K. John Smith

Size and charge requirements for kinetic modulation and actin binding by alkali 1-type myosin essential light chains.

The alkali 1-type isoforms of myosin essential light chains from vertebrate striated muscles have an additional 40 or so amino acids at their N terminus compared with the alkali 2-type. Consequently two light chain isoenzymes of myosin subfragment-1 can be isolated. Using synthesized peptide mimics of the N-terminal region of alkali 1-type essential light chains, we have found by1H NMR that the major actin binding region occurred in the N-terminal four residues, APKK. . . . . These results were confirmed by mutating this region of the human atrial essential light chain, resulting in altered actin-activated MgATPase kinetics when the recombinant light chains were hybridized into rabbit skeletal subfragment 1. Substitution of either Lys3 or Lys4 with Ala resulted in increased K m and k cat and decreased actin binding (as judged by chemical cross-linking). Replacement of Lys4 with Asp reduced actin binding and increased K m andk cat still further. Alteration of Ala1 to Val did not alter the kinetic parameters of the hybrid subfragment 1 or the essential light chain’s ability to bind actin. Furthermore, we found a significant correlation between the apparent K m for actin and thek cat for MgATP turnover for each mutant hybrid, strengthening our belief that the binding of actin by alkali 1-type essential light chains results directly in modulation of the myosin motor.

Solution structure of the NADP(H)-binding component (dIII) of proton-translocating transhydrogenase from Rhodospirillum rubrum

Transhydrogenase is a proton pump found in the membranes of bacteria and animal mitochondria. The solution structure of the expressed, 21.5 kDa, NADP(H)-binding component (dIII) of transhydrogenase from Rhodospirillum rubrum has been solved by NMR methods. This is the first description of the structure of dIII from a bacterial source. The protein adopts a Rossmann fold: an open, twisted, parallel beta-sheet, flanked by helices. However, the binding of NADP(+) to dIII is profoundly different to that seen in other Rossmann structures, in that its orientation is reversed: the adenosine moiety interacts with the first betaalphabetaalphabeta motif, and the nicotinamide with the second. Features in the structure that might be responsible for changes in nucleotide-binding affinity during catalysis, and for interaction with other components of the enzyme, are identified. The results are compared with the recently determined, high-resolution crystal structures of human and bovine dIII which also show the reversed nucleotide orientation.

The mobile loop region of the NAD(H) binding component (dI) of proton-translocating nicotinamide nucleotide transhydrogenase from Rhodospirillum rubrum: complete NMR assignment and effects of bound nucleotides

The dI component of transhydrogenase binds NAD+ and NADH. A mobile loop region of dI plays an important role in the nucleotide binding process, and mutations in this region result in impaired hydride transfer in the complete enzyme. We have previously employed one-dimensional 1H-NMR spectroscopy to study wild-type and mutant dI proteins of Rhodospirillum rubrum and the effects of nucleotide binding. Here, we utilise two- and three-dimensional NMR experiments to assign the signals from virtually all of the backbone and side-chain protons of the loop residues. The mobile loop region encompasses 17 residues: Asp223-Met239. The assignments also provide a much strengthened basis for interpreting the structural changes occurring upon nucleotide binding, when the loop closes down onto the surface of the protein and loses mobility. The role of the mobile loop region in catalysis is discussed with particular reference to a newly-developed model of the dI protein, based on its homology with alanine dehydrogenase.

Letter to the Editor: Sequence-specific resonance assignments for the NADP(H)-binding component (domain III) of proton- translocating transhydrogenase from Rhodospisrillum rubrum

Transhydrogenase is a proton pump, found in the inner membrane of animal mitochondria, and the cytoplasmic membrane of bacteria. It has a tripartite structure. Domains I and III protrude from the membrane (on the cytoplasmic side in bacteria, and on the matrix side in mitochondria). The domain II component spans the membrane, and serves as a channel for proton conduction. Transhydrogenase couples the transfer of reducing equivalents (hydride ion equivalents) between NAD(H) and NADP(H) to the translocation of protons across the membrane (reviewed by Jackson et al., 1998),

Motoring down the highways of the cell

All eukaryotic cells contain large numbers of motor proteins (kinesins, dyneins and myosins), each of which appears to carry out a specialized force-generating function within the cell. They are known to have roles in muscle contraction, ciliary movement, organelle and vesicle transport, mitosis and cytokinesis. These motor proteins operate on different cytoskeletal filaments; myosins move along actin filaments, and kinesins and dyneins along microtubules. Recently published crystal structures of the motor domains of two members of the kinesin superfamily reveal that they share the same overall fold that is also found at the core of the larger myosin motor. This suggests that they may share a common mechanism as well as a common ancestry.

The Use of Peptide Mimetics to Define the Actin-Binding Sites on the Head of the Myosin Molecule

Definition of the molecular mechanisms in muscle contraction and its regulation entails a description of how the components of the organised assembly of proteins first dock with their substrates/partners, then interact and transmit information through the molecular array. Crosslinking studies and experiments with proteolysed fragments of the myosin head (subfragment 1, S1) from a variety of laboratories [e.g. Chaussepied et al., 1986a; Sutoh, 1983] have indicated the approximate regions of the molecule involved in complex formation. In order to define precisely the exact locations of these interfaces we have synthesized peptides based on the S1 sequence and tested these for their ability to bind to actin and influence its biological properties. Such chemical synthesis allows small regions of the parent protein, usually not obtainable by proteolytic or chemical digestion, to be examined in isolation.

Proton-Translocating Transhydrogenase from Rhodospirillum rubrum: Hydride Transfer in Recombinant Domains

Transhydrogenase couples the transfer of reducing equivalents (hydride ion equivalents) between NAD(H) and NADP(H) to the translocation of protons across a membrane.