Myopathy-Sensitive G-Actin Segment 227-235 Is Involved in Salt-Induced Stabilization of Contacts within the Actin Filament.

Formation of stable actin filaments, critically important for actin functions, is determined by the ionic strength of the solution. However, not much is known about the elements of the actin fold involved in ionic-strength-dependent filament stabilization. In this work, F-actin was destabilized by Cu2+ binding to Cys374, and the effects of solvent conditions on the dynamic properties of F-actin were correlated with the involvement of Segment 227-235 in filament stabilization. The results of our work show that the presence of Mg2+ at the high-affinity cation binding site of Cu-modified actin polymerized with MgCl2 strongly enhances the rate of filament subunit exchange and promotes the filament instability. In the presence of 0.1 M KCl, the filament subunit exchange was 2-3-fold lower than that in the MgCl2-polymerized F-actin. This effect correlates with the reduced accessibility of the D-loop and Segment 227-235 on opposite filament strands, consistent with an ionic-strength-dependent conformational change that modulates involvement of Segment 227-235 in stabilization of the intermonomer interface. KCl may restrict the mobility of the α-helix encompassing part of Segment 227-235 and/or be bound to Asp236 at the boundary of Segment 227-235. These results provide experimental evidence for the involvement of Segment 227-235 in salt-induced stabilization of contacts within the actin filament and suggest that they can be weakened by mutations characteristic of actin-associated myopathies.

Interactions between motor domains in kinesin-14 Ncd - a molecular dynamics study.

Minus-end directed, non-processive kinesin-14 Ncd is a dimeric protein with C-terminally located motor domains (heads). Generation of the power-stroke by Ncd consists of a lever-like rotation of a long superhelical 'stalk' segment while one of the kinesin's heads is bound to the microtubule. The last ∼30 amino acids of Ncd head play a crucial but still poorly understood role in this process. Here, we used accelerated molecular dynamics simulations to explore the conformational dynamics of several systems built upon two crystal structures of Ncd, the asymmetrical T436S mutant in pre-stroke/post-stroke conformations of two partner subunits and the symmetrical wild-type protein in pre-stroke conformation of both subunits. The results revealed a new conformational state forming following the inward motion of the subunits and stabilized with several hydrogen bonds to residues located on the border or within the C-terminal linker, i.e. a modeled extension of the C-terminus by residues 675-683. Forming of this new, compact Ncd conformation critically depends on the length of the C-terminus extending to at least residue 681. Moreover, the associative motion leading to the compact conformation is accompanied by a partial lateral rotation of the stalk. We propose that the stable compact conformation of Ncd may represent an initial state of the working stroke.

A simple and reproducible protocol of glass surface silanization for TIRF microscopy imaging.

We describe a simple and reproducible protocol for the preparation of microscope glass slides for in vitro motility assays that use total internal reflection fluorescence microscopy. The developed method utilizes trimethylchlorosilane (TMCS) as a silanizing reagent, which in the presence of imidazole as a catalyst and under optimized conditions enables reproducible preparation of high-quality hydrophobic glass surfaces. This method presents a simplification and improvement in reproducibility over the commonly applied protocol utilizing dichlorodimethylsilane (DDS) as a silanizing agent. We demonstrate the applicability of the new method by performing the analysis of the interactions of a molecular motor, kinesin-1 with microtubules.

Working stroke of the kinesin-14, ncd, comprises two substeps of different direction.

Single-molecule experiments have been used with great success to explore the mechanochemical cycles of processive motor proteins such as kinesin-1, but it has proven difficult to apply these approaches to nonprocessive motors. Therefore, the mechanochemical cycle of kinesin-14 (ncd) is still under debate. Here, we use the readout from the collective activity of multiple motors to derive information about the mechanochemical cycle of individual ncd motors. In gliding motility assays we performed 3D imaging based on fluorescence interference contrast microscopy combined with nanometer tracking to simultaneously study the translation and rotation of microtubules. Microtubules gliding on ncd-coated surfaces rotated around their longitudinal axes in an [ATP]- and [ADP]-dependent manner. Combined with a simple mechanical model, these observations suggest that the working stroke of ncd consists of an initial small movement of its stalk in a lateral direction when ADP is released and a second, main component of the working stroke, in a longitudinal direction upon ATP binding.

Insights into the process of EB1-dependent tip-tracking of kinesin-14 Ncd. The role of the microtubule.

End-binding proteins are capable of tracking the plus-ends of growing microtubules (MTs). The motor protein Ncd, a member of the kinesin-14 family, interacts with EB1 protein and becomes a non-autonomous tip-tracker. Here, we attempted to find out whether at least for Ncd, the efficient EB1-mediated tip-tracking involves the interaction of the kinesin with the MT surface. We prepared a series of Ncd tail mutants in which the MT-binding sites were altered or eliminated. Using TIRF microscopy, we characterized their behavior as tip-trackers and measured the dwell times of single molecules of EB1 and Ncd tail or its mutated forms. The mutated forms of Ncd tail exhibited tip-tracking in the presence of EB1 and the effectiveness of this process was proportional to the affinity of the mutant's tail to MT. Even though the interaction of Ncd with EB1 was weak (Kd∼9µM) the half saturating concentration of EB1 for tip-tracking was 7nM. The dwell time of Ncd tail in the presence of EB1 was ∼1s. The dwell time of EB1 alone was shorter (∼0.3s) and increased considerably in the presence of a large excess of Ncd tail. We demonstrated that tip-tracking of kinesin-14 occurs through several concurrent mechanisms: binding of kinesin only to EB1 located at the MT end, interaction of the kinesin molecules with a composite site formed by EB1 and the MT tip, and probably surface diffusion of the tail along MT. The second mechanism seems to play a crucial role in efficient tip-tracking.

The mechanism of calcium-induced inhibition of muscle fructose 1,6-bisphosphatase and destabilization of glyconeogenic complex.

The mechanism by which calcium inhibits the activity of muscle fructose 1,6-bisphosphatase (FBPase) and destabilizes its interaction with aldolase, regulating glycogen synthesis from non-carbohydrates in skeletal muscle is poorly understood. In the current paper, we demonstrate evidence that Ca(2+) affects conformation of the catalytic loop 52-72 of muscle FBPase and inhibits its activity by competing with activatory divalent cations, e.g. Mg(2+) and Zn(2+). We also propose the molecular mechanism of Ca(2+)-induced destabilization of the aldolase-FBPase interaction, showing that aldolase associates with FBPase in its active form, i.e. with loop 52-72 in the engaged conformation, while Ca(2+) stabilizes the disengaged-like form of the loop.

The human kinesin-14 HSET tracks the tips of growing microtubules in vitro.

Tip-tracking of kinesin-14 motor proteins is believed to be crucial for the assembly and maintenance of dynamic microtubule arrays. However, in contrast to other members of the kinesin-14 family, H. sapiens kinesin-14 HSET has so far never been observed to be prominently located at microtubule plus ends. Here, using an in vitro microtubule dynamics reconstitution assay we observe tip-tracking of GFP-HSET in the presence of H. sapiens EB1 (hsEB1). Tip-tracking depended on the SxIP-like motif in HSET as well as on the EB homology domain in hsEB1. D. melanogaster Ncd and S. pombe Klp2 tip-tracking reconstitution assays accompanied by kinesin-14 amino acid sequence comparisons suggest that SxIP-like motif mediated tip-tracking dependent on EB family proteins is conserved in the kinesin-14 family of molecular motors.

Prion protein impairs kinesin-driven transport.

Our previous studies have demonstrated that prion protein (PrP) leads to disassembly of microtubular cytoskeleton through binding to tubulin and its oligomerization. Here we found that PrP-treated cells exhibited improper morphology of mitotic spindles. Formation of aberrant spindles may result not only from altered microtubule dynamics - as expected from PrP-induced tubulin oligomerization - but also from impairing the function of molecular motors. Therefore we checked whether binding of PrP to microtubules affected movement generated by Ncd - a kinesin responsible for the proper organization of division spindles. We found that PrP inhibited Ncd-driven transport of microtubules. Most probably, the inhibition of the microtubule movement resulted from PrP-induced changes in the microtubule structure since Ncd-microtubule binding was reduced already at low PrP to tubulin molar ratios. This study suggests another plausible mechanism of PrP cytotoxicity related to the interaction with tubulin, namely impeding microtubule-dependent transport.

Functional effects of congenital myopathy-related mutations in gamma-tropomyosin gene.

Missense mutations in human TPM3 gene encoding γ-tropomyosin expressed in slow muscle type 1 fibers, were associated with three types of congenital myopathies-nemaline myopathy, cap disease and congenital fiber type disproportion. Functional effects of the following substitutions: Leu100Met, Ala156Thr, Arg168His, Arg168Cys, Arg168Gly, Lys169Glu, and Arg245Gly, were examined in biochemical assays using recombinant tropomyosin mutants and native proteins isolated from skeletal muscle. Most, but not all, mutations decreased the affinity of tropomyosin for actin alone and in complex with troponin (±Ca(2+)). All studied tropomyosin mutants reduced Ca-induced activation but had no effect on the inhibition of actomyosin cross-bridges. Ca(2+)-sensitivity of the actomyosin interactions, as well as cooperativity of myosin-induced activation of the thin filament was affected by individual tropomyosin mutants with various degrees. Decreased motility of the reconstructed thin filaments was a result of combined functional defects caused by myopathy-related tropomyosin mutants. We conclude that muscle weakness and structural abnormalities observed in TPM3-related congenital myopathies result from reduced capability of the thin filament to fully activate actin-myosin cross-bridges.

The C-terminus of kinesin-14 Ncd is a crucial component of the force generating mechanism.

Ncd, a member of kinesin-14 family motors, uses the power stroke, a lever-like pivoting action of a long and stiff element, to exert force and generate movement. To better understand the role of the Ncd C-terminus in this process we produced four Ncd mutants in which this segment was altered or deleted. For these proteins we measured their affinity to the microtubule, steady-state ATPase and gliding velocity in multiple motor assays. The mutations had a dramatic effect on all three parameters measured, suggesting that the C-terminal residues of Ncd play an important role in modulating the interaction of the motor with the microtubule.

Interactions between subunits in heterodimeric Ncd molecules.

The nonprocessive minus-end-directed kinesin-14 Ncd is involved in the organization of the microtubule (MT) network during mitosis. Only one of the two motor domains is involved in the interaction with the MT. The other head is tethered to the bound one. Here we prepared, purified, and characterized mutated Ncd molecules carrying point mutations in one of the heads, thus producing heterodimeric motors. The mutations tested included substitutions in Switch I and II: R552A, E585A, and E585D; the decoupling mutant N600K; and a deletion in the motor domain in one of the subunits resulting in a single-headed molecule (NcN). These proteins were isolated by two sequential affinity chromatography steps, followed by measurements of their affinities to MT, enzymatic properties, and the velocity of the microtubule gliding test in vitro. A striking observation is a low affinity of the single-headed NcN for MT both without nucleotides and in the presence of 5'-adenylyl-beta,gamma-imidodiphosphate, implying that the tethered head has a profound effect on the structure of the Ncd-MT complex. Mutated homodimers had no MT-activated ATPase and no motility, whereas NcN had motility comparable with that of the wild type Ncd. Although the heterodimers had one fully active and one inactive head, the ATPase and motility of Ncd heterodimers varied dramatically, clearly demonstrating that interactions between motor domains exist in Ncd. We also show that the bulk property of dimeric proteins that interact with the filament with only one of its heads depends also on the distribution of the filament-interacting subunits.

[Plus end tracking proteins and their role in mitotic spindle organization]. / Bialka sledzace koniec plus mikrotubul oraz ich udzial w organizacji wrzeciona mitotycznego.

Plus end tracking proteins (+TIPs) form a diverse protein family, members of which were found in all eukaryotes. Their characteristic feature is the ability of localisation on dynamically growing plus ends of microtubules. +TIPs perform many important functions during mitosis: they control microtubule growth, the recruitment of other proteins to the microtubule plus end and promote the interaction of microtubules with other elements of the spindle. In the article we describe the structure of main +TIPs groups, focusing especially on domains responsible for plus end tracking. We also discuss several mechanisms for plus-end accumulation, the influence of these proteins on microtubule dynamics and their importance in the spindle organization.

The mitotic kinesin-14 Ncd drives directional microtubule-microtubule sliding.

During mitosis and meiosis, the bipolar spindle facilitates chromosome segregation through microtubule sliding as well as microtubule growth and shrinkage. Kinesin-14, one of the motors involved, causes spindle collapse in the absence of kinesin-5 (Refs 2, 3), participates in spindle assembly and modulates spindle length. However, the molecular mechanisms underlying these activities are not known. Here, we report that Drosophila melanogaster kinesin-14 (Ncd) alone causes sliding of anti-parallel microtubules but locks together (that is, statically crosslinks) those that are parallel. Using single molecule imaging we show that Ncd diffuses along microtubules in a tail-dependent manner and switches its orientation between sliding microtubules. Our results show that kinesin-14 causes sliding and expansion of an anti-parallel microtubule array by dynamic interactions through the motor domain on the one side and the tail domain on the other. This mechanism accounts for the roles of kinesin-14 in spindle organization.

The use of FRET in the analysis of motor protein structure.

Fluorescence resonance energy transfer (FRET) is a spectroscopic phenomenon that consists of long-range dipole-dipole interaction between two chromophores. This method can be employed to gain quantitative distance information on macromolecules. FRET is particularly useful to characterize structural states of motor proteins, because the spatial relationship between various mechanical elements of the motor undergoing its mechanical cycle is essential to understand how force and movement are generated. In this chapter, we describe the technique, including the equations, methods of introducing fluorescence probes in specific loci of the protein, and data analysis. Practical guidelines and hints are also provided for protein preparation, labeling, and measuring FRET efficiency. The protocol is presented for interhead distance measurements in the dimeric kinesin-like motor, Ncd. However, it can easily be adapted to many other motor proteins.

Subunits interactions in kinesin motors.

Kinesins form a large and diverse superfamily of proteins involved in numerous important cellular processes. The majority of them are molecular motors moving along microtubules. Conversion of chemical energy into mechanical work is accomplished in a sequence of events involving both biochemical and conformational alternation of the motor structure called the mechanochemical cycle. Different members of the kinesin superfamily can either perform their function in large groups or act as single molecules. Conventional kinesin, a member of the kinesin-1 subfamily, exemplifies the second type of motor which requires tight coordination of the mechanochemical cycle in two identical subunits to accomplish processive movement toward the microtubule plus end. Recent results strongly support an asymmetric hand-over-hand model of "walking" for this protein. Conformational strain between two subunits at the stage of the cycle where both heads are attached to the microtubule seems to be a major factor in intersubunit coordination, although molecular and kinetic details of this phenomenon are not yet deciphered. We discuss also current knowledge concerning intersubunit coordination in other kinesin subfamilies. Members of the kinesin-3 class use at least three different mechanisms of movement and can translocate in monomeric or dimeric forms. It is not known to what extent intersubunit coordination takes place in Ncd, a dimeric member of the kinesin-14 subfamily which, unlike conventional kinesin, exercises a power-stroke toward the microtubule minus end. Eg5, a member of the kinesin-5 subfamily is a homotetrameric protein with two kinesin-1-like dimeric halves controlled by their relative orientation on two microtubules. It seems that diversity of subunit organization, quaternary structures and cellular functions in the kinesin superfamily are reflected also by the divergent extent and mechanism of intersubunit coordination during kinesin movement along microtubules.

Biotemplated nanopatterning of planar surfaces with molecular motors.

We report on the generation of nanometer-wide, non-topographical patterns of proteins on planar surfaces. In particular, we used the regular lattice of reconstituted microtubules as template structures to specifically bind and transfer kinesin-1 and nonclaret disjunctional motor proteins. The generated tracks, which comprise dense and structurally oriented arrays of functional motor proteins, proved to be highly efficient for the guiding of microtubule transporters.

Spatial relationship between heads of dimeric Ncd in the presence of nucleotides and microtubules.

Kinesins are molecular motors that produce mechanical work at the expense of ATP hydrolysis. Here, we studied Ncd (non-claret disjunctional), a (-)-end-directed member of this superfamily. To gain insight into the mechanism by which Ncd generates force and movement, we measured distances between the heads in dimeric Ncd-250-700 using fluorescence resonance energy transfer (FRET). About 5% of Ncd heads were labeled with 1,5-IAEDANS (donor), and the remaining thiol groups were modified with QSY35-iodoacetamide (acceptor). Several lines of experimental evidence suggest that the probes were conjugated to Cys-670 in each head of the dimer. The measured donor-acceptor distance was about 35 A. Nucleotides (ADP, ATP, and AMP-PNP) in the presence and absence of microtubules had only small effects on the interhead distances. Similar results were obtained for bidirectional Ncd mutant in which Asn-340 was replaced by a lysine. The results argue against models of Ncd movement in which the heads undergo large spatial rearrangements during mechanochemical cycle and suggest Gly-347 as a possible pivot point for the head rotation.

Ca2+ binding to myosin regulatory light chain affects the conformation of the N-terminus of essential light chain and its binding to actin.

We prepared a new type of skeletal myosin subfragment 1 (S1-MLC1F) containing both, the essential and the regulatory light chains, intact, by exchanging the essential light chains of papain S1 with bacterially expressed longer isoform (MLC1F) of this light chain. We then compared the enzymatic and structural properties of chymotryptic S1, papain S1, and S1-MLC1F in the presence and in the absence of Ca(2+) ions bound to the regulatory light chain. In the presence of Ca(2+), subfragment 1 containing both intact light chains exhibited lower V(max) and lower K(m) for actin activation of S1 ATPase. When S1-MLC1F was cross-linked to actin via the N-terminus of the essential light chain, the yield was much higher when Ca(2+) ions saturated the regulatory light chain. Limited proteolysis of the essential light chain in S1-MLC1F was significantly inhibited in the presence of calcium as compared to chymotryptic S1. We conclude that the effect of binding of Ca(2+) to the regulatory light chain is transmitted to the N-terminal extension of the longer isoform of the essential light chain. The resulting structure of the N-terminus is less susceptible to proteolytic digestion, binds tighter to actin, and has an inhibitory effect on actin-activated myosin ATPase. This new conformation of the N-terminus may be responsible for calcium induced myosin-linked modulation of striated muscle contraction.

A two-plasmid system for independent genetic manipulation of subunits of homodimeric proteins and selective isolation of chimeric dimers.

We have designed and tested a modular two-plasmid expression system which allows coexpression of two different subunits of recombinant dimeric protein in Escherichia coli and selective purification of heterodimers. We have constructed a new expression vector, pBIOEx, with p15a replication origin which allows its stable coexistence with different ColE1 group plasmids. The expression cassette of this plasmid under control of the T7 promoter contains cloning site, followed by a short sequence coding for the C-terminal extension of the recombinant protein which is a target of the in vivo biotinylation by BirA protein. The expression unit is bicistronic, the second expressed protein being BirA. We have used this plasmid together with pET30a to clone kinesin heavy-chain fragment and coexpressed the two polypeptide chains differing by tags on their C-termini and we purified heterodimers made of two recombinant molecules. The heterodimeric protein had a normal biochemical activity. There was no discrimination against heterodimer formation at the dimerization step. The system is a powerful tool in studies of different aspects of interactions between subunits of the homodimeric proteins since it makes possible separate genetic manipulations on each subunit of the dimer.

Directionality of kinesin motors.

Kinesins are molecular motors that transport various cargoes along microtubule tracks using energy derived from ATP hydrolysis. Although the motor domains of kinesins are structurally similar, the family contains members that move on microtubules in opposite directions. Recent biochemical and biophysical studies of several kinesins make it possible to identify structural elements responsible for the different directionality, suggesting that reversal of the motor movement can be achieved through small, local changes in the protein structure.