Salp15 is an Ixodes scapularis salivary protein that inhibits CD4+ T cell activation through the repression of TCR ligation-triggered calcium fluxes and IL-2 production. We show in this study that Salp15 binds specifically to the CD4 coreceptor on mammalian host T cells. Salp15 specifically associates through its C-terminal residues with the outermost two extracellular domains of CD4. Upon binding to CD4, Salp15 inhibits the subsequent TCR ligation-induced T cell signaling at the earliest steps including tyrosine phosphorylation of the Src kinase Lck, downstream effector proteins, and lipid raft reorganization. These results provide a molecular basis to understanding the immunosuppressive activity of Salp15 and its specificity for CD4+ T cells.

Myosin IIIA is specifically expressed in photoreceptors and cochlea and is important for the phototransduction and hearing processes. In addition, myosin IIIA contains a unique N-terminal kinase domain and C-terminal tail actin-binding motif. We examined the kinetic properties of baculovirus expressed human myosin IIIA containing the kinase, motor, and two IQ domains. The maximum actin-activated ATPase rate is relatively slow (k cat ‫؍‬ 0.77 ؎ 0.08 s ؊1 ), and high actin concentrations are required to fully activate the ATPase rate (K ATPase ‫؍‬ 34 ؎ 11 M). However, actin co-sedimentation assays suggest that myosin III has a relatively high steady-state affinity for actin in the presence of ATP (K actin ϳ 7 M). The rate of ATP binding to the motor domain is quite slow both in the presence and absence of actin (K 1 k ؉2 ‫؍‬ 0.020 and 0.001 M ؊1 ⅐s ؊1 , respectively). The rate of actin-activated phosphate release is more than 100-fold faster (85 s ؊1 ) than the k cat , whereas ADP release in the presence of actin follows a two-step mechanism (7.0 and 0.6 s ؊1 ). Thus, our data suggest a transition between two actomyosin-ADP states is the rate-limiting step in the actomyosin III ATPase cycle. Our data also suggest the myosin III motor spends a large fraction of its cycle in an actomyosin ADP state that has an intermediate affinity for actin (K d ϳ 5 M). The long lived actomyosin-ADP state may be important for the ability of myosin III to function as a cellular transporter and actin crosslinker in the actin bundles of sensory cells.

Myosin is an actin-based motor protein that generates force by cycling between actin-attached (strong binding: ADP or rigor) and actin-detached (weak binding: ATP or ADP⅐P i ) states during its ATPase cycle. However, it remains unclear what specific conformational changes in the actin binding site take place on binding to actin, and how these structural changes lead to product release and the production of force and motion. We studied the dynamics of the actin binding region of myosin V by using fluorescence resonance energy transfer ( actin ͉ energy transfer ͉ motor proteins ͉ myosin ͉ structural dynamics A current challenge in biophysics is to understand the mechanism of how motor proteins such as myosin convert chemical energy into mechanical work through a cyclic interaction with actin filaments. Numerous structure/function studies have converged on a structural model for force generation, where conformational changes in the active site are coupled to a large rotation of the light-chain binding region, also known as the lever arm hypothesis (1, 2). Myosin alters its affinity for actin in a nucleotide-dependent manner, from strong actin binding states (ADP or rigor state) to weak actin binding states (ATP or ADP⅐P i state), and thus phosphate release is believed to be associated with a large increase in actin affinity. Based on the high-resolution x-ray structure of myosin II (3) it was predicted that the actin binding cleft may open during ATP-induced dissociation of actomyosin and close during the release of the hydrolysis products induced by actin binding. The crystal structure of myosin V in the absence of nucleotide (rigor) demonstrated a closed conformation of the actin binding cleft (4, 5), and this structure fit quite well into the electron microscopy image reconstructions of the actomyosin rigor complex (6). Further studies of myosin II (7) and myosin V (8) by electron microscopy image reconstruction have demonstrated conformational changes in the actin binding cleft in different nucleotide states. Recently, the closed-cleft conformation was also observed in crystallographic studies of molluscan myosin II, wherein a ''counterclockwise'' orientation of the cleft rather than the extent of its closure was proposed to be critical for forming the strong binding rigor conformation (9).By placing fluorescent probes in the actin binding cleft it was directly demonstrated that the cleft opens during ATP-induced dissociation of actomyosin (10, 11). However, there is currently no direct evidence that describes the kinetics of actin binding cleft closure in relationship to actin-activated phosphate release. In addition, there is a lack of information about how conformational changes in the cleft are coupled to structural changes in the nucleotide binding region. Most models of the actomyosin cross-bridge cycle suggest that myosin binds to actin through both ionic and hydrophobic interactions that stabilize a structural change in the actin binding region, such as closure of the actin binding cleft (1, 2). This st...

Myosins consist of a superfamily of molecular motor proteins that use the energy from ATP hydrolysis to generate force and motion through a cyclic interaction with actin filaments (1). The structural details of how conformational changes in myosin are coupled to specific steps in the actomyosin ATPase cycle have yet to be fully elucidated. The current working model suggests that small changes in the nucleotide-binding region (switch I and switch II) are communicated to the lever arm region and amplified to generate force and motion (2). One key question that remains is how myosin changes conformation to alter its affinity for actin in a nucleotide-dependent manner. The myosin V crystal structures (3, 4) as well as previous structural and biochemical data (5-10) suggest that the large cleft that separates the actin-binding region and extends from the nucleotide-binding site may change conformation in a nucleotide-dependent manner. However, it is unclear how the coupling between the nucleotide-binding region and actin-binding cleft occurs.The structure of myosin subfragment 1 can be described by three domains generated by trypsin cleavage: a 25-kDa N-terminal domain, a central 50-kDa domain, and a 20-kDa C-terminal domain (Fig. 1). The actin-binding domain is separated into the upper and lower 50-kDa domains by a 50-kDa cleft, also known as the actin-binding cleft. The actin-binding cleft extends from near the active site to the actin-binding region, making it a logical candidate for communication between the nucleotide-and actin-binding domains (9, 10). The atomic structures demonstrate a rotation of the upper 50-kDa region in the nucleotidefree structure of myosin V, which results in closure of the actin-binding cleft, compared with the near rigor and ATP-bound states (4). In addition, cryoelectron microscopy demonstrated a structural change in the upper 50-kDa region of smooth muscle myosin when bound to actin in the presence and absence of ADP (6). Another study demonstrated that a tryptophan residue in the upper 50-kDa region of the cleft changed conformation with kinetics identical to that of ATP-induced dissociation from actin (7). Finally, a previous study that placed pyrene probes on either side of the cleft monitored a change in pyrene excimer fluorescence upon ATP-induced dissociation from actin (8). Thus, the cleft appears to play a role in an ATP-dependent conformational change that results in a large reduction in affinity for actin. It has been proposed that the upper 50-kDa rotation occurs by distortion of a highly conserved ␤ sheet, which may be a conformational change similar to that observed in the sequential product release of F1-ATPase (4, 11). However, little is known about how the cleft changes conformation upon binding to actin in the ADP⅐P i state and if the cleft plays a role in the actin-activated P i and ADP release. Studies in which the dynamics of the actin-binding cleft can be measured in the presence of actin during the product release steps are critical to answering these questions....

Myosin IIIA is unique among myosin proteins in that it contains an N-terminal kinase domain capable of autophosphorylating sites on the motor domain. A construct of myosin IIIA lacking the kinase domain localizes more efficiently to the stereocilia tips and alters the morphology of the tips in inner ear hair cells. Therefore, we performed a kinetic analysis of myosin IIIA without the kinase domain (MIII DeltaK) and compared these results with our reported analysis of myosin IIIA containing the kinase domain (MIII). The steady-state kinetic properties of MIII DeltaK indicate that it has a 2-fold higher maximum actin-activated ATPase rate (kcat = 1.5 +/- 0.1 s-1) and a 5-fold tighter actin affinity (KATPase = 6.0 +/- 1.4 microM, and KActin = 1.4 +/- 0.4 microM) compared to MIII. The rate of ATP binding to the motor domain is enhanced in MIII DeltaK (K1k+2 approximately 0.10 +/- 0.01 microM-1.s-1) to a level similar to the rate of binding to MIII in the presence of actin. The rate of ATP hydrolysis in the absence of actin is slow and may be rate limiting. Actin-activated phosphate release is identical with and without the kinase domain. The transition between actomyosin.ADP states, which is rate limiting in MIII, is enhanced in MIII DeltaK. MIII DeltaK accumulates more efficiently at the tips of filopodia in HeLa cells. Our results suggest a model in which the activity and concentration of myosin IIIA localized to the tips of actin bundles mediates the morphology of the tips in sensory cells.