Nikos Pinotsis

Palindromic assembly of the giant muscle protein titin in the sarcomeric Z-disk

The Z-disk of striated and cardiac muscle sarcomeres is one of the most densely packed cellular structures in eukaryotic cells. It provides the architectural framework for assembling and anchoring the largest known muscle filament systems by an extensive network of protein–protein interactions, requiring an extraordinary level of mechanical stability. Here we show, using X-ray crystallography, how the amino terminus of the longest filament component, the giant muscle protein titin, is assembled into an antiparallel (2:1) sandwich complex by the Z-disk ligand telethonin. The pseudosymmetric structure of telethonin mediates a unique palindromic arrangement of two titin filaments, a type of molecular assembly previously found only in protein–DNA complexes. We have confirmed its unique architecture in vivo by protein complementation assays, and in vitro by experiments using fluorescence resonance energy transfer. The model proposed may provide a molecular paradigm of how major sarcomeric filaments are crosslinked, anchored and aligned within complex cytoskeletal networks.

The binding of β- and γ-cyclodextrins to glycogen phosphorylase b: Kinetic and crystallographic studies

A number of regulatory binding sites of glycogen phosphorylase (GP), such as the catalytic, the inhibitor, and the new allosteric sites are currently under investigation as targets for inhibition of hepatic glycogenolysis under high glucose concentrations; in some cases specific inhibitors are under evaluation in human clinical trials for therapeutic intervention in type 2 diabetes. In an attempt to investigate whether the storage site can be exploited as target for modulating hepatic glucose production, α‐, β‐, and γ‐cyclodextrins were identified as moderate mixed‐type competitive inhibitors of GPb (with respect to glycogen) with Ki values of 47.1, 14.1, and 7.4 mM, respectively. To elucidate the structural basis of inhibition, we determined the structure of GPb complexed with β‐ and γ‐cyclodextrins at 1.94 Å and 2.3 Å resolution, respectively. The structures of the two complexes reveal that the inhibitors can be accommodated in the glycogen storage site of T‐state GPb with very little change of the tertiary structure and provide a basis for understanding their potency and subsite specificity. Structural comparisons of the two complexes with GPb in complex with either maltopentaose (G5) or maltoheptaose (G7) show that β‐ andγ‐cyclodextrins bind in a mode analogous to the G5 and G7 binding with only some differences imposed by their cyclic conformations. It appears that the binding energy for stabilization of enzyme complexes derives from hydrogen bonding and van der Waals contacts to protein residues. The binding of α‐cyclodextrin and octakis (2,3,6‐tri‐O‐methyl)‐γ‐cyclodextrin was also investigated, but none of them was bound in the crystal; moreover, the latter did not inhibit the phosphorylase reaction.

Molecular basis of the C-terminal tail-to-tail assembly of the sarcomeric filament protein myomesin.

Sarcomeric filament proteins display extraordinary properties in terms of protein length and mechanical elasticity, requiring specific anchoring and assembly mechanisms. To establish the molecular basis of terminal filament assembly, we have selected the sarcomeric M‐band protein myomesin as a prototypic filament model. The crystal structure of the myomesin C‐terminus, comprising a tandem array of two immunoglobulin (Ig) domains My12 and My13, reveals a dimeric end‐to‐end filament of 14.3 nm length. Although the two domains share the same fold, an unexpected rearrangement of one β‐strand reveals how they are evolved into unrelated functions, terminal filament assembly (My13) and filament propagation (My12). The two domains are connected by a six‐turn α‐helix, of which two turns are void of any interactions with other protein parts. Thus, the overall structure of the assembled myomesin C‐terminus resembles a three‐body beads‐on‐the‐string model with potentially elastic properties. We predict that the found My12‐helix‐My13 domain topology may provide a structural template for the filament architecture of the entire C‐terminal Ig domain array My9–My13 of myomesin.

Fast-folding α-helices as reversible strain absorbers in the muscle protein myomesin

The highly oriented filamentous protein network of muscle constantly experiences significant mechanical load during muscle operation. The dimeric protein myomesin has been identified as an important M-band component supporting the mechanical integrity of the entire sarcomere. Recent structural studies have revealed a long α-helical linker between the C-terminal immunoglobulin (Ig) domains My12 and My13 of myomesin. In this paper, we have used single-molecule force spectroscopy in combination with molecular dynamics simulations to characterize the mechanics of the myomesin dimer comprising immunoglobulin domains My12–My13. We find that at forces of approximately 30 pN the α-helical linker reversibly elongates allowing the molecule to extend by more than the folded extension of a full domain. High-resolution measurements directly reveal the equilibrium folding/unfolding kinetics of the individual helix. We show that α-helix unfolding mechanically protects the molecule homodimerization from dissociation at physiologically relevant forces. As fast and reversible molecular springs the myomesin α-helical linkers are an essential component for the structural integrity of the M band.

The structure of the 2[4Fe-4S] ferredoxin from Pseudomonas aeruginosa at 1.32-A resolution: comparison with other high-resolution structures of ferredoxins and contributing structural features to reduction potential values.

The structure of the 2[4Fe–4S] ferredoxin (PaFd) from Pseudomonas aeruginosa, which belongs to the Allochromatium vinosum (Alvin) subfamily, has been determined by X-ray crystallography at 1.32-Å resolution, which is the highest up to now for a member of this subfamily of Fds. The main structural features of PaFd are similar to those of AlvinFd. However, the significantly higher resolution of the PaFd structure makes possible a reliable comparison with available high-resolution structures of [4Fe–4S]-containing Fds, in an effort to rationalize the unusual electrochemical properties of Alvin-like Fds. Three major factors contributing to the reduction potential values of [4Fe–4S]2+/+ clusters of Fds, namely, the surface accessibility of the clusters, the N–H···S hydrogen-bonding network, and the volume of the cavities hosting the clusters, are extensively discussed. The volume of the cavities is introduced in the present work for the first time, and can in part explain the very negative potential of cluster I of Alvin-like Fds.

Terminal assembly of sarcomeric filaments by intermolecular β-sheet formation

The contraction-relaxation cycle of muscle cells translates into large movements of several filament systems in sarcomeres, requiring special molecular mechanisms to maintain their structural integrity. Recent structural and functional data from three filaments harboring extensive arrays of immunoglobulin-like domains - titin, filamin and myomesin--have, for the first time, unraveled a common function of their terminal domains: assembly and anchoring of the respective filaments. In each case, the protein-protein interactions are mediated by antiparallel dimerization modules via intermolecular beta-sheets. These observations on terminal filament assembly indicate an attractive model for several other filament proteins that require structural characterization.

Structure of muscle α-actinin: Insights into its regulation and Z-disk assembly

1 University of Vienna, Max F. Perutz Laboratories, Department of Structural and Computational Biology, Vienna, Austria, 2 King’s College London BHF Centre for Research Excellence, Randall Division for Cell and Molecular Biophysics and Cardiovascular Division, London, UK, 3 EMBL-Hamburg c/o DESY, Hamburg, Germany, 4 Norwegian University of Science and Technology, Department of Biotechnology, Norway, 5 ETH-Hönggerberg, Laboratory of Physical Chemistry, Zurich, Switzerland, 6 University of Natural Resources and Life Sciences, Division of Biochemistry, Department of Chemistry, Austria, 7 University of Ljubljana, Department of Biochemistry, Faculty of Chemistry and Chemical Technology, Ljubljana, Slovenia