A regulatory component of the human ryanodine receptor 2 N-terminus

Human cardiac ryanodine receptor (hRyR2) is a channel mediating Ca2+ release from the sarcoplasmic reticulum during excitation-contraction coupling. The N-terminal (1-655) and central (2100-2500) regions of hRyR2 are thought to be involved in regulating channel gating. Mutations linked to several heart diseases are clustered within these two, as well as in the channel pore-containing C-terminal regions. High resolution structures of key regions involved in the regulation of RyR2 activity could further the understanding of the gating mechanism of hRyR2 and of its malfunction in disease

Manipulating Conserved Heme Cavity Residues of Chlorite Dismutase: Effect on Structure, Redox Chemistry and Reactivity

Chlorite dismutases (Clds) are heme b containing oxidoreductases that convert chlorite to chloride and molecular oxygen. In order to elucidate the role of conserved heme cavity residues in the catalysis of this reaction comprehensive mutational and biochemical analyses of Cld from \u201cCandidatus Nitrospira defluvii\u201d (NdCld) were performed. Particularly, point mutations of the cavity-forming residues R173, K141, W145, W146, and E210 were performed. The effect of manipulation in 12 single and double mutants was probed by UV\u2013vis spectroscopy, spectroelectrochemistry, pre-steady-state and steady-state kinetics, and X-ray crystallography. Resulting biochemical data are discussed with respect to the known crystal structure of wild-type NdCld and the variants R173A and R173K as well as the structures of R173E, W145V, W145F, and the R173Q/W146Y solved in this work. The findings allow a critical analysis of the role of these heme cavity residues in the reaction mechanism of chlorite degradation that is proposed to involve hypohalous acid as transient intermediate and formation of an O\u2550O bond. The distal R173 is shown to be important (but not fully essential) for the reaction with chlorite, and, upon addition of cyanide, it acts as a proton acceptor in the formation of the resulting low-spin complex. The proximal H-bonding network including K141-E210-H160 keeps the enzyme in its ferric (E\ub0\u2032 = 12113 mV) and mainly five-coordinated high-spin state and is very susceptible to perturbation

The Structure and Regulation of Human Muscle α-Actinin

SummaryThe spectrin superfamily of proteins plays key roles in assembling the actin cytoskeleton in various cell types, crosslinks actin filaments, and acts as scaffolds for the assembly of large protein complexes involved in structural integrity and mechanosensation, as well as cell signaling. α-actinins in particular are the major actin crosslinkers in muscle Z-disks, focal adhesions, and actin stress fibers. We report a complete high-resolution structure of the 200 kDa α-actinin-2 dimer from striated muscle and explore its functional implications on the biochemical and cellular level. The structure provides insight into the phosphoinositide-based mechanism controlling its interaction with sarcomeric proteins such as titin, lays a foundation for studying the impact of pathogenic mutations at molecular resolution, and is likely to be broadly relevant for the regulation of spectrin-like proteins

Heads decide: Structural and functional characterization of selected plectin isoform-specific sequences

Plektin ist ein großes und höchst vielseitiges Protein, welches Aktin, Tubulin und einige andere Intermediärfilamente querverlinkt, und dadurch einen großen Einfluss auf die zeitlichen und örtlichen Arragements und Dynamiken nimmt. Aufgrund von alternativem Splicing wird Plektin in verschiedenen Isoformen exprimiert, die sich im N-terminalen Bereich unterscheiden, welcher wiederum die subzelluläre Verwendungsstellen diktiert. Allerdings versteht man die Mechanismen noch wenig, wie der N-terminale Bereich dieses riesige Protein zu verschiedenen Orten und/oder Bindungspartner dirigiert und dadurch die Funktion abstimmt; und diese Mechanismen müssen dementsprechend noch aufgeklärt werden. Um nun die strukturellen Eigenschaften von Plektin Isoformen 1a- und 1c-spezifischen Sequenzen zu untersuchen und diese in Verbindung mit ihrer Funktion zu bringen, haben wir Methoden zur Aufreingung von Plektinfragmenten etabliert, bestehend aus diesen Sequenzen fusioniert mit der ABD von Plektin oder mit längeren N-terminalen Bereichen von Plektin, unter anderem mit dessen Plakin Domäne. Die Kristallisation dieser Fragmente war nicht erfolgreich, was darauf hindeutet, dass die für Plektin Isoformen 1a und 1c spezifischen Sequenzen sehr flexible oder ungeordnet sind, zumindest im ungebundenen Zustand. Im zweiten Teil wurden biochemische und zellbiologische Herangehensweisen benutzt, um neue Bindungspartner zu identifizieren, die bevorzugt an den N-terminalen Teil binden. Eines der beiden neu identifizierten Bindungspartnern, Vimentin, ein Intermediärfilament, bindet den N-terminalen Bereich der ABD von Plektin in seiner löslichen tetrameren Form. Zusätzlich wurde eine isoformspezifische Interaktion von Plektin mit Calmodulin, ein Ca2+ wahrnehmendes Protein, charakterisiert. Wir zeigen, dass Calmodulin an die CH1 Domäne von Plektin Isoform 1a in einer Ca2+ abhängigen Weise bindet. Weiters wurde gezeigt, dass die Bindung von Calmodulin/Ca2+ an Plektin seine Bindung an F-Aktin und Integrin β4 verhindert. Interessanterweise spielt die Interaktion von Integrin α6β4 mit Plektin eine große Rolle in der Ausbildung von Hemidesmosomen, das sind Adhäsionskomplexe multipler Proteine, die Epithelzellen mit der basalen Matrix verbinden. Darüberhinaus war die bisher gängige Meinung zur Loslösung der Epithelzellen von der basalen Matrix während der Wundheilung und die Differenzierung von Keratinozyten, welche die Disassemblierung der Hemidesmosomen benötigen, dass diese nur durch Phosphorylierung von Integrin β4 durch Proteinkinasen bewerkstelligt werden. Unsere Resultate erlauben uns ein neues Modell für die Disassemblierung der Hemidesmosomen während der Differenzierung der Keratinozyten vorzuschlagen, welches sowohl die Bindung von Calmodulin an Plektin 1a als auch die Phosphorylierung von Integrin β4 benötigt, um den Komplex von Integrin α6β4 mit Plektin unterbricht.Plectin is a large and highly versatile cytolinker protein, which cross-links actin, tubulin, and various intermediate filament proteins, having a great impact on their spatial-temporal arrangements and dynamics. Due to alternative splicing, plectin is expressed as various isoforms with differing N-terminal heads, which dictate subcellular targeting of the protein. However, detailed mechanism(s) how plectin’s heads direct the giant protein to different location and/or binding partners and thus modulate its function is not well understood yet, and needs to be, elucidated. To investigate structural properties of plectin 1a and plectin 1c isoform specific sequences and relate them to their function, we established method(s) for purification of tagged and un-tagged plectin fragments comprising these sequences fused to plectin’s ABD or to longer N-terminal parts of plectin including its plakin domain as well. Crystallization of these fragments was not successful, and indicated that plectin 1a and plectin 1c isoform specific sequences might be highly flexible or disordered, at least in their unbound state. In the second part biochemical and cell biological approaches were used to identify novel binding partners of plectin interacting preferentially with its N-terminal part. Of the two novel binding partners identified, vimentin, an intermediate filament protein, was found to interact with the N-terminal part of plectin’s ABD in its soluble “tetrameric” form. In addition, isoform specific interaction of plectin with calmodulin, a Ca2+-sensing protein, has been characterized. Calmodulin was showed to bind to the CH1 domain of plectin 1a in a Ca2+- and isoform-dependent fashion. Furthermore, binding of Ca2+/calmodulin to plectin 1a was found to prevent its binding to F-actin, and integrin β4. Interestingly, the interaction of integrin α6β4 with plectin 1a plays a major role in the formation of hemidesmosomes, multiprotein adhesion complexes, connecting epithelial cells to the basal matrix. Moreover, the detachment of epithelial cells from the basal matrix during wound healing and differentiation of keratinocytes requires the disassembly of the hemidesmosomal complexes, which formerly was believed to be mediated only by phosphorylation of integrin β4 by protein kinases. Thus our results allowed us to propose a novel model for the disassembly of hemidesmosomes during keratinocyte differentiation, where both, binding of calmodulin to plectin 1a and phosphorylation of integrin β4, are required for disruption of the integrin α6β4- plectin complex

Structures of three MORN repeat proteins and a re-evaluation of the proposed lipid-binding properties of MORN repeats

UK Research and Innovation | Medical Research Council (MRC):Terry K. Smith MR/Mo20118/1.MORN (Membrane Occupation and Recognition Nexus) repeat proteins have a wide taxonomic distribution, being found in both prokaryotes and eukaryotes. Despite this ubiquity, they remain poorly characterised at both a structural and a functional level compared to other common repeats. In functional terms, they are often assumed to be lipid-binding modules that mediate membrane targeting. We addressed this putative activity by focusing on a protein composed solely of MORN repeats—Trypanosoma brucei MORN1. Surprisingly, no evidence for binding to membranes or lipid vesicles by TbMORN1 could be obtained either in vivo or in vitro. Conversely, TbMORN1 did interact with individual phospholipids. High- and low-resolution structures of the MORN1 protein from Trypanosoma brucei and homologous proteins from the parasites Toxoplasma gondii and Plasmodium falciparum were obtained using a combination of macromolecular crystallography, small-angle X-ray scattering, and electron microscopy. This enabled a first structure-based definition of the MORN repeat itself. Furthermore, all three structures dimerised via their C-termini in an antiparallel configuration. The dimers could form extended or V-shaped quaternary structures depending on the presence of specific interface residues. This work provides a new perspective on MORN repeats, showing that they are protein-protein interaction modules capable of mediating both dimerisation and oligomerisation.Publisher PDFPeer reviewe

The structure and DNA-binding properties of Mgm101 from a yeast with a linear mitochondrial genome

To study the mechanisms involved in the maintenance of a linear mitochondrial genome we investigated the biochemical properties of the recombination protein Mgm101 from Candida parapsilosis. We show that CpMgm101 complements defects associated with the Saccharomyces cerevisiae mgm101-1$^{ts}$ mutation and that it is present in both the nucleus and mitochondrial nucleoids of C. parapsilosis. Unlike its S. cerevisiae counterpart, CpMgm101 is associated with the entire nucleoid population and is able to bind to a broad range of DNA substrates in a non-sequence specific manner. CpMgm101 is also able to catalyze strand annealing and D-loop formation. CpMgm101 forms a roughly C-shaped trimer in solution according to SAXS. Electron microscopy of a complex of CpMgm101 with a model mitochondrial telomere revealed homogeneous, ring-shaped structures at the telomeric single-stranded overhangs. The DNA-binding properties of CpMgm101, together with its DNA recombination properties, suggest that it can play a number of possible roles in the replication of the mitochondrial genome and the maintenance of its telomeres