leishmaniasis caused by Leishmania donovani The latter is usually fatal if not treated. Persistence of L. major in the mammalian host depends upon maintaining gene-regulatory programs to support essential parasite metabolic functions. These include expression and assembly of mitochondrial L. major cytochrome c oxidase (LmCOX) subunits, important for Leishmania ATP production. Significantly, under mammalian conditions, WT levels of LmCOX subunits require threshold levels of the Leishmania ribosome-associated scaffold protein, LACK. Unexpectedly, we find that although disruption of LACK's putative ribosome-binding motif does not grossly perturb ribosome association or global protein synthesis, it nonetheless impairs COX subunit expression, mitochondrial function, and virulence. Our data indicate that the quality of LACK's interaction with Leishmania ribosomes is critical for LmCOX subunit expression and parasite mitochondrial function in the mammalian host. Collectively, these findings validate LACK's ribosomal interactions as a potential therapeutic target.

Characterization of kinesin switch I mutations that cause hereditary spastic paraplegia.

Kif5A is a neuronally-enriched isoform of the Kinesin-1 family of cellular transport motors. 23 separate mutations in the motor domain of Kif5A have been identified in patients with the complicated form of hereditary spastic paraplegia (HSP). We performed in vitro assays on dimeric recombinant Kif5A with HSP-causing mutations in the Switch I domain, which participates in the coordination and hydrolysis of ATP by kinesin. We observed a variety of significantly reduced catalytic and mechanical activities as a result of each mutation, with the shared phenotype from each that motility was significantly reduced. Substitution of Mn2+ for Mg2+ in our reaction buffers provides a dose-dependent rescue in both the catalytic and ensemble mechanical properties of the S203C mutant. This work provides mechanistic insight into the cause of HSP in patients with these mutations and points to future experiments to further dissect the root cause of this disease.

Small molecule screen for candidate antimalarials targeting Plasmodium Kinesin-5.

Plasmodium falciparum and vivax are responsible for the majority of malaria infections worldwide, resulting in over a million deaths annually. Malaria parasites now show measured resistance to all currently utilized drugs. Novel antimalarial drugs are urgently needed. The Plasmodium Kinesin-5 mechanoenzyme is a suitable "next generation" target. Discovered via small molecule screen experiments, the human Kinesin-5 has multiple allosteric sites that are "druggable." One site in particular, unique in its sequence divergence across all homologs in the superfamily and even within the same family, exhibits exquisite drug specificity. We propose that Plasmodium Kinesin-5 shares this allosteric site and likewise can be targeted to uncover inhibitors with high specificity. To test this idea, we performed a screen for inhibitors selective for Plasmodium Kinesin-5 ATPase activity in parallel with human Kinesin-5. Our screen of nearly 2000 compounds successfully identified compounds that selectively inhibit both P. vivax and falciparum Kinesin-5 motor domains but, as anticipated, do not impact human Kinesin-5 activity. Of note is a candidate drug that did not biochemically compete with the ATP substrate for the conserved active site or disrupt the microtubule-binding site. Together, our experiments identified MMV666693 as a selective allosteric inhibitor of Plasmodium Kinesin-5; this is the first identified protein target for the Medicines of Malaria Venture validated collection of parasite proliferation inhibitors. This work demonstrates that chemical screens against human kinesins are adaptable to homologs in disease organisms and, as such, extendable to strategies to combat infectious disease.

Kinesin-5: cross-bridging mechanism to targeted clinical therapy.

Kinesin motor proteins comprise an ATPase superfamily that works hand in hand with microtubules in every eukaryote. The mitotic kinesins, by virtue of their potential therapeutic role in cancerous cells, have been a major focus of research for the past 28 years since the discovery of the canonical Kinesin-1 heavy chain. Perhaps the simplest player in mitotic spindle assembly, Kinesin-5 (also known as Kif11, Eg5, or kinesin spindle protein, KSP) is a plus-end-directed motor localized to interpolar spindle microtubules and to the spindle poles. Comprised of a homotetramer complex, its function primarily is to slide anti-parallel microtubules apart from one another. Based on multi-faceted analyses of this motor from numerous laboratories over the years, we have learned a great deal about the function of this motor at the atomic level for catalysis and as an integrated element of the cytoskeleton. These data have, in turn, informed the function of motile kinesins on the whole, as well as spearheaded integrative models of the mitotic apparatus in particular and regulation of the microtubule cytoskeleton in general. We review what is known about how this nanomotor works, its place inside the cytoskeleton of cells, and its small-molecule inhibitors that provide a toolbox for understanding motor function and for anticancer treatment in the clinic.

Loop 5-directed compounds inhibit chimeric kinesin-5 motors: implications for conserved allosteric mechanisms.

The human Eg5 (HsEg5) protein is unique in its sensitivity to allosteric agents even among phylogenetic kin. For example, S-trityl-l-cysteine (STC) and monastrol are HsEg5 inhibitors that bind to a surface pocket created by the L5 loop, but neither compound inhibits the Drosophila Kinesin-5 homologue (Klp61F). Herein we ask whether or not drug sensitivity can be designed into Klp61F. Two chimeric Klp61F motor domains were engineered, bacterially expressed, and purified to test this idea. We report that effector binding can elicit a robust allosteric response comparable with HsEg5 in both motor domain chimeras. Furthermore, isothermal titration calorimetry confirms that the Klp61F chimeras have de novo binding affinities for both STC and monastrol. These data show that the mechanism of intramolecular communication between the three ligand binding sites is conserved in the Kinesin-5 family, and reconstitution of a drug binding cassette within the L5 pocket is sufficient to restore allosteric inhibition. However, the two compounds were not equivalent in their allosteric inhibition. This surprising disparity in the response between the chimeras to monastrol and STC suggests that there is more than one allosteric communication network for these effectors.

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

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.

ATP hydrolysis in Eg5 kinesin involves a catalytic two-water mechanism.

Motor proteins couple steps in ATP binding and hydrolysis to conformational switching both in and remote from the active site. In our kinesin.AMPPPNP crystal structure, closure of the active site results in structural transformations appropriate for microtubule binding and organizes an orthosteric two-water cluster. We conclude that a proton is shared between the lytic water, positioned for gamma-phosphate attack, and a second water that serves as a general base. To our knowledge, this is the first experimental detection of the catalytic base for any ATPase. Deprotonation of the second water by switch residues likely triggers subsequent large scale structural rearrangements. Therefore, the catalytic base is responsible for initiating nucleophilic attack of ATP and for relaying the positive charge over long distances to initiate mechanotransduction. Coordination of switch movements via sequential proton transfer along paired water clusters may be universal for nucleotide triphosphatases with conserved active sites, such as myosins and G-proteins.

NSC 622124 inhibits human Eg5 and other kinesins via interaction with the conserved microtubule-binding site.

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 for probing mechanochemical transduction and as anticancer 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 [(14)C]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 alpha3 and alpha5 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.

A mitotic role for GSK-3beta kinase in Drosophila.

Glycogen synthase kinase-3beta (GSK-3beta) is involved in a wide variety of cellular processes, and implicated in a growing list of human diseases. Recent drug inhibition studies have suggested a role for GSK-3beta in mitosis in animals. Here, we take an alternative approach to understanding GSK-3beta function in mitosis by genetic mutational analysis in Drosophila. GSK-3beta function is well conserved between Drosophila (Zw3) and humans, frequently operating similarly in pathways, as diverse as the Wnt signaling and circadian rhythm pathways, and sharing a key role in the development of the neuromuscular junction. Unlike drug inhibitor studies, we find that loss of function mutations of zw3 result in markedly curved, or bent, metaphase spindles that exhibit metaphase delay. These defects do not routinely result in mitotic catastrophe, and argue that Zw3 plays a role in the maintenance of the mitotic spindle, rather than an essential role in spindle morphogenesis. Consistent with a mitotic function, we observe a complex and dynamic localization of Zw3 during cell division. These studies provide genetic data that validate and extend drug inhibition studies on a novel mitotic role for glycogen synthase kinase in the maintenance of the mitotic spindle.

The generation and analysis of deficiencies within a small genomic region on the X chromosome of Drosophila melanogaster containing two genes, enhancer of rudimentary and CG15352.

The enhancer of rudimentary gene, e(r), encodes a 104-amino-acid, highly conserved transcription cofactor. Hypomorphic mutations of e(r) show an enhancement of a hypomorphic rudimentary mutant wing phenotype. These mutants in a wild-type background are viable, fertile, and morphologically wild-type. Since the only mutant alleles were hypomorphic, it was important to isolate null mutations to determine if any other phenotypes might be associated with a loss-of-function of e(r). We utilized a marked P element, P{SUPor-P, y(+)}, located 895 bp upstream of the start of transcription of e(r) to generate nineteen deficiencies in the region. Deficiencies of e(r) enhance the mutant wing phenotype of a hypomorphic rudimentary allele, r(hd1). In a wild-type background, the deficiencies of e(r), unlike the hypomorphic alleles, have a low viability and females have low fertility. The expression of e(r) in the nurse cells of the ovary is consistent with the low fertility, and suggests an ovarian function for e(r). Deficiencies of CG15352, the gene directly upstream of e(r), are not associated with any obvious mutant phenotypes and present the possibility that it encodes a nonvital or redundant function.

Disparity in allosteric interactions of monastrol with Eg5 in the presence of ADP and ATP: a difference FT-IR investigation.

Eg5 is a kinesin-like motor protein required for mitotic progression in higher eukaryotes. It is thought to cross-link antiparallel microtubules, and provides a force required for the formation of a bipolar spindle. Monastrol causes the catastrophic collapse of the mitotic spindle through the allosteric inhibition of Eg5. Utilizing a truncated Eg5 protein, we employ difference infrared spectroscopy to probe structural changes that occur in the motor protein with monastrol in the presence of either ADP or ATP. Difference FT-IR spectra of Eg5-monastrol-nucleotide complexes demonstrate that there are triggered conformational changes corresponding to an interconversion of secondary structural elements in the motor upon interaction with nucleotides. Notably, conformational changes elicited in the presence of ADP are different from those in the presence of ATP. In Eg5-monastrol complexes, exchange of ADP is associated with a decrease in random structure and an increase in alpha-helical content. In contrast, formation of the Eg5-monastrol-ATP complex is associated with a decrease in alpha-helical content and a concomitant increase in beta-sheet content. One or more carboxylic acid residues in Eg5 undergo unique changes when ATP, but not ADP, interacts with the motor domain in the presence of monastrol. This first direct dissection of inhibitor-protein interactions, using these methods, demonstrates a clear disparity in the structural consequences of monastrol in the presence of ADP versus ATP.