Collagens have long been believed to adopt a triple-stranded molecular structure with a 10/3 symmetry (ten triplet units in three turns) and an axial repeat of 29 Å. This belief even persisted after an alternative structure with a 7/2 symmetry (seven triplet units in two turns) with an axial repeat of 20 Å had been proposed. The uncertainty regarding the helical symmetry of collagens is attributed to inadequate X-ray fiber diffraction data. Therefore, for better understanding of the collagen helix, single-crystal analyses of peptides with simplified characteristic amino acid sequences and similar compositions to collagens have long been awaited. Here we report the crystal structure of (Gly-Pro-Hyp)(9) peptide at a resolution of 1.45 Å. The repeating unit of this peptide, Gly-Pro-Hyp, is the most typical sequence present in collagens, and it has been used as a basic repeating unit in fiber diffraction analyses of collagen. The (Gly-Pro-Hyp)(9) peptide adopts a triple-stranded structure with an average helical symmetry close to the ideal 7/2 helical model for collagen. This observation strongly suggests that the average molecular structure of collagen is not the accepted Rich and Crick 10/3 helical model but is a 7/2 helical conformation.

Background: Hyperelastosis cutis in horses is caused by a homozygous mutation in cyclophilin B (PPIB). Results: This mutation changes protein-protein interactions of CypB and delays folding of collagen. Conclusion: Mutant CypB has PPIase activity, but the synthesized collagen contains less post-translational modifications of lysine residues. Significance: CypB has other important functions besides its PPIase activity. The formation of these interactions is crucial for a correct biosynthesis of collagen.

Escherichia coli apomanganese superoxide dismutase, prepared by removing the native metal ion under denaturing conditions, exhibits thermally triggered metal uptake behavior previously observed for thermophilic and hyperthermophilic superoxide dismutases but over a lower temperature range. Superoxide dismutases (SODs) 1 (E.C. 1.15.1.1) are important antioxidant metalloenzymes protecting cells against oxidative stress arising from reactive oxygen species (1-4). SODs are ubiquitous, and multiple forms often exist within a single organism or cell. Four major forms of SODs (Mn, Fe, Cu/Zn, and Ni) have been identified, distinguished by the protein fold and by the nature of the catalytic metal ion (5-8). Mn-and Fe-SODs share extensive homology in protein structure, whereas the Ni and Cu/Zn enzymes are structurally distinct. Despite their similar structures (9), the Mn-and Fe-SODs exhibit a strict specificity for the catalytic metal ion (manganese or iron). Much lower selectivity is generally observed for metal binding, and the dimeric Mn-SOD from Escherichia coli is typically isolated as a mixture of Mn 2 -, (Mn,Fe)-, Fe 2 -, and half-apoforms (10, 11). Both the manganese-and iron-replete forms may be enriched by supplementing the culture medium with either manganese or iron salts (12). Apo-Mn-SOD binds metals nonselectively in the presence of guanidinium denaturants, and this method has been used to prepare manganese-, iron-, and cobalt-reconstituted forms (13-17).Each subunit of the E. coli Mn-SOD homodimer is composed of two domains, a predominantly ␣-helical N-terminal domain and a mixed ␣/␤ C-terminal domain (18). The metal-binding site lies on the interface between these two domains buried in the interior of the protein. The mononuclear metal ion is coordinated by four amino acid side chains (Fig. 1), two arising from the N-terminal domain (His-26 and His-81) and two from the C-terminal domain (Asp-167 and His-171), resulting in a metal ion cross-link between the two domains. A buried solvent molecule serves as a fifth ligand forming a hydrogen bond in the outer sphere of the metal complex with the amido head group of Glu-146 (E. coli Mn-SOD sequence numbering). The metal ion appears to be very tightly bound, being released only under denaturing conditions and resisting extraction by metal chelators like EDTA (13).Earlier work has shown that metal-free apo-SOD is produced during expression of recombinant thermophilic SODs in the mesophilic host, E. coli. The purified apoprotein has been shown to efficiently take up metal ion in vitro at elevated temperatures, and the binding is essentially irreversible under these conditions. At lower temperatures, metal uptake is not detected (19,20). The sigmoidal temperature profile for metalation of the recombinant thermophilic apo-SODs suggests that a two-state activated process is involved. The requirements for denaturing conditions to remove the metal and thermally triggered metal uptake imply the existence of large activation barriers for changes in the metalation state ...

X-linked ectodermal dysplasia and immunodeficiency (XL-EDA-ID) is an X-linked recessive disease caused by a mutation in the nuclear factor-B (NF-B) essential modulator (NEMO). IntroductionEctodermal dysplasia and immunodeficiency (EDA-ID) is a disease whose clinical features include hypohidrosis, delay of eruption of teeth, coarse hair, and immunodeficiency associated with frequent bacterial infections. [1][2][3][4][5] Two genes responsible for EDA-ID have been identified: nuclear factor-B (NF-B) essential modulator (NEMO; in X-linked-EDA-ID [XL-EDA-ID]) [6][7][8] and IB (in autosomal-dominant EDA-ID). 9 NEMO is necessary for the function of IB kinase, which phosphorylates and degrades IB to activate NF-B. 10 Thus, the defect in NEMO causes various kinds of abnormalities in signal transduction involving NF-B, the interleukin 1 (IL-1) family protein receptors, the Toll-like receptors, vascular endothelial growth factor receptor-3 (VEGFR-3), receptor activator of nuclear factor B (RANK), the ectodysplasin-A receptor, CD40, and the tumor necrosis factor (TNF) receptor. 11 NEMO is also responsible for X-linked-dominant incontinentia pigmenti (IP). 12 Males with IP usually die before birth. The XL-EDA-ID cases reported so far have also been male (with one exception 13 ) but have only one mutated NEMO allele. Residual NEMO activity in XL-EDA-ID and a total lack of NEMO activity in IP can explain the phenotype differences in these 2 populations of males with NEMO defects. 7,8,12 Furthermore, the majority of NEMO mutations in IP patients are large deletions due to recombination, while XL-EDA-ID patients have small mutations such as missense mutations, early stop codons, and stop codon mutations. 12,14 The immunologic features of XL-EDA-ID reported so far consist of dysregulated immunoglobulin synthesis or hyperimmunoglobulin M (hyper-IgM) syndrome, defective antipolysaccharide antibody synthesis (antipneumococcal antibody and isohemagglutinin), reduced lipopolysaccharide (LPS) and IL-1 family protein responses, and defective natural killer (NK) cell activity. 3,4,[6][7][8][15][16][17][18][19] Complete loss of NEMO function is lethal in mice due to liver failure, 20 but studies using conditional knockout mice or recombination-activating gene (RAG) chimera reconstitution have suggested that T and B cells do not develop in the complete absence of NEMO in the mouse. [21][22][23][24] Although XL-EDA-ID is phenotypically different in individuals with different NEMO mutations, there have been no XL-EDA-ID cases reported so far that show a role for NEMO in T-cell development and survival. Now we report a patient with a novel type of XL-EDA-ID whose NEMO expression varied among cell lineages due to reversion mosaicism of a 4.4-kb duplication of a portion of the NEMO gene. The patient provided us with a unique opportunity to elucidate NEMO biology in humans because in this patient we could correlate the NEMO level with cell function in various cell types. In Reprints: Tatsutoshi Nakahata, 54 Shogoin Kawahara-cho, Sakyo, Kyoto 606...