Spinal muscular atrophies (SMA, also known as hereditary motor neuropathies) and hereditary motor and sensory neuropathies (HMSN) are clinically and genetically heterogeneous disorders of the peripheral nervous system. Here we report that mutations in the TRPV4 gene cause congenital © 2009 Nature America, Inc. All rights reserved.Correspondence should be addressed to M.A.-G. (michaela.auergrumbach@medunigraz.at).. METHODS: Methods and any associated references are available in the online version of the paper at http://www.nature.com/ naturegenetics/. Accession codes. GenBank: human TRPV4 cDNA, NM_021625; human TRPV4, NP_067638 IsoA. Pfam: ankyrin repeat, PF00023.Note: Supplementary information is available on the Nature Genetics website. AUTHOR CONTRIBUTIONS: M.A.-G., S.U., J.S., M.E.M., A.H.C., K.J.D., C.M.A.v.R.-A., N.E.A., H.L., B.S.-W., R.P., C.L., G.W.P., H.J.S., H.K. and T.R.P. recruited the study participants, acquired clinical data, conducted neurological and neurophysiological evaluations and performed linkage analysis. M.A.-G, C.G., L.P. and C.F. carried out the Affymetrix array linkage studies and identified the mutations. A.O., Z.B. and B.T. designed, carried out and analyzed the electrophysiological and Ca 2+ -imaging studies. E.F. conducted immunofluorescence and immunohistochemistry studies. H.S. conducted fluorescence-activated cell sorting (FACS) and biotinylation studies. A.K. performed structural biology and biocomputing analyses. A.H.C., M.E.M. and H.K. participated in the data analysis and reviewed the manuscript. M.A.-G. and C.G. analyzed the data, designed and supervised the study and wrote the manuscript. Supplementary Fig. 1) and observed linkage to three chromosomal regions with log 10 of odds (lod) scores >2 for several SNP markers, including the chromosome 12q23-24 region (data not shown). We constructed haplotypes by including additional distantly related family members (right branch of the pedigree; Supplementary Fig. 1). The genetic interval transmitted with the disease resides between SNPs rs2374688 and rs35426 (Chr. 12: 106,197,054,429 bp; Supplementary Table 1) and overlaps with the intervals reported for risk of congenital distal SMA, SPSMA and HMSN2C 2-4 . Europe PMC Funders GroupIn an affected individual from family FAM_1, we began sequencing all protein-coding exons and exon-intron boundaries of 19 genes but initially observed only known SNPs (Supplementary Table 2). However, sequencing of all protein-coding exons of TRPV4 (transient receptor potential vanilloid 4; chr. 12: 108,705,277-108,755,595; reverse strand) revealed a heterozygous C-to-T nucleotide change at position 943 in exon 6 (Supplementary Fig. 2a), which is predicted to cause the substitution of arginine with tryptophan at position 315 of TRPV4 (R315W). We then screened DNA samples from additional families showing one of the phenotypes described above, including two families previously reported 1,3,4 . All affected individuals from the chromosome 12q23-24-linked family (here called FAM_2) described by...

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The alpha-1,4-D-glucan phosphorylase from gram-positive Corynebacterium callunae has been isolated and characterized. The enzyme is inducible approx. 2-fold by maltose, but remarkably not repressed by D-glucose. The phosphorylase is a homodimer with a stoichiometric content of the cofactor pyridoxal 5'-phosphate per 88-kDa protein subunit. The specificity constants (kcat/Km, glucan) in the directions of glucan synthesis and degradation are used for the classification of the enzyme as the first bacterial starch phosphorylase. A preference for large over small substrates is determined by variations in the apparent binding constants rather than catalytic-centre activities. The contribution of substrate chain length to binding energy is explained assuming two glucan binding sites in C. callunae phosphorylase: an oligosaccharide binding site composed of five subsites and a high-affinity polysaccharide site separated from the active site. A structural model of the molecular shape of the phosphorylase was obtained from small-angle solution X-ray scattering measurements. A flat, slightly elongated, ellipsoidal model with the three axes related to each other as 1:(0.87-0.95):0.43 showed scattering equivalence with the enzyme molecule. The model of C. callunae phosphorylase differs from the structurally well-characterized rabbit-muscle phosphorylase in size and axial dimensions.

Microtubules are essential cytoskeletal structures that mediate several dynamic processes in a cell. To shed light on the structural processes relating to microtubule formation and dynamic instability, we investigated microtubules composed of 15 protofilaments using cryo-electron microscopy, helical image reconstruction and computational modelling. Analysis of the configuration of the ab-tubulin heterodimer shows distinct structural differences in both subunits, and illustrates that the tubulin subunits have different roles in the microtubule lattice. Our modelling data suggest that after GTP hydrolysis microtubules, adopt a conformational state somewhere between a straight protofilament conformation-as found in zinc-induced tubulin sheets-and an outward curved conformation-as found in tubulin-stathmin complexes. The tendency towards a curved conformation seems to be mediated mostly by b-tubulin, whereas a-tubulin resembles a state more related to the straight structure. Our data suggest a possible explanation of dynamic instability of microtubules, and for nucleotide-sensitive microtubule-binding properties of microtubule-associated proteins and molecular motors.

G-protein-coupled receptors are integral membrane proteins that respond to environmental signals and initiate signal transduction pathways, which activate cellular processes. Rhodopsin, a well known member of the G-protein-coupled receptor family, is located in the disk membranes of the rod outer segment, where it is responsible for the visualization of dim light. Rhodopsin is the most extensively studied G-protein-coupled receptor, and knowledge about its structure serves as a template for other related receptors. We have gained detailed structural knowledge from the crystal structure (1), which was solved by x-ray crystallography in 2000 using three-dimensional crystals. Here we report a three-dimensional density map of bovine rhodopsin determined by electron cryomicroscopy of two-dimensional crystals with p22 1 2 1 symmetry. The usage of relatively small and disordered crystals made the process of structure determination challenging. Special attention was paid to the extraction of amplitudes and phases, since usable raw data were limited to a maximum tilt of 45°. In the refinement process, an improved unbending procedure was applied. This led to a final resolution of 5.5 Å in the membrane plane and ϳ13 Å perpendicular to it, making our electron density map the most accurate map of a G-protein-coupled receptor currently available by electron microscopy. Most important is the information we gain about the center of the membrane plane and the orientation of the molecule relative to the bilayer. This information cannot be retrieved from the three-dimensional crystals. In our electron density map, all seven transmembrane helices were identified, and their arrangement is in agreement with the arrangement known from the crystal structure (1). In the retinal binding pocket, a density peak adjacent to helix 3 suggests the position of the ␤-ionine ring of the chromophore, and in its vicinity several of the bigger amino acids can be identified. G-protein-coupled receptors (GPCRs)1 are a large group of integral membrane proteins that provide molecular links between extracellular signals and intracellular processes (2-7). Many neurotransmitters, hormones, and drugs produce their intracellular signaling through the mediation of G-protein-coupled receptors. The binding of a hormone or neurotransmitter causes a change in the structure of the receptor, which then activates a G-protein. Different members of the receptor family respond to different ligands, and the binding site for these ligands is in the membrane-embedded part of the protein (8, 9).One of the most widely studied GPCRs is rhodopsin (1, 10 -18). Rhodopsin, located in the retina disc membrane of the eye, is responsible for the visualization of dim light. It is composed of the protein opsin (ϳ40 kDa) covalently linked to 11-cis-retinal through Lys 296 of helix 7 (19). The chromophore 11-cis-retinal isomerizes upon light activation to its all-trans conformation, which changes the arrangement of the transmembrane helices (20). This triggers the signal transduction c...