Christine R. Cremo

The kinesin-1 motor protein is regulated by a direct interaction of its head and tail

Kinesin-1 is a molecular motor protein that transports cargo along microtubules. Inside cells, the vast majority of kinesin-1 is regulated to conserve ATP and to ensure its proper intracellular distribution and coordination with other molecular motors. Regulated kinesin-1 folds in half at a hinge in its coiled-coil stalk. Interactions between coiled-coil regions near the enzymatically active heads at the N terminus and the regulatory tails at the C terminus bring these globular elements in proximity and stabilize the folded conformation. However, it has remained a mystery how kinesin-1s microtubule-stimulated ATPase activity is regulated in this folded conformation. Here, we present evidence for a direct interaction between the kinesin-1 head and tail. We photochemically cross-linked heads and tails and produced an 8-Å cryoEM reconstruction of the cross-linked head–tail complex on microtubules. These data demonstrate that a conserved essential regulatory element in the kinesin-1 tail interacts directly and specifically with the enzymatically critical Switch I region of the head. This interaction suggests a mechanism for tail-mediated regulation of the ATPase activity of kinesin-1. In our structure, the tail makes simultaneous contacts with the kinesin-1 head and the microtubule, suggesting the tail may both regulate kinesin-1 in solution and hold it in a paused state with high ADP affinity on microtubules. The interaction of the Switch I region of the kinesin-1 head with the tail is strikingly similar to the interactions of small GTPases with their regulators, indicating that other kinesin motors may share similar regulatory mechanisms.

ADP release produces a rotation of the neck region of smooth myosin but not skeletal myosin.

Current theories of muscle cross-bridge function suggest that force is generated by a change in the orientation of the myosin neck region. We attached a paramagnetic probe to a subunit in the neck region and measured the orientation of the probe using electron paramagnetic resonance spectroscopy. The angle of the probes on smooth myosin S1 were changed by 20°±4° on addition of ADP (50% effect at 5±2 μM), but ADP produced little effect on skeletal S1. The orientation of smooth myosin, +ADP, resembled that of skeletal myosin, ±ADP, suggesting that the release of ADP generates an extra rotation of the neck region in smooth muscle at the end of its power stroke.

Plant chimeric Ca2+/Calmodulin-dependent protein kinase. Role of the neural visinin-like domain in regulating autophosphorylation and calmodulin affinity

Chimeric Ca2+/calmodulin-dependent protein kinase (CCaMK) is characterized by a serine-threonine kinase domain, an autoinhibitory domain, a calmodulin-binding domain and a neural visinin-like domain with three EF-hands. The neural visinin-like Ca2+-binding domain at the C-terminal end of the CaM-binding domain makes CCaMK unique among all the known calmodulin-dependent kinases. Biological functions of the plant visinin-like proteins or visinin-like domains in plant proteins are not well known. Using EF-hand deletions in the visinin-like domain, we found that the visinin-like domain regulated Ca2+-stimulated autophosphorylation of CCaMK. To investigate the effects of Ca2+-stimulated autophosphorylation on the interaction with calmodulin, the equilibrium binding constants of CCaMK were measured by fluorescence emission anisotropy using dansylated calmodulin. Binding was 8-fold tighter after Ca2+-stimulated autophosphorylation. This shift in affinity did not occur in CCaMK deletion mutants lacking Ca2+-stimulated autophosphorylation. A variable calmodulin affinity regulated by Ca2+-stimulated autophosphorylation mediated through the visinin-like domain is a new regulatory mechanism for CCaMK activation and calmodulin-dependent protein kinases. Our experiments demonstrate the existence of two functional molecular switches in a protein kinase regulating the kinase activity, namely a visinin-like domain acting as a Ca2+-triggered switch and a CaM-binding domain acting as an autophosphorylation-triggered molecular switch.

Biochemistry of smooth muscle myosin light chain kinase

The smooth muscle isoform of myosin light chain kinase (MLCK) is a Ca(2+)-calmodulin-activated kinase that is found in many tissues. It is particularly important for regulating smooth muscle contraction by phosphorylation of myosin. This review summarizes selected aspects of recent biochemical work on MLCK that pertains to its function in smooth muscle. In general, the focus of the review is on new findings, unresolved issues, and areas with the potential for high physiological significance that need further study. The review includes a concise summary of the structure, substrates, and enzyme activity, followed by a discussion of the factors that may limit the effective activity of MLCK in the muscle. The interactions of each of the many domains of MLCK with the proteins of the contractile apparatus, and the multi-domain interactions of MLCK that may control its behaviors in the cell are summarized. Finally, new in vitro approaches to studying the mechanism of phosphorylation of myosin are introduced.

Structure and Function of the 10 S Conformation of Smooth Muscle Myosin

Smooth myosin regulatory light chain (RLC) was exchanged with RLC labeled with benzophenone-4-iodoacetamide at Cys-108. Irradiation under conditions that favor the folded (10 S) conformation resulted in 10 S cross-linked myosin that could not unfold. Purified 10 S cross-linked myosin was cross-linked between the RLC of one head to light meromyosin between leucine 1554 and glutamate 1583, adjacent to a predicted noncoiled region, approximately 60 nm from the tip of the tail. At high ionic strength without actin, product release from one-half of the heads was slow (like 10 S) whereas the other half were activated. This suggests that tail binding to the RLC carboxyl-terminal domain stabilizes ionic interactions important to slow nucleotide release. With actin, product release from both (un)phosphorylated 10 S cross-linked myosin was from one slow population similar to unphosphorylated filaments. 10 S cross-linked myosin weakly bound actin (dissociation constant > 500 μM) and did not move actin in vitro. Single-headed myosin did not fold or trap nucleotide. These and other data suggest that “trapping” occurs only with both heads and the tail binds to a newly formed site, which includes the RLC carboxyl-terminal domain, once trapping has occurred.

Structural Model of the Regulatory Domain of Smooth Muscle Heavy Meromyosin

The goal of this study was to provide structural information about the regulatory domains of double-headed smooth muscle heavy meromyosin, including the N terminus of the regulatory light chain, in both the phosphorylated and unphosphorylated states. We extended our previous photo-cross-linking studies (Wu, X., Clack, B. A., Zhi, G., Stull, J. T., and Cremo, C. R. (1999)J. Biol. Chem. 274, 20328–20335) to determine regions of the regulatory light chain that are cross-linked by a cross-linker attached to Cys108 on the partner regulatory light chain. For this purpose, we have synthesized two new biotinylated sulfhydryl reactive photo-cross-linking reagents, benzophenone, 4-(N-iodoacetamido)-4′-(N-biotinylamido) and benzophenone, 4-(N-maleimido)-4′-(N-biotinylamido). Cross-linked peptides were purified by avidin affinity chromatography and characterized by Edman sequencing and mass spectrometry. Labeled Cys108 from one regulatory light chain cross-linked to71GMMSEAPGPIN81, a loop in the N-terminal half of the regulatory light chain, and to4RAKAKTTKKRPQR16, a region for which there is no atomic resolution data. Both cross-links were to the partner regulatory light chain and occurred in unphosphorylated but not phosphorylated heavy meromyosin. Using these data, data from our previous study, and atomic coordinates from various myosin isoforms, we have constructed a structural model of the regulatory domain in an unphosphorylated double-headed molecule that predicts the general location of the N terminus. The implications for the structural basis of the phosphorylation-mediated regulatory mechanism are discussed.

Direct evidence for functional smooth muscle myosin II in the 10S self-inhibited monomeric conformation in airway smooth muscle cells

The 10S self-inhibited monomeric conformation of myosin II has been characterized extensively in vitro. Based upon its structural and functional characteristics, it has been proposed to be an assembly-competent myosin pool in equilibrium with filaments in cells. It is known that myosin filaments can assemble and disassemble in nonmuscle cells, and in some smooth muscle cells, but whether or not the disassembled pool contains functional 10S myosin has not been determined. Here we address this question using human airway smooth muscle cells (hASMCs). Using two antibodies against different epitopes on smooth muscle myosin II (SMM), two distinct pools of SMM, diffuse, and stress-fiber–associated, were visualized by immunocytochemical staining. The two SMM pools were functional in that they could be interconverted in two ways: (i) by exposure to 10S- versus filament-promoting buffer conditions, and (ii) by exposure to a peptide that shifts the filament-10S equilibrium toward filaments in vitro by a known mechanism that requires the presence of the 10S conformation. The effect of the peptide was not due to a trivial increase in SMM phosphorylation, and its specificity was demonstrated by use of a scrambled peptide, which had no effect. Based upon these data, we conclude that hASMCs contain a significant pool of functional SMM in the 10S conformation that can assemble into filaments upon changing cellular conditions. This study provides unique direct evidence for the presence of a significant pool of functional myosin in the 10S conformation in cells.

Role of the tail in the regulated state of myosin 2

Myosin 2 from vertebrate smooth muscle or non-muscle sources is in equilibrium between compact, inactive monomers and thick filaments under physiological conditions. In the inactive monomer, the two heads pack compactly together, and the long tail is folded into three closely packed segments that are associated chiefly with one of the heads. The molecular basis of the folding of the tail remains unexplained. By using electron microscopy, we show that compact monomers of smooth muscle myosin 2 have the same structure in both the native state and following specific, intramolecular photo-cross-linking between Cys109 of the regulatory light chain (RLC) and segment 3 of the tail. Nonspecific cross-linking between lysine residues of the folded monomer by glutaraldehyde also does not perturb the compact conformation and stabilizes it against unfolding at high ionic strength. Sequence comparisons across phyla and myosin 2 isoforms suggest that the folding of the tail is stabilized by ionic interactions between the positively charged N-terminal sequence of the RLC and a negatively charged region near the start of tail segment 3 and that phosphorylation of the RLC could perturb these interactions. Our results support the view that interactions between the heads and the distal tail perform a critical role in regulating activity of myosin 2 molecules through stabilizing the compact monomer conformation.

Characterization of Tightly Associated Smooth Muscle Myosin–Myosin Light-Chain Kinase–Calmodulin Complexes

A current popular model to explain phosphorylation of smooth muscle myosin (SMM) by myosin light-chain kinase (MLCK) proposes that MLCK is bound tightly to actin but weakly to SMM. We found that MLCK and calmodulin (CaM) co-purify with unphosphorylated SMM from chicken gizzard, suggesting that they are tightly bound. Although the MLCK:SMM molar ratio in SMM preparations was well below stoichiometric (1:73+/-9), the ratio was approximately 23-37% of that in gizzard tissue. Fifteen to 30% of MLCK was associated with CaM at approximately 1 nM free [Ca(2+)]. There were two MLCK pools that bound unphosphorylated SMM with K(d) approximately 10 and 0.2 microM and phosphorylated SMM with K(d) approximately 20 and 0.2 microM. Using an in vitro motility assay to measure actin sliding velocities, we showed that the co-purifying MLCK-CaM was activated by Ca(2+) and phosphorylation of SMM occurred at a pCa(50) of 6.1 and at a Hill coefficient of 0.9. Similar properties were observed from reconstituted MLCK-CaM-SMM. Using motility assays, co-sedimentation assays, and on-coverslip enzyme-linked immunosorbent assays to quantify proteins on the motility assay coverslip, we provide strong evidence that most of the MLCK is bound directly to SMM through the telokin domain and some may also be bound to both SMM and to co-purifying actin through the N-terminal actin-binding domain. These results suggest that this MLCK may play a role in the initiation of contraction.

Vanadate-mediated photocleavage of myosin

Publisher Summary Skeletal myosin is known to form a very stable transition state-like complex with MgADP and vanadate ions (V i ). During the irradiation ADP and V i are released simultaneously with a concomitant four-fold increase in the Ca 2+ -ATPase activity and an increase in the UV absorbance of myosin subfragment 1 (S1) over unirradiated controls. This modification is the result of the vanadate-promoted photooxidation of the fl-hydroxymethyl group of a serine to an aldehyde. This aldehyde can tautomerize to an enol. Photocleavage of myosin at active site yields a stable photomodified myosin, which, in the presence of excess V i and MgADP, will reform a new stable MgADP-V i complex at the active site. After purification of the new complex by removal of excess V i and MgADP by centrifugal gel filtration, irradiation leads to specific cleavage of the heavy chain. The heavy chain of S1 is cleaved at two sites when irradiated in the presence of miUimolar vanadate (in the absence of Mg 2+ or ADP. Mocz 6 has also reported a third site of vanadate-mediated photocleavage near the COOH terminus of the myosin heavy chain. An important consideration in photocleavage studies is that various polyvanadates, such as di-, tetra-, and pentavanadate, are in rapid equilibria with monovanadate at total vanadate concentrations in the millimolar range.