Background:Myosins convert the energy of ATP hydrolysis into force production. Results: Substitution of the metal-interacting serine in switch-1 with cysteine renders the motor sensitive to manganese. Conclusion: This technology provides a reversible structural linkage between the nucleotide pocket and actin-binding region in the myosin motor domain. Significance: Understanding the ATPase mechanism requires a description of allosteric communication in molecular motors.

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Formins are a class of proteins that influence the rate of actin filament nucleation and elongation. Mammals possess 15 formin isoforms, providing a myriad of possibilities for regulating actin-based structures in cells. The dimeric formin homology 2 (FH2) domain is capable of accelerating the nucleation rate of new actin filaments and subsequently influences filament elongation via direct interaction with the barbed end of an actin filament. The FH2 domain moves processively with the barbed end as the filament elongates. A subset of formins, including FRL2, can also bundle filaments. Our goal in this study is to examine FRL2's interaction with actin in detail. A portion of FRL2 containing the FH2 domain forms a stable interaction with tetramethylrhodamine-malamide labeled actin (TMR-actin) or Latrunculin B-bound actin, two forms of actin that are unable to polymerize. The actin/FRL2 complex is mono-disperse, as judged by analytical ultracentrifugation and gel filtration. FH2 domain-containing constructs from three other mammalian formins (mDia1, mDia2, and INF2) do not show an equivalent interaction. A mutation in the FH2 domain of FRL2 that prevents barbed-end binding also prevents interaction with TMR-actin, suggesting that the interaction interface is similar to that of FRL2 with the filament barbed end. These properties make the actin/FRL2 complex an ideal system for a structural study of actin/formin interactions. Both biochemical and structural experiments are being carried out with FRL2 constructs and both LatB treated and TMR labeled actin in order to characterize the details of their binding interactions.

Skeletal muscle myosin class II proteins are molecular machines that convert the chemical energy derived from the hydrolysis of ATP into mechanical work used to power muscle contraction. Drosophila melanogaster contains one gene encoding all striated muscle myosin II isoforms. The objective of this study is to gain insight into how alternative exon selection imparts myosin protein isoform biochemical and biophysical specificity. To this end, two His-tagged recombinant proteins encoding an indirect flight muscle myosin isoform (IFI) and an embryonic body wall myosin isoform (EMB) were expressed in and purified from the indirect flight muscles (IFM) of engineered fly lines lacking endogenous IFM myosin (Caldwell et al., Methods, 2012). The purified His-tagged myosins retain ATPase activity similar to their corresponding untagged isoforms. The myosin subfragment-1 (S1) contains the myosin heavy chain motor domain and the essential light chain. We determined the three-dimensional structure of IFI S1 at 2.5 Å resolution and EMB S1 at 2.2 Å resolution. They are the first insect myosin protein structures determined by X-ray crystallography (EMB is PDB 4QBD). Both structures are in the post rigor enzymatic state, as determined by comparisons with known myosin structures. For the EMB structure, two copies of the myosin molecule with slight conformational differences were resolved in the asymmetric unit. The electron density revealed a citrate molecule (contained in the crystallization solution) bound in the nucleotidebinding pocket. For the IFI structure, there is one myosin molecule in the asymmetric unit. The nucleotide-binding pocket contains an ADP molecule. Differences in conformation within regions of the proteins encoded by alternative exons suggest a source of the observed physiological differences in the embryonic and adult flight muscle fibers. (Funded by NIH R01GM32443 to SIB).