Characterization of the pre-force-generation state in the actomyosin cross-bridge cycle.

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 ADP.P(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 (FRET) to monitor conformational changes in the upper-50-kDa domain of the actin binding cleft in the weak and strong actin binding states. Steady-state and lifetime data monitoring the FRET signal suggest that the cleft is in a more open conformation in the weak actin binding states. Transient kinetic experiments suggest that a rapid conformational change occurs, which is consistent with cleft closure before actin-activated phosphate release. Our results have identified a pre-force-generation actomyosin ADP.P(i) state, and suggest force generation may occur from a state not yet seen by crystallography in which the actin binding cleft and the nucleotide binding pocket are closed. Computational modeling uncovers dramatic changes in the rigidity of the upper-50-kDa domain in different nucleotide states, which suggests that the intrinsic flexibility of this domain allows myosin motors to accomplish simultaneous tight nucleotide binding (closed nucleotide binding pocket) and high-affinity actin binding (closed actin binding cleft).

The kinase domain alters the kinetic properties of the myosin IIIA motor.

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.

Human deafness mutation E385D disrupts the mechanochemical coupling and subcellular targeting of myosin-1a.

Missense mutations in the membrane-binding actin-based motor protein, myosin-1a (Myo1a), have recently been linked to sensorineural deafness in humans. One of these mutations, E385D, impacts a residue in the switch II region of the motor domain that is present in virtually all members of the myosin superfamily. We sought to examine the impact of E385D on the function of Myo1a, both in terms of mechanochemical activity and ability to target to actin-rich microvilli in polarized epithelial cells. While E385D-Myo1a demonstrated actin-activated ATPase activity, the V(MAX) was reduced threefold relative to wild-type. Despite maintaining an active mechanochemical cycle, E385D-Myo1a was unable to move actin in the sliding filament assay. Intriguingly, when an enhanced-green-fluorescent-protein-tagged form of E385D-Myo1a was stably expressed in polarized epithelial cells, this mutation abolished the microvillar targeting normally demonstrated by wild-type Myo1a. Notably, these data are the first to suggest that mechanical activity is essential for proper localization of Myo1a in microvilli. These studies also provide a unique example of how even the most mild substitution of invariant switch II residues can effectively uncouple enzymatic and mechanical activity of the myosin motor domain.

Kinetic mechanism of human myosin IIIA.

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 microm). 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) approximately 7 microm). 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 microm(-1).s(-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) approximately 5 microm). The long lived actomyosin-ADP state may be important for the ability of myosin III to function as a cellular transporter and actin cross-linker in the actin bundles of sensory cells.

Cutting edge: CD4 is the receptor for the tick saliva immunosuppressor, Salp15.

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.

Dynamics of the upper 50-kDa domain of myosin V examined with fluorescence resonance energy transfer.

The upper 50-kDa region of myosin may be critical for coupling between the nucleotide- and actin-binding regions. We introduced a tetracysteine motif in the upper 50-kDa domain (residues 292-297) of myosin V containing a single IQ domain (MV 1IQ), allowing us to label this site with the fluorescein biarscenical hairpin-binding dye (FlAsH) (MV 1IQ FlAsH). The enzymatic properties of MV 1IQ FlAsH were similar to those of unlabeled MV 1IQ except for a 3-fold reduced ADP-release rate. MV 1IQ FlAsH was also capable of moving actin filaments in the in vitro motility assay. To examine rotation of the upper 50-kDa region, we determined the difference in the degree of energy transfer from N-methylanthraniloyl (mant)-labeled nucleotides to FlAsH in both steady-state and transient kinetic experiments. The energy transfer efficiency was higher with mant-ATP (0.65 +/- 0.02) compared with mant-ADP (0.55 +/- 0.02) in the absence of actin. Stopped-flow measurements suggested that the energy transfer efficiency decreased with phosphate release (0.04 s(-1)) in the absence of actin. In contrast, upon mixing MV 1IQ FlAsH in the ADP.P(i) state with actin, a decrease in the energy transfer signal was observed at a rate of 13 s(-1), similar to the ADP release rate. Our results demonstrate there was no change in the energy transfer signal upon actin-activated phosphate release and suggest that actin binding alters the dynamics of the upper 50-kDa region, which may be critical for the ability of myosin to bind tightly to both ADP and actin.

Delivery of the immunosuppressive antigen Salp15 to antigen-presenting cells by Salmonella enterica serovar Typhimurium aroA mutants.

A Salmonella enterica serovar Typhimurium aroA-deficient delivery system was used to target the immunosuppressive protein Salp15 to antigen-presenting cells. In vitro and in vivo infections with Salp15-containing Salmonella resulted in an impaired CD4(+)-T-cell activation, suggesting that the protein was produced by antigen-presenting cells in a physiologically active form.

Cyclooxygenase 2 activity modulates the severity of murine Lyme arthritis.

Cyclooxygenase (Cox) is a key enzyme in the biosynthetic metabolism of prostaglandins. The inducible isoform of Cox-2 has been implicated in inflammation and its specific inhibition can be used to treat noninfectious inflammatory diseases, such as rheumatoid arthritis. Borrelia burgdorferi, the agent of Lyme disease, can induce joint inflammation. Here we show that B. burgdorferi induced the upregulation of cox-2 gene expression in murine joints at the onset of arthritis in infected mice. The level of mRNA expression correlated with the degree of inflammation. The specific inhibition of Cox-2 diminished the degree of joint inflammation, without affecting B. burgdorferi-specific antibody or cytokine responses. Cox-2 activity is therefore associated with the genesis of infectious arthritis caused by B. burgdorferi.