The Trp-cage, as the smallest miniprotein, remains the subject of numerous computational and experimental studies of protein folding dynamics and pathways. The original Trp-cage (NLYIQWLKDGGPSSGRPPPS, Tm = 42 degrees C) can be significantly stabilized by mutations; melting points as high as 64 degrees C are reported. In helical portions of the structure, each allowed replacement of Leu, Ile, Lys or Ser residues by Ala results in a 1.5 (+/-0.35) kJ/mol fold stabilization. No changes in structure or fluxionality of the core results upon stabilization. Contrary to the initial hypothesis, specific Pro/Trp interactions are not essential for core formation. The entropic advantage of Pro versus Ala (DeltaDeltaS(U) = 11 +/- 2 J/mol K) was measured at the solvent-exposed P17 site. Pro-Ala mutations at two of the three prolines (P12 and P18) that encage the indole ring result in less fold destabilization (2.3-3.4 kJ/mol). However, a P19A mutation reduces fold stability by 16 kJ/mol reflecting a favorable Y3/P19 interaction as well as Trp burial. The Y3/P19 hydrophobic staple interaction defines the folding motif as an 18-residue unit. Other stabilizing features that have been identified include a solvent-exposed Arg/Asp salt bridge (3.4-6 kJ/mol) and a buried H-bonded Ser side chain ( approximately 10 kJ/mol).

Omecamtiv mecarbil (OM) is a positive cardiac inotrope in phase-3 clinical trials for treatment of heart failure. Although initially described as a direct myosin activator, subsequent studies are at odds with this description and do not explain OM-mediated increases in cardiac performance. Here we show, via single-molecule, biophysical experiments on cardiac myosin, that OM suppresses myosin’s working stroke and prolongs actomyosin attachment 5-fold, which explains inhibitory actions of the drug observed in vitro. OM also causes the actin-detachment rate to become independent of both applied load and ATP concentration. Surprisingly, increased myocardial force output in the presence of OM can be explained by cooperative thin-filament activation by OM-inhibited myosin molecules. Selective suppression of myosin is an unanticipated route to muscle activation that may guide future development of therapeutic drugs.

Tropomyosin (Tm) is a two-chained, α-helical coiled-coil protein that associates end-to-end to form a continuous strand along actin filaments and regulates the functions and stability of actin in eukaryotic muscle and nonmuscle cells. Mutations in Tm cause skeletal and cardiac myopathies. We applied a neoteric molecular evolution approach to gain insight into the fundamental unresolved question of what makes the Tm coiled coil an actin binding protein. We carried out a phylogenetic analysis of 70 coding sequences of Tm genes from 26 animal species, from cnidarians to chordates, and evaluated the substitution rates (ω) at individual codons to identify conserved sites. The most conserved residues at surface b, c, f heptad repeat positions were mutated in rat striated muscle αTm and expressed in Escherichia coli. Each mutant had 3-4 sites mutated to Ala within the first half or the second half of periods 2-6. Actin affinity and thermodynamic stability were determined in vitro. Mutations in the first half of periods 2, 4, and 5 resulted in the largest reduction in actin affinity (>4-fold), indicating these mutations include residues in actin-binding sites. Mutations in the second half of the periods had a ≤2-fold effect on affinity indicating these residues may be involved in other conserved regulatory functions. The structural relevance of these results was assessed by constructing molecular models for the actin-Tm filament. Molecular evolution analysis is a general approach that may be used to identify potential binding sites of a protein for a conserved protein.cytoskeleton | muscle regulation | protein evolution | actin filament structure

Cooperative activation of actin-myosin interaction by tropomyosin (Tm) is central to regulation of contraction in muscle cells and cellular and intracellular movements in nonmuscle cells. The steric blocking model of muscle regulation proposed 40 y ago has been substantiated at both the kinetic and structural levels. Even with atomic resolution structures of the major players, how Tm binds and is designed for regulatory function has remained a mystery. Here we show that a set of periodically distributed evolutionarily conserved surface residues of Tm is required for cooperative regulation of actomyosin. Based on our results, we propose a model of Tm on a structure of actin-Tm-myosin in the "open" (on) state showing potential electrostatic interactions of the residues with both actin and myosin. The sites alternate with a second set of conserved surface residues that are important for actin binding in the inhibitory state in the absence of myosin. The transition from the closed to open states requires the sites identified here, even when troponin + Ca 2+ is present. The evolutionarily conserved residues are important for actomyosin regulation, a universal function of Tm that has a common structural basis and mechanism.actin filament structure | cell motility | cytoskeleton | muscle contraction | motility assay A ctin and myosin are found in almost all eukaryotic cells and are required for diverse cellular functions, including muscle contraction, cell motility, cell adhesion, cytokinesis, and organelle transport (1). The actin-myosin interaction produces two types of movements: force generation between actin filaments leading to contractions, such as in muscle contraction, cell motility, and cytokinesis; and transport of subcellular organelles and macromolecular complexes by myosin motors along actin filaments. The actomyosin contractile apparatus is best characterized in striated muscle contraction, which is regulated in a Ca 2+ -dependent manner by the proteins, tropomyosin (Tm) and troponin (Tn).Tm is a universal regulator of the actin cytoskeleton. It is a twochained, α-helical coiled-coil actin binding protein that associates end-to-end to form a continuous strand along both sides of actin filaments and regulates the functions and stability of actin filaments (2, 3). Tm regulates the interaction of actin filaments with actin binding proteins, including myosin, Tn, ADF-cofilin, Arp2/3, formin, and tropomodulin. Tm can activate or inhibit actomyosin, depending on the myosin and Tm isoforms (4-7). The Tm-dependent regulation is widespread, having been reported in fission yeast and mammalian muscle and nonmuscle systems. The regulation of actomyosin by Tm may be viewed as universal, although there is little understanding of the mechanism or structural basis, the focus of the present work.In striated muscle, regulation of myosin binding to the thin filament (actin-Tm-Tn) is described by a three-state model, where the three kinetic states are thought to correspond to three positions of Tm on the actin filament (8-1...

Background:The interface of actin with tropomyosin, the universal regulator of the actin filament, is unknown. Results: Mutagenesis of actin and tropomyosin revealed a pattern of residues required for complex formation in the closed state. Conclusion:The results support models of the actin-tropomyosin filament in the absence of myosin and troponin. Significance: A validated actin-tropomyosin model is required to understand regulation and disease mechanisms.

Tropomyosin (Tm) is a coiled-coil protein that binds to filamentous actin (F-actin) and regulates its interactions with actin-binding proteins like myosin by moving between three positions on F-actin (the blocked, closed, and open positions). To elucidate the molecular details of Tm flexibility in relation to its binding to F-actin, we conducted extensive molecular dynamics simulations for both Tm alone and Tm-F-actin complex in the presence of explicit solvent (total simulation time >400 ns). Based on the simulations, we systematically analyzed the local flexibility of the Tm coiled coil using multiple parameters. We found a good correlation between the regions with high local flexibility and a number of destabilizing regions in Tm, including six clusters of core alanines. Despite the stabilization by F-actin binding, the distribution of local flexibility in Tm is largely unchanged in the absence and presence of F-actin. Our simulations showed variable fluctuations of individual Tm periods from the closed position toward the open position. In addition, we performed Tm-F-actin binding calculations based on the simulation trajectories, which support the importance of Tm flexibility to Tm-F-actin binding. We identified key residues of Tm involved in its dynamic interactions with F-actin, many of which have been found in recent mutational studies to be functionally important, and the rest of which will make promising targets for future mutational experiments.

Temperature sensitive (ts) mutants are widely used to reversibly modulate protein function in vivo and to understand functions of essential genes. Despite this, little is known about the protein structural features and mechanisms responsible for generating a ts phenotype. Also, such mutants are often difficult to isolate, limiting their use. In this study, a library consisting of 75% of all possible single-site mutants of the 101-residue, homodimeric Escherichia coli toxin CcdB was constructed. Mutants were characterized in terms of their activity at two different temperatures and at six different expression levels. Of the total of 1430 single-site mutants that were screened, 231 (16%) mutants showed a ts phenotype. The bulk of these consisted of 120 ts mutants found at all 22 buried sites and 34 ts mutants at all seven active site residues involved in binding DNA gyrase. Of the remaining ts mutants, 16 were found at residues in van der Waals contact with active site residues, 36 were at partially buried residues, and 30 resulted from introduction of Pro. Thus virtually all ts mutants could be rationalized in terms of the structure of the native protein and without knowledge of folding pathways. Data were analyzed to obtain insights into molecular features responsible for the ts phenotype and to outline structure- and sequence-based criteria for designing ts mutants of any globular protein. The criteria were validated by successful prediction of ts mutants of three other unrelated proteins, TBP, T4 lysozyme, and Gal4.

Toxin-antitoxin (TA) systems are found on both bacterial plasmids and chromosomes, but in most cases their functional role is unclear. Gene knockouts often yield limited insights into functions of individual TA systems because of their redundancy. The well-characterized F-plasmid–based CcdAB TA system is important for F-plasmid maintenance. We have isolated several point mutants of the toxin CcdB that fail to bind to its cellular target, DNA gyrase, but retain binding to the antitoxin, CcdA. Expression of such mutants is shown to result in release of the WT toxin from a functional preexisting TA complex as well as derepression of the TA operon. One such inactive, active-site mutant of CcdB was used to demonstrate the contribution of CcdB to antibiotic persistence. Transient activation of WT CcdB either by coexpression of the mutant or by antibiotic/heat stress was shown to enhance the generation of drug-tolerant persisters in a process dependent on Lon protease and RecA. An F-plasmid containing a ccd locus can, therefore, function as a transmissible persistence factor.