Structural anisotropy results in mechano-directional transport of proteins across nuclear pores.

The nuclear pore complex regulates nucleocytoplasmic transport by means of a tightly synchronized suite of biochemical reactions. The physicochemical properties of the translocating cargos are emerging as master regulators of their shuttling dynamics. As well as being affected by molecular weight and surface-exposed amino acids, the kinetics of the nuclear translocation of protein cargos also depend on their nanomechanical properties, yet the mechanisms underpinning the mechanoselectivity of the nuclear pore complex are unclear. Here we show that proteins with locally soft regions in the vicinity of the nuclear-localization sequence exhibit higher nuclear-import rates, and that such mechanoselectivity is specifically impaired upon knocking down nucleoporin 153, a key protein in the nuclear pore complex. This allows us to design a short, easy-to-express and chemically inert unstructured peptide tag that accelerates the nuclear-import rate of stiff protein cargos. We also show that U2OS osteosarcoma cells expressing the peptide-tagged myocardin-related transcription factor import this mechanosensitive protein to the nucleus at higher rates and display faster motility. Locally unstructured regions lower the free-energy barrier of protein translocation and might offer a control mechanism for nuclear mechanotransduction.

The mechanical stability of proteins regulates their translocation rate into the cell nucleus.

The translocation of mechanosensitive transcription factors (TFs) across the nuclear envelope is a crucial step in cellular mechanotransduction. Yet the molecular mechanisms by which external mechanical cues control the nuclear shuttling dynamics of TFs through the nuclear pore complex (NPC) to activate gene expression are poorly understood. Here, we show that the nuclear import rate of myocardin-related transcription factor A (MRTFA) - a protein that regulates cytoskeletal dynamics via the activation of the TF serum response factor (SRF) - inversely correlates with the protein's nanomechanical stability and does not relate to its thermodynamic stability. Tagging MRTFA with mechanically resistant proteins results in the downregulation of SRF-mediated myosin light-chain 9 (MYL9) gene expression and subsequent slowing down of cell migration. We conclude that the mechanical unfolding of proteins regulates their nuclear translocation rate through the NPC, and highlight the role of the NPC as a selective mechanosensor able to discriminate forces as low as ~10 pN. The modulation of the mechanical stability of TFs may represent a new strategy for the control of gene expression.

Solution NMR assignment of the C-terminal domain of human chTOG.

The microtubule regulatory protein colonic and hepatic tumor overexpressed gene (chTOG), also known as cytoskeleleton associated protein 5 (CKAP5) plays an important role in organizing the cytoskeleton and in particular in the assembly of k-fibres in mitosis. Recently, we dissected the hitherto poorly understood C-terminus of this protein by discovering two new domains-a cryptic TOG domain (TOG6) and a smaller, helical domain at the very C-terminus. It was shown that the C-terminal domain is important for the interaction with the TACC domain in TACC3 during the assembly of k-fibres in a ternary complex that also includes clathrin. Here we now present the solution NMR assignment of the chTOG C-terminal domain which confirms our earlier prediction that it is mainly made of α-helices. However, the appearance of the 1H-15N HSQC spectrum is indicative of the presence of a considerable amount of unstructured and possibly flexible portions of protein in the domain.

Solution NMR assignment of the heavy chain complex of the human cardiac myosin regulatory light chain.

The regulatory light chain (RLC) of striated and cardiac muscle myosin plays a complex role in muscle function and regulation. Together with the essential light chain it provides stability to the lever arm, which is essential for force generation. Furthermore, phosphorylation and interaction with myosin binding protein C (MyBP-C) suggest an additional role in the regulation of muscle contraction. The former is of particular importance in the heart, where RLC phosphorylation appears to be correlated to the wringing motion of heart contraction. To address these questions and because of a lack of mammalian RLC structures, we initiated an NMR study of the human cardiac regulatory myosin light chain.

Structure and interactions of myosin-binding protein C domain C0: cardiac-specific regulation of myosin at its neck?

Myosin-binding protein C (MyBP-C) is a multidomain protein present in the thick filaments of striated muscles and is involved in both sarcomere formation and contraction regulation. The latter function is believed to be located at the N terminus, which is close to the motor domain of myosin. The cardiac isoform of MyBP-C is linked to hypertrophic cardiomyopathy. Here, we use NMR spectroscopy and biophysical and biochemical assays to study the three-dimensional structure and interactions of the cardiac-specific Ig-like domain C0, a part of cardiac MyBP-C of which little is known. The structure confirmed that C0 is a member of the IgI class of proteins, showing many of the characteristic features of this fold. Moreover, we identify a novel interaction between C0 and the regulatory light chain of myosin, thus placing the N terminus of the protein in proximity to the motor domain of myosin. This novel interaction is disrupted by several cardiomyopathy-linked mutations in the MYBPC3 gene. These results provide new insights into how cardiac MyBP-C incorporates in the sarcomere and how it can contribute to the regulation of muscle contraction.

Myosin binding protein C positioned to play a key role in regulation of muscle contraction: structure and interactions of domain C1.

Myosin binding protein C (MyBP-C) is a thick filament protein involved in the regulation of muscle contraction. Mutations in the gene for MyBP-C are the second most frequent cause of hypertrophic cardiomyopathy. MyBP-C binds to myosin with two binding sites, one at its C-terminus and another at its N-terminus. The N-terminal binding site, consisting of immunoglobulin domains C1 and C2 connected by a flexible linker, interacts with the S2 segment of myosin in a phosphorylation-regulated manner. It is assumed that the function of MyBP-C is to act as a tether that fixes the S1 heads in a resting position and that phosphorylation releases the S1 heads into an active state. Here, we report the structure and binding properties of domain C1. Using a combination of site-directed mutagenesis and NMR interaction experiments, we identified the binding site of domain C1 in the immediate vicinity of the S1-S2 hinge, very close to the light chains. In addition, we identified a zinc binding site on domain C1 in close proximity to the S2 binding site. Its zinc binding affinity (K(d) of approximately 10-20 microM) might not be sufficient for a physiological effect. However, the familial hypertrophic cardiomyopathy-related mutation of one of the zinc ligands, glutamine 210 to histidine, will significantly increase the binding affinity, suggesting that this mutation may affect S2 binding. The close proximity of the C1 binding site to the hinge, the light chains and the S1 heads also provides an explanation for recent observations that (a) shorter fragments of MyBP-C unable to act as a tether still have an effect on the actomyosin ATPase and (b) as to why the myosin head positions in phosphorylated wild-type mice and MyBP-C knockout mice are so different: Domain C1 bound to the S1-S2 hinge is able to manipulate S1 head positions, thus influencing force generation without tether. The potentially extensive extra interactions of C1 are expected to keep it in place, while phosphorylation dislodges the C1-C2 linker and domain C2. As a result, the myosin heads would always be attached to a tether that has phosphorylation-dependent length regulation.

Activation of myocardial contraction by the N-terminal domains of myosin binding protein-C.

Myosin binding protein-C (MyBP-C) is a poorly understood component of the thick filament in striated muscle sarcomeres. Its C terminus binds tightly to myosin, whereas the N terminus contains binding sites for myosin S2 and possibly for the thin filament. To study the role of the N-terminal domains of cardiac MyBP-C (cMyBP-C), we added human N-terminal peptide fragments to human and rodent skinned ventricular myocytes. At concentrations >10 micromol/L, the N-terminal C0C2 peptide activated force production in the absence of calcium (pCa 9). Force at the optimal concentration (80 micromol/L) of C0C2 was approximately 60% of that in maximal Ca2+ (pCa 4.5), but the rate constant of tension redevelopment (ktr) matched or exceeded (by up to 80%) that produced by Ca2+ alone. Experiments using different N-terminal peptides suggested that this activating effect of C0C2 resulted from binding by the pro/ala-rich C0-C1 linker region, rather than the terminal C0 domain. At a lower concentration (1 micromol/L), exogenous C0C2 strongly sensitized cardiac myofibrils to Ca2+ at a sarcomere length (SL) of 1.9 microm but had no significant effect at SL 2.3 microm. This differential effect caused the normal SL dependence of myofibrillar Ca2+ sensitivity to be reduced by 80% (mouse myocytes) or abolished (human myocytes) in 1 micromol/L C0C2. These results suggest that cMyBP-C provides a regulatory pathway by which the thick filament can influence the activation of the thin filament, separately from its regulation by Ca2+. Furthermore, the N-terminal region of cMyBP-C can influence the SL-tension (Frank-Starling) relationship in cardiac muscle.

The kinase domain of titin controls muscle gene expression and protein turnover.

The giant sarcomeric protein titin contains a protein kinase domain (TK) ideally positioned to sense mechanical load. We identified a signaling complex where TK interacts with the zinc-finger protein nbr1 through a mechanically inducible conformation. Nbr1 targets the ubiquitin-associated p62/SQSTM1 to sarcomeres, and p62 in turn interacts with MuRF2, a muscle-specific RING-B-box E3 ligase and ligand of the transactivation domain of the serum response transcription factor (SRF). Nuclear translocation of MuRF2 was induced by mechanical inactivity and caused reduction of nuclear SRF and repression of transcription. A human mutation in the titin protein kinase domain causes hereditary muscle disease by disrupting this pathway.

Binding of myosin binding protein-C to myosin subfragment S2 affects contractility independent of a tether mechanism.

Mutations in the cardiac myosin binding protein-C gene (cMyBP-C) are among the most prevalent causes of inherited hypertrophic cardiomyopathy. Although most cMyBP-C mutations cause reading frameshifts that are predicted to encode truncated peptides, it is not known if or how expression of these peptides causes disease. One possibility is that because the N-terminus contains a unique binding site for the S2 subfragment of myosin, shortened cMyBP-C peptides could directly affect myosin contraction by binding to S2. To test this hypothesis, we compared the effects of a C1C2 protein containing the myosin S2 binding site on contractile properties in permeabilized myocytes from wild-type and cMyBP-C knockout mice. In wild-type myocytes, the C1C2 protein reversibly increased myofilament Ca2+ sensitivity of tension, but had no effect on resting tension. Identical results were observed in cMyBP-C knockout myocytes where C1C2 increased Ca2+ sensitivity of tension with the half-maximal response elicited at approximately 5 micromol/L C1C2. Maximum force was not affected by C1C2. However, phosphorylation of C1C2 by cAMP-dependent protein kinase reduced its ability to increase Ca2+ sensitivity. These results demonstrate that binding of the C1C2 peptide to S2 alone is sufficient to affect myosin contractile function and suggest that regulated binding of cMyBP-C to myosin S2 by phosphorylation directly influences myofilament Ca2+ sensitivity.

Chemo-Enzymatic Synthesis of Fluorescent Rab 7 Proteins: Tools to Study Vesicular Trafficking in Cells.

The N-methylanthraniloylisoprenoid diphosphate derivatives 1 and 2 bind to RabGGTase II and are enzymatically transferred to Rab 7 (the Rab proteins are small G-proteins that control events of docking and fusion of intracellular vesicles). The fluorescent Rab 7 proteins thus obtained may become important tools for further biological studies on vesicular trafficking in cells.