Background: Tau inhibits kinesin on GDP-microtubules in vitro, but the physiological significance in neurons is unclear. Results: On GTP-microtubules, Tau loses its inhibitory effect, and kinesin becomes less processive. Conclusion:The nucleotide-binding state of the microtubule influences the behavior of both kinesin and Tau. Significance: Tau has different functions, both inhibitory and non-inhibitory, in regulating axonal transport.

Elucidation of the molecular details of the cyclic actomyosin interaction requires the ability to examine structural changes at specific sites in the actin-binding interface of myosin. To study these changes dynamically, we have expressed two mutants of a truncated fragment of chicken gizzard smooth muscle myosin, which includes the motor domain and essential light chain (MDE). These mutants were engineered to contain a single tryptophan at (Trp-546) or near (Trp-625) the putative actin-binding interface. Both 546-and 625-MDE exhibited actin-activated ATPase and actin-binding activities similar to wild-type MDE. Fluorescence emission spectra and acrylamide quenching of 546-and 625-MDE suggest that Trp-546 is nearly fully exposed to solvent and Trp-625 is less than 50% exposed in the presence and absence of ATP, in good agreement with the available crystal structure data. The spectrum of 625-MDE bound to actin was quite similar to the unbound spectrum indicating that, although Trp-625 is located near the 50͞20-kDa loop and the 50-kDa cleft of myosin, its conformation does not change upon actin binding. However, a 10-nm blue shift in the peak emission wavelength of 546-MDE observed in the presence of actin indicates that Trp-546, located in the A-site of the lower 50-kDa subdomain of myosin, exists in a more buried environment and may directly interact with actin in the rigor acto-S1 complex. This change in the spectrum of Trp-546 constitutes direct evidence for a specific molecular interaction between residues in the A-site of myosin and actin.

The putative actin-binding interface of myosin is separated by a large cleft that extends into the base of the nucleotide binding pocket, suggesting that it may be important for mediating the nucleotide-dependent changes in the affinity for myosin on actin. We have genetically engineered a truncated version of smooth muscle myosin containing the motor domain and the essential light chain-binding region ( At the molecular level, muscle contraction is driven by the hydrolysis of MgATP and the cyclic interaction of two proteins, actin and myosin. Myosin possesses two striking functional features that allow it to act as a molecular motor protein, namely its ability to 1) generate force and motion with actin filaments through a putative powerstroke and 2) alter its affinity for actin by more than 4 orders of magnitude at different stages of its enzymatic MgATPase cycle. The myosin MgATPase cycle can be described by a kinetic scheme (1, 2), where A, M, and AM represent actin, myosin, and the actomyosin complex, respectively (Scheme 1).In Scheme 1, myosin binds strongly to actin with nanomolar affinity in the presence of MgADP or in the absence of nucleotide, and weakly to actin with micromolar affinity in the ATP and ADP⅐P i states of the contractile cycle. Thus binding of MgATP to myosin is thought to initiate the transition to the weakly bound states of the MgATPase cycle, and phosphate release is believed to be associated with the transition to the strongly bound states of the MgATPase cycle and the subsequent powerstroke. Although the biochemical basis for the ability of myosin to interact with actin in a nucleotide-dependent manner is well established (3), the structural basis for its enzymatic and motile functions are less well understood.Crystal structures of myosin (see Fig. 1) solved in different nucleotide states (4 -9), as well as other studies (3), have identified key structural rearrangements within the light chainbinding region of myosin that suggest how it may generate force and motion. However, the domain motions within the actin-binding region of myosin that are responsible for its ability to cycle between weak and strong actin-binding states during its MgATPase cycle are still unclear. Thus, examining the conformational changes that mediate the large changes in the affinity of myosin for actin during the contractile cycle are important to fully understand the molecular details of the acto-myosin interaction.Within the putative actin-binding interface of myosin there are three main regions that are thought to interact with actin (Ref. 10; see Fig. 1) (amino acid residues refer to smooth muscle myosin sequence, see Ref. 11 for comparison of myosin isoforms). First, there is a highly charged surface loop (residues 628 -657) known as loop 2 or the actin-binding loop that is not visible in the crystal structures of myosin, but has been crosslinked to actin (12) and contains a proteolytic site that is protected in the presence of actin (13). Second, a helix-loophelix motif (residues 531-561) located in the...

The helix-loop-helix (A-site) and myopathy loop (R-site) are located on opposite sides of the cleft that separates the proposed actin-binding interface of myosin. To investigate the structural features of the A- and R-sites, we engineered two mutants of the smooth muscle myosin motor domain with the essential light chain (MDE), containing a single tryptophan located either in the A-site (W546-MDE) or in the R-site (V413W MDE). W546- and V413W-MDE display actin-activated ATPase and actin-binding properties similar to those of wild-type MDE. The steady-state fluorescence properties of W546-MDE [emission peak (lambda(max)) = 344, quantum yield = 0.20, and acrylamide bimolecular quenching constant (k(q)) = 6.4 M(-)(1). ns(-)(1)] and V413W-MDE [lambda(max) = 338, quantum yield = 0.27, and k(q) = 3.6 M(-)(1).ns(-)(1)] demonstrate that Trp-546 and Trp-413 are nearly fully exposed to solvent, in agreement with the crystallographic data on these residues. In the presence of actin, Trp-546 shifts to a more buried environment in both the ADP-bound and nucleotide-free (rigor) actomyosin complexes, as indicated by an average lambda(max) of 337 or 336 nm, respectively, and protection from dimethyl(2-hydroxy-5-nitrobenzyl)sulfonium bromide (DHNBS) oxidation. In contrast, Trp-413 has a single conformation with an average lambda(max) of 338 nm in the ADP-bound complex, but in the rigor complex it is 50% more accessible to DHNBS oxidation and can adopt a range of possible conformations (lambda(max) = 341-347 nm). Our results suggest a structural model in which the A-site remains tightly bound to actin and the R-site adopts a more flexible and solvent-exposed conformation upon ADP release.

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