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

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, α4-α5-loop 13, α1, and β4-β6-β7. 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 β-sheet residues and α1, leading to a dynamic decoupling of nucleotide, microtubule, and neck-linker binding sites. Additional analyses of point mutations, including P131 (loop 5), Q78/I79 (α1), E166 (loop 7), and K272/I273 (β7) 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.     

Mapping the Structural and Dynamical Features of Kinesin Motor Domains

Kinesin motor proteins drive intracellular transport by coupling ATP hydrolysis to conformational changes that mediate directed movement along microtubules. Characterizing these distinct conformations and their interconversion mechanism is essential to determining an atomic-level model of kinesin action. Here we report a comprehensive principal component analysis of 114 experimental structures along with the results of conventional and accelerated molecular dynamics simulations that together map the structural dynamics of the kinesin motor domain. All experimental structures were found to reside in one of three distinct conformational clusters (ATP-like, ADP-like and Eg5 inhibitor-bound). These groups differ in the orientation of key functional elements, most notably the microtubule binding α4–α5, loop8 subdomain and α2b-β4-β6-β7 motor domain tip. Group membership was found not to correlate with the nature of the bound nucleotide in a given structure. However, groupings were coincident with distinct neck-linker orientations. Accelerated molecular dynamics simulations of ATP, ADP and nucleotide free Eg5 indicate that all three nucleotide states could sample the major crystallographically observed conformations. Differences in the dynamic coupling of distal sites were also evident. In multiple ATP bound simulations, the neck-linker, loop8 and the α4–α5 subdomain display correlated motions that are absent in ADP bound simulations. Further dissection of these couplings provides evidence for a network of dynamic communication between the active site, microtubule-binding interface and neck-linker via loop7 and loop13. Additional simulations indicate that the mutations G325A and G326A in loop13 reduce the flexibility of these regions and disrupt their couplings. Our combined results indicate that the reported ATP and ADP-like conformations of kinesin are intrinsically accessible regardless of nucleotide state and support a model where neck-linker docking leads to a tighter coupling of the microtubule and nucleotide binding regions. Furthermore, simulations highlight sites critical for large-scale conformational changes and the allosteric coupling between distal functional sites.     

Exploring a potential allosteric inhibition mechanism in the motor domain of human Eg-5

Kinesin-5 (Eg-5), microtubule motor protein, is one of the emerging drug targets in cancer research. Several inhibitors have been reported to bind the hEg-5 “motor domain” in two different locations that are potentially allosteric. Interestingly, the crystal structure of Eg-5 bound to benzimidazole unveils two chemically different allosteric pockets (PDB ID: 3ZCW). The allosteric modulators inhibit Eg-5 activity by causing conformational changes that affect nucleotide turnover rate. In the present work, three allosteric inhibitors were simulated along with the substrate nucleotides (ADP and ATP) to capture conformation changes induced by the allosteric inhibitors. To analyze the allosteric inhibition mechanism, we used dynamics cross-correlation, principal component analysis (PCA), and enthalpic calculations. The loop L5 interaction is determined by the type of substrate bind at the nucleotide binding site. The SW-II flexibility increased upon dual allosteric inhibition by SB-743921 and 6a. The ionic interaction between R221-E116 is observed only in the presence of two allosteric inhibitors. Also, we noticed that the α2/α3 helical orientation is responsible for the SW-1 loop position and substrate binding. Our simulation data suggest the critical chemical features required to block the motor domain by the allosteric inhibitors. The results summarized in this work will help the researchers to design better therapeutic agents targeting hEg-5.

Communicated by Ramaswamy H. Sarma     

Altered Nucleotide-Microtubule Coupling and Increased Mechanical Output by a Kinesin Mutant

Kinesin motors hydrolyze ATP to produce force and do work in the cell – how the motors do this is not fully understood, but is thought to depend on the coupling of ATP hydrolysis to microtubule binding by the motor. Transmittal of conformational changes from the microtubule- to the nucleotide-binding site has been proposed to involve the central β-sheet, which could undergo large structural changes important for force production. We show here that mutation of an invariant residue in loop L7 of the central β-sheet of the Drosophila kinesin-14 Ncd motor alters both nucleotide and microtubule binding, although the mutated residue is not present in either site. Mutants show weak-ADP/tight-microtubule binding, instead of tight-ADP/weak-microtubule binding like wild type – they hydrolyze ATP faster than wild type, move faster in motility assays, and assemble long spindles with greatly elongated poles, which are also produced by simulations of assembly with tighter microtubule binding and faster sliding. The mutated residue acts like a mechanochemical coupling element – it transmits changes between the microtubule-binding and active sites, and can switch the state of the motor, increasing mechanical output by the motor. One possibility, based on our findings, is that movements by the residue and the loop that contains it could bend or distort the central β-sheet, mediating free energy changes that lead to force production.     

Configuration of the two kinesin motor domains during ATP hydrolysis

To understand the mechanism of kinesin movement we have investigated the relative configuration of the two kinesin motor domains during ATP hydrolysis using fluorescence polarization microscopy of ensemble and single molecules. We found that: (i) in nucleotide states that induce strong microtubule binding, both motor domains are bound to the microtubule with similar orientations; (ii) this orientation is maintained during processive motion in the presence of ATP; (iii) the neck-linker region of the motor domain has distinct configurations for each nucleotide condition tested. Our results fit well with a hand-over-hand type movement mechanism and suggest how the ATPase cycle in the two motor domains is coordinated. We propose that the motor neck-linker domain configuration controls ADP release.     

Inhibitors of sterol biosynthesis. Syntheses of 14α-alkyl substituted 15-oxygenated sterols

The chemical syntheses of a number of 14α-alkyl substituted 15-oxygenated sterols have been pursued to permit evaluation of their activity in the inhibition of the biosynthesis of cholesterol and other biological effects. Described herein are the first chemical syntheses of 14α-ethyl-5α-cholest-7-en-3β-ol-15-one, bis-3β,15α-acetoxy-14α-ethyl-5α-cholest-7-ene, 3β-acetoxy-14α-ethyl-5α-cholest-7-en-15β-ol, 14α-ethyl-5α-cholest-7-en-3β,15β-diol, 14α-ethyl-5α-cholest-7-en-3β,15α-diol, 3β-hexadecanoyloxy-14α-ethyl-5α-cholest-7-en-15α-ol, 3β-hexadecanoyloxy-14α-ethyl-5α-cholest-7-en-15β-ol, bis-3β,15α-hexadecanoyloxy-14α-ethyl-5α-cholest-7-ene, 3β-hexadecanoyloxy-14α-ethyl-5α-cholest-7-en-15-one, 3α-benzoyloxy-14α-ethyl-5α-cholest-7-en-15-one, 14α-ethyl-5α-cholest-7-en-3α-ol-15-one, 14α-ethyl-5α-cholest-7-en-15-on-3β-yl pyridinium sulfate, 14α-ethyl-5α-cholest-7-en-15-on-3β-yl potassium sulfate (monohydrate), 14α-ethyl-5α-cholest-7-en-15-on-3α-yl pyridinium sulfate, 14α-ethyl-5α-cholest-7-en-15-on-3α-yl potassium sulfate (monohydrate), 3β-ethoxy-14α-ethyl-5α-cholest-7-en-15-one, 3β-acetoxy-14α-n-propyl-5α-cholest-7-en-15-one, 14α-n-propyl-5α-cholest-7-en-3β-ol-15-one, bis-3β, 15α-acetoxy-14α-n-propyl-5α-cholest-7-ene, 3β-acetoxy-14α-n-propyl-5α-cholest-7-en-15β-ol, 14α-n-propyl-5α-cholest-7-en-3β, 15α-diol, 14α-n-propyl-5α-cholest-7-en-3β, 15β-diol, 14α-n-butyl-5α-cholest-7-en-3β-ol-15-one, 3β-acetoxy-14-α-n-butyl-5α-cholest-7-en-15-one, bis-3β,15α-acetoxy-14α-n-butyl-5α-cholest-7-ene, 3β-acetoxy-14α-n-butyl-5α-cholest-7-en-15β-ol, 14α-n-butyl-5β-cholest-7-en-3β, 15β-diol, and 14α-n-butyl-5α-cholest-7-en-3β, 15α-diol.     

Cooperative Changes in Solvent Exposure Identify Cryptic Pockets,Switches, and Allosteric Coupling

Proteins are dynamic molecules that undergo conformational changes to a broad spectrum of different excited states. Unfortunately, the small populations of these states make it difficult to determine their structures or functional implications. Computer simulations are an increasingly powerful means to identify and characterize functionally relevant excited states. However, this advance has uncovered a further challenge: it can be extremely difficult to identify the most salient features of large simulation data sets. We reasoned that many functionally relevant conformational changes are likely to involve large, cooperative changes to the surfaces that are available to interact with potential binding partners. To examine this hypothesis, we introduce a method that returns a prioritized list of potentially functional conformational changes by segmenting protein structures into clusters of residues that undergo cooperative changes in their solvent exposure, along with the hierarchy of interactions between these groups. We term these groups exposons to distinguish them from other types of clusters that arise in this analysis and others. We demonstrate, using three different model systems, that this method identifies experimentally validated and functionally relevant conformational changes, including conformational switches, allosteric coupling, and cryptic pockets. Our results suggest that key functional sites are hubs in the network of exposons. As a further test of the predictive power of this approach, we apply it to discover cryptic allosteric sites in two different β-lactamase enzymes that are widespread sources of antibiotic resistance. Experimental tests confirm our predictions for both systems. Importantly, we provide the first evidence, to our knowledge, for a cryptic allosteric site in CTX-M-9 β-lactamase. Experimentally testing this prediction did not require any mutations and revealed that this site exerts the most potent allosteric control over activity of any pockets found in β-lactamases to date. Discovery of a similar pocket that was previously overlooked in the well-studied TEM-1 β-lactamase demonstrates the utility of exposons.     

Cryo-EM Structure (4.5-Å) of Yeast Kinesin-5–Microtubule Complex Reveals a Distinct Binding Footprint and Mechanism of Drug Resistance

Kinesin-5s are microtubule-dependent motors that drive spindle pole separation during mitosis. We used cryo-electron microscopy to determine the 4.5-Å resolution structure of the motor domain of the fission yeast kinesin-5 Cut7 bound to fission yeast microtubules and explored the topology of the motor–microtubule interface and the susceptibility of the complex to drug binding. Despite their non-canonical architecture and mechanochemistry, Schizosaccharomyces pombe microtubules were stabilized by epothilone at the taxane binding pocket. The overall Cut7 footprint on the S. pombe microtubule surface is altered compared to mammalian tubulin microtubules because of their different polymer architectures. However, the core motor–microtubule interaction is tightly conserved, reflected in similar Cut7 ATPase activities on each microtubule type. AMPPNP-bound Cut7 adopts a kinesin-conserved ATP-like conformation including cover neck bundle formation. However, the Cut7 ATPase is not blocked by a mammalian-specific kinesin-5 inhibitor, consistent with the non-conserved sequence and structure of its loop5 insertion.     

Unique flavonoid glycosides from the new zealand white pine, Dacrycarpus dacrydioides

A number of new flavonoid glycosides have been isolated from foliage of the New Zealand white pine, Dacrycarpus dacrydioides. These include tricetin 3′,5′-di-O-β-glucopyranoside; the 3′-O-β-xylopyranoside, 7-O-α-rhamnopyranoside and 7-O-α-rhamnopyranoside-3′-O-β-xylopyranoside of 3-O-methylmyricetin; the 3′-O-β-xylopyranoside, 7-O-α-rhamnopyranoside and 7-O-α-rhamnopyranoside-3′-O-β-xylopyranoside of 3-O-methyl-quercetin, and the 3′-O-β-xylopyranoside and 7-O-α-rhamnopyranoside-3′-O-β-xylopyranoside of 3,4′-di-O-methylmyricetin. The accumulation of 3-methoxyflavones and B-ring trioxygenated flavonoids appears to distinguish D. dacrydioides from all other New Zealand members of the classical genus Podocarpus. Support for De Laubenfels' proposed separation of Dacrycarpus from this genus is seen in the present work.     

Unusual RNA binding of FUS RRM studied by molecular dynamics simulation and enhanced sampling method

Amyotrophic lateral sclerosis (ALS) and frontotemporal lobe degeneration (FTLD) are two inter-related intractable diseases of motor neuron degeneration. Fused in sarcoma (FUS) is found in cytoplasmic accumulation of ALS and FTLD patients, which readily link the protein with the diseases. The RNA recognition motif (RRM) of FUS has the canonical α-β folds along with an unusual lysine-rich loop (KK-loop) between α1 and β2. This KK-loop is highly conserved among FET family proteins. Another contrasting feature of FUS RRM is the absence of critical binding residues, which are otherwise highly conserved in canonical RRMs. These residues in FUS RRM are Thr286, Glu336, Thr338, and Ser367, which are substitutions of lysine, phenylalanine, phenylalanine, and lysine, respectively, in other RRMs. Considering the importance of FUS in RNA regulation and metabolism, and its implication in ALS and FTLD, it is important to elucidate the underlying molecular mechanism of RNA recognition. In this study, we have performed molecular dynamics simulation with enhanced sampling to understand the conformational dynamics of noncanonical FUS RRM and its binding with RNA. We studied two sets of mutations: one with alanine mutation of KK-loop and another with KK-loop mutations along with critical binding residues mutated back to their canonical form. We find that concerted movement of KK-loop and loop between β2 and β3 facilitates the folding of the partner RNA, indicating an induced-fit mechanism of RNA binding. Flexibility of the RRM is highly restricted upon mutating the lysine residues of the KK-loop, resulting in weaker binding with the RNA. Our results also suggest that absence of the canonical residues in FUS RRM along with the KK-loop is equally important in regulating its binding dynamics. This study provides a significant structural insight into the binding of FUS RRM with its cognate RNA, which may further help in designing potential drugs targeting noncanonical RNA recognition.     

Nucleotide-dependent movements of the kinesin motor domain predicted by simulated annealing.

The structure of an ATP-bound kinesin motor domain is predicted and conformational differences relative to the known ADP-bound form of the protein are identified. The differences should be attributed to force-producing ATP hydrolysis. Candidate ATP-kinesin structures were obtained by simulated annealing, by placement of the ATP gamma-phosphate in the crystal structure of ADP-kinesin, and by interatomic distance constraints. The choice of such constraints was based on mutagenesis experiments, which identified Gly-234 as one of the gamma-phosphate sensing residues, as well as on structural comparison of kinesin with the homologous nonclaret disjunctional (ncd) motor and with G-proteins. The prediction of nucleotide-dependent conformational differences reveals an allosteric coupling between the nucleotide pocket and the microtubule binding site of kinesin. Interactions of ATP with Gly-234 and Ser-202 trigger structural changes in the motor domain, the nucleotide acting as an allosteric modifier of kinesin's microtubule-binding state. We suggest that in the presence of ATP kinesin's putative microtubule binding regions L8, L12, L11, alpha4, alpha5, and alpha6 form a face complementary in shape to the microtubule surface; in the presence of ADP, the microtubule binding face adopts a more convex shape relative to the ATP-bound form, reducing kinesin's affinity to the microtubule.     

Nucleotide binding and phosphorylation in microtubule assembly in vitro.

Two non-hydrolyzable analogs of GTP, guanylyl-β,γ-methylene diphosphonate and guanylyl imidodiphosphate, have been found to induce rapid and efficient microtubule assembly in vitro by binding at the exchangeable site (E-site) on tubulin. Characterization of microtubule polymerization by several criteria, including polymerization kinetics, nucleotide binding to depolymerized and polymerized microtubules, and microtubule stability, reveals strong similarities between microtubule assembly induced by GTP and non-hydrolyzable GTP analogs. Nucleoside triphosphates which bind weakly or not at all to tubulin, such as ATP, UTP and CTP, are shown to induce microtubule assembly by means of a nucleoside diphosphate kinase (NDP-kinase, EC 2.7.4.6.) activity which is not intrinsic to tubulin. The NDP-kinase mediates microtubule polymerization by phosphorylating tubulin-bound GDP in situ at the E-site. Although hydrolysis of exchangeably bound GTP occurs, it is found to be uncoupled from the polymerization reaction. The non-exchangeable nucleotide binding site on tubulin (N-site) is not directly involved in microtubule assembly in vitro. The N-site is shown to contain almost exclusively GTP which is not hydrolyzed during microtubule assembly. A scheme is presented in which GTP acts as an allosteric effector at the E-site during microtubule assembly in vitro.     

Feedback of the kinesin-1 neck-linker position on the catalytic site

Kinesin-1 motor proteins step along microtubules by a mechanism in which the heads cycle through microtubule-bound and unbound states in an interlaced fashion. An important contribution to head-head coordination arises from the action of the neck-linker that docks onto the core motor domain upon ATP binding. We show here that the docked neck-linker not only guides the microtubule-unbound head to the next microtubule binding site but also signals its position to the head to which it is attached. Cross-linking studies on mutated kinesin constructs reveal that residues at the interface motor core/docked neck-linker, among them most importantly a conserved tyrosine, are involved in this feedback. The primary effect of the docked neck-linker is a reduced microtubule binding affinity in the ADP state.     

Concerning the structure of 7α,15β-dichloro-5α-cholest-8(14)-en-3β-ol,a novel inhibitor of cholesterol biosynthesis

Treatment of 3β-p-bromobenzoyloxy-14α, 15α-epoxy-5α-cholest-7-ene with gaseous HCI in chloroform at ?25°C gave 3β-p-bromobenzoyloxy-7α, 15β-dichloro-5α-cholest-8(14)-ene in 93% yield. The structure of the latter compound was unequivocally established by the results of X-ray crystallographic analysis.     

Two new guaiane sesquiterpenes from Datura metel L. with anti-inflammatory activity

Chemical investigation of an acidic methanol extract of the whole plants of Datura metel resulted in the isolation of two new guainane sesquiterpenes, 1β,5α,7β-guaiane-4β,10α,11-triol (1) and 1α,5α,7α-11-guaiene-2α,3β,4α,10α,13-pentaol (2), along with eight known compounds: pterodontriol B (3), disciferitriol (4), scopolamine (5), kaempferol 3-O-β-d-glucosyl(1 → 2)-β-d-galactoside 7-O-β-d-glucoside (6), kaempferol 3-O-β-glucopyranosyl(1 → 2)-β-glucopyranoside-7-O-α-rhamnopyranoside (7), pinoresinol 4′′-O-β-d-glucopyranoside (8), (7R,8S,7′S,8′R)-4,9,4′,7′-tetrahydroxy-3,3′-dimethoxy-7,9′-epoxy-lignan-4-O-β-d-glucopyranoside (9), and (7S,8R,7′S,8′S)-4,9,4′,7′-tetrahydroxy-3,3′-dimethoxy-7,9′-epoxylignan-4-O-β-d-glucopyranoside (10). Their structures were elucidated by extensive spectroscopic methods, including 1D and 2D NMR and MS spectra. Compounds 2-4 and 6-10 were shown to have modest anti-inflammatory effects through inhibition of NO production in LPS-stimulated BV cells.     

Dynamic Coupling and Allosteric Networks in the α Subunit of Heterotrimeric G Proteins

G protein α subunits cycle between active and inactive conformations to regulate a multitude of intracellular signaling cascades. Important structural transitions occurring during this cycle have been characterized from extensive crystallographic studies. However, the link between observed conformations and the allosteric regulation of binding events at distal sites critical for signaling through G proteins remain unclear. Here we describe molecular dynamics simulations, bioinformatics analysis, and experimental mutagenesis that identifies residues involved in mediating the allosteric coupling of receptor, nucleotide, and helical domain interfaces of Gα_i. Most notably, we predict and characterize novel allosteric decoupling mutants, which display enhanced helical domain opening, increased rates of nucleotide exchange, and constitutive activity in the absence of receptor activation. Collectively, our results provide a framework for explaining how binding events and mutations can alter internal dynamic couplings critical for G protein function.     

Melongosides n,o and p: steroidal saponins from seeds of solanum melongena

Three new saponins, melongosides N, O and P, have been isolated from the methanolic extract of seeds of Solanum melongena and their structures elucidated. Melongoside N is 3-O-[β-D-glucopyranosy l-(1 → 2)-β-D-glucopyranosyl]-26-O-(β-D-glucopyranosyl)-(25R)-5α-furostan-3β,22 α,26-triol, whereas melongoside O is 3-O-[β-D-glucopyranosyl-(1 → 2)β-D-glucopyranosyl]- 26-O-(β-D-glucopyranosyl)-(25R)-furost-5-en-3β,22α,26-triol and melongoside P is 3-O- [β-D-glucopyranosyl-(1 → 2)]-[α-L-rhamnopyranosyl-(1 → 3)]-β-D-glucopyranosyl)-26-O- (β-D-glucopyranosyl)-(25 R)-5α-furostan-3β,22α,26-triol.     

Side chain functionalization of cholesterol in the biosynthesis of solasodine in Solanum laciniatum

(25R)-26-Amino-cholesterol-[7α-³H], (25R)-26-amino-5-cholestene-3β,16β-diol-[7α-³H] and (25R)-26-acetylamino-5-cholestene-3β,16β-diol-[7α-³H] administered to Solanum laciniatum were converted into solasodine. The results indicate that in the biosynthesis of solasodine the introduction of nitrogen occurs immediately after the hydroxylation at C-26 and before a further oxidation of the side chain of cholesterol. The next step after the amination at C-26 is not hydroxylation at the 16β-position but probably the functionalization of C-22.     

Role of the DELSEED Loop in Torque Transmission of F1-ATPase

F₁-ATPase is an ATP-driven rotary motor that generates torque at the interface between the catalytic β-subunits and the rotor γ-subunit. The β-subunit inwardly rotates the C-terminal domain upon nucleotide binding/dissociation; hence, the region of the C-terminal domain that is in direct contact with γ—termed the DELSEED loop—is thought to play a critical role in torque transmission. We substituted all the DELSEED loop residues with alanine to diminish specific DELSEED loop-γ interactions and with glycine to disrupt the loop structure. All the mutants rotated unidirectionally with kinetic parameters comparable to those of the wild-type F₁, suggesting that the specific interactions between DELSEED loop and γ is not involved in cooperative interplays between the catalytic β-subunits. Glycine substitution mutants generated half the torque of the wild-type F₁, whereas the alanine mutant generated comparable torque. Fluctuation analyses of the glycine/alanine mutants revealed that the γ-subunit was less tightly held in the α₃β₃-stator ring of the glycine mutant than in the wild-type F₁ and the alanine mutant. Molecular dynamics simulation showed that the DELSEED loop was disordered by the glycine substitution, whereas it formed an α-helix in the alanine mutant. Our results emphasize the importance of loop rigidity for efficient torque transmissions.