Loop 1, a flexible surface loop in the myosin motor domain, comprises in part the transducer region that lies near the nucleotide-binding site and is proposed from structural studies to be responsible for the kinetic tuning of product release following ATP hydrolysis (1). Biochemical studies have shown that loop 1 affects the affinity of actin-myosin-II for ADP, motility and the V max of the actin-activated Mg 2؉ -ATPase activity, possibly through P i release (2-8). To test the influence of loop 1 on the mammalian class I myosin, Myo1b, chimeric molecules in which (i) loop 1 of a truncated form of Myo1b, Myo1b 1IQ , was replaced with either loop 1 from other myosins; (ii) loop 1 was replaced with glycine; or (iii) some amino acids in the loop were substituted with alanine and were expressed in baculovirus, and their interactions with actin and nucleotide were evaluated. The steady-state actin-activated ATPase activity; rate of ATP-induced dissociation of actin from Myo1b 1IQ ; rate of ADP release from actin-Myo1b 1IQ ; and the affinity of actin for Myo1b 1IQ and Myo1b 1IQ ⅐ADP differed in the chimeras versus wild type, indicating that loop 1 has a much wider range of effects on the coupling between actin and nucleotide binding events than previously thought. In particular, the biphasic ATP-induced dissociation of actin from actin-Myo1b 1IQ was significantly altered in the chimeras. This provided evidence that loop 1 contributes to the accessibility of the nucleotide pocket and is involved in the integration of information from the actin-, nucleotide-, ␥-P i -, and calmodulin-binding sites and predicts that loop 1 modulates the load dependence of the motor.Myo1b (aka 130-kDa myosin I, MYR 1, and MM1␣) is a mammalian class I myosin that is expressed in many different tissues (9 -11). It is associated with the plasma membrane (12) and in cell protrusions such as lamellipodia and membrane ruffles, suggesting its role in some aspects of cell motility (13). Overproduction of Myo1b or non-functional truncated Myo1b affects the distribution of endocytic compartments, suggesting its role in membrane trafficking (14). Furthermore, evidence indicates that Myo1b mediates lysosome movement (15).The ATP-induced dissociation of actin from actin-Myo1b is much slower than from most other myosins, and unlike skeletal muscle, myosin II is biphasic, consisting of both a fast and a slow phase (16). The fast phase is dependent on ATP concentration and is eliminated by preincubation with ADP. The slow phase is independent of ATP concentration and shares the same rate constant as ADP release but cannot be eliminated by decreasing ADP concentration. We have interpreted these results in conjunction with results from two other approaches: (i) single molecule studies demonstrating that Myo1b exhibits a two-part power stroke (17) and (ii) cryo-electron microscopy studies showing that Myo1b exhibits an ADP-induced conformational change. 1The biphasic nature of the ATP-induced dissociation of actinMyo1b (16, 19); the two-part power stroke (1...

Structural studies of the class I myosin, MyoE, led to the predictions that loop 4, a surface loop near the actin-binding region that is longer in class I myosins than in other myosin subclasses, might limit binding of myosins I to actin when actin-binding proteins, like tropomyosin, are present, and might account for the exclusion of myosin I from stress fibers. To test these hypotheses, mutant molecules of the related mammalian class I myosin, Myo1b, in which loop 4 was truncated (from an amino acid sequence of RMNGLDES to NGLD) or replaced with the shorter and distinct loop 4 found in Dictyostelium myosin II (GAGEGA), were expressed in vitro and their interaction with actin and with actin-tropomyosin was tested. Saturating amounts of expressed fibroblast tropomyosin-2 resulted in a decrease in the maximum actin-activated Mg2+-ATPase activity of wild-type Myo1b but had little or no effect on the actin-activated Mg2+-ATPase activity of the two mutants. In motility assays, few actin filaments bound tightly to Myo1b-WT-coated cover slips when tropomyosin-2 was present, whereas actin filaments both bound and were translocated by Myo1b-NGLD or Myo1b-GAGEGA in both the presence and absence of tropomyosin-2. When expressed in mammalian cells, like the wild type, the mutant myosins were largely excluded from tropomyosin-containing actin filaments, indicating that in the cell additional factors besides loop 4 determine targeting of myosins I to specific subpopulations of actin filaments.

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Phenol soluble modulin (PSM) α3, the most toxic member of α-toxin in <i>Staphylococcus aureus</i> bacteria, has been recently found to form cross-α amyloid fibrils and is selectively toxic to the mammalian cell membranes. In this work, it has been discovered that hydrophobic interactions play a major role in fibril formation of PSM-α3 strands, with<br>stabilization energy of 28.7 kCal/mol. We considered two model bilayers mimicking mammalian and bacterial cell membranes, and found that single α-helix strand penetration is energetically unfavourable in both of them. Hence, we propose a simple model using energetics to understand the reason for selective toxicity of the peptide to the mammalian cell membrane. This study, besides enhancing the understanding of PSM-α3, can also act as a stepping stone in future drug development against <i>​S. aureus.</i> ​<br><br>