Structural representative of the protein family PF14466 has a new fold and establishes links with the C2 and PLAT domains from the widely distant Pfams PF00168 and PF01477

The domain of unknown function (DUF) YP_001302112.1, a protein secreted by the human intestinal microbita, has been determined by NMR and represents the first structure for the Pfam PF14466. Its NMR structure is classified as a new fold, which, nonetheless, shows limited similarities with representatives of the PLAT/LH2 domains from PF01477 and the C2 domains from PF00168, both of which bind Ca²⁺ for their physiological functions. Further experiments revealed affinity of YP_001302112.1 for Ca²⁺, and the NMR structure in the presence of CaCl₂ was better defined than that of the apo‐protein. Overall, these NMR structures establish a new connection between structural representatives from two widely different Pfams that include the calcium‐binding domain of a sialidase from Vibrio cholerae and the α‐toxin from Clostridium perfrigens, whereby these two proteins have only 7% sequence identity. Furthermore, it provides information toward the functional annotation of YP_001302112.1, based on its capacity to bind Ca²⁺, and thus adds to the structural and functional coverage of the protein sequence universe. © 2013 The Protein Society     

Genome-Wide Identification of DUF26 Domain-Containing Genes in Dongxiang Wild Rice and Analysis of Their Expression Responses under Submergence

The DUF26 domain-containing protein is an extracellular structural protein, which plays an important role in signal transduction. Dongxiang wild rice (Oryza rufipogon Griff.) is the northern-most common wild rice in China. Using domain analysis, 85 DUF26 domain-containing genes were identified in Dongxiang wild rice (DXWR) and further divided into four categories. The DUF26 domain-containing genes were unevenly distributed on chromosomes, and there were 18 pairs of tandem repeats. Gene sequence analysis showed that there were significant differences in the gene structure and motif distribution of the DUF26 domain in different categories. Motifs 3, 8, 9, 13, 14, 16, and 18 were highly conserved in all categories. It was also found that there were eight plasmodesmata localization proteins (PDLPs) with a unique motif 19. Collinearity analysis showed that DXWR had a large number of orthologous genes with wheat, maize, sorghum and zizania, of which 17 DUF26 domain-containing genes were conserved in five gramineous crops. Under the stress of anaerobic germination and seedling submergence treatment, 33 DUF26 domain-containing genes were differentially expressed in varying degrees. Further correlation analysis with the expression of known submergence tolerance genes showed that these DUF26 domain-containing genes may jointly regulate the submergence tolerance process with these known submergence tolerance genes in DXWR.     

Exclusion of glucosyl-hydroxymethylcytosine DNA containing bacteriophages is overcome by the injected protein inhibitor IPI*

The Escherichia coli isolate CT596 excludes infection by the Myoviridae T4 ip1(-) phage that lacks the encapsidated IPI* protein normally injected into the host with the phage DNA. Screening of a CT596 genomic library identified adjacent genes responsible for this exclusion, gmrS (942 bp) and gmrD (708 bp) that are encoded by a cryptic prophage DNA. The two genes are necessary and sufficient to confer upon a host the ability to exclude infection by T4 ip1(-) phage and other glucosyl-hydroxymethylcytosine (glc-HMC) Tevens lacking the ip1 gene, yet allow infection by phages with non-glucoslyated cytosine (C) DNA that lack the ip1 gene. A plasmid expressing the ip1 gene product, IPI*, allows growth of Tevens lacking ip1 on E. coli strains carrying the cloned gmrS/gmrD genes. Members of the Teven family carry a diverse and, in some cases, expanded set of ip1 locus genes. In vivo analysis suggests a family of gmr genes that specifically target sugar-HMC modified DNA have evolved to exclude Teven phages, and these exclusion genes have in turn been countered by a family of injected exclusion inhibitors that likely help determine the host range of different glc-HMC phages.     

The photoconvertible water-soluble chlorophyll-binding protein of Chenopodium album is a member of DUF538, a superfamily that distributes in Embryophyta

Various plants possess hydrophilic chlorophyll (Chl) proteins known as water-soluble Chl-binding proteins (WSCPs). WSCPs exist in two forms: Class I and Class II, of which Class I alone exhibits unique photoconvertibility. Although numerous genes encoding Class II WSCPs have been identified and the molecular properties of their recombinant proteins have been well characterized, no Class I WSCP gene has been identified to date. In this study, we cloned the cDNA and a gene encoding the Class I WSCP of Chenopodium album (CaWSCP). Sequence analyses revealed that CaWSCP comprises a single exon corresponding to 585 bp of an open reading frame encoding 195 amino acid residues. The CaWSCP protein sequence possesses a signature of DUF538, a protein superfamily of unknown function found almost exclusively in Embryophyta. The recombinant CaWSCP was expressed in Escherichia coli as a hexa-histidine fusion protein (CaWSCP-His) that removes Chls from the thylakoid. Under visible light illumination, the reconstituted CaWSCP-His was successfully photoconverted into a different pigment with an absorption spectrum identical to that of native CaWSCP. Interestingly, while CaWSCP-His could bind both Chl a and Chl b, photoconversion occurred only in CaWSCP-His reconstituted with Chl a.     

Crystal structure of the conserved protein TT1542 from Thermus thermophilus HB8

The TT1542 protein from Thermus thermophilus HB8 is annotated as a conserved hypothetical protein, and belongs to the DUF158 family in the Pfam database. A BLAST search revealed that homologs of TT1542 are present in a wide range of organisms. The TT1542 homologs in eukaryotes, PIG-L in mammals, and GPI12 in yeast and protozoa, have N-acetylglucosaminylphosphatidylinositol (GlcNAc-PI) de-N-acetylase activity. Although most of the homologs in prokaryotes are hypothetical and have no known function, Rv1082 and Rv1170 from Mycobacterium tuberculosis are enzymes involved in the mycothiol detoxification pathway. Here we report the crystal structure of the TT1542 protein at 2.0 A resolution, which represents the first structure for this superfamily of proteins. The structure of the TT1542 monomer consists of a twisted beta-sheet composed of six parallel beta-strands and one antiparallel beta-strand (with the strand order 3-2-1-4-5-7-6) sandwiched between six alpha-helices. The N-terminal five beta-strands and four alpha-helices form an incomplete Rossmann fold-like structure. The structure shares some similarity to the sugar-processing enzymes with Rossmann fold-like domains, especially those of the GPGTF (glycogen phosphorylase/glycosyl transferase) superfamily, and also to the NAD(P)-binding Rossmann fold domains. TT1542 is a homohexamer in the crystal and in solution, the six monomers forming a cylindrical structure. Putative active sites are suggested by the structure and conserved amino acid residues.     

Structure and Function of the DUF2233 Domain in Bacteria and in the Human Mannose 6-Phosphate Uncovering Enzyme

DUF2233, a domain of unknown function (DUF), is present in many bacterial and several viral proteins and was also identified in the mammalian transmembrane glycoprotein N-acetylglucosamine-1-phosphodiester α-N-acetylglucosaminidase (“uncovering enzyme” (UCE)). We report the crystal structure of BACOVA_00430, a 315-residue protein from the human gut bacterium Bacteroides ovatus that is the first structural representative of the DUF2233 protein family. A notable feature of this structure is the presence of a surface cavity that is populated by residues that are highly conserved across the entire family. The crystal structure was used to model the luminal portion of human UCE (hUCE), which is involved in targeting of lysosomal enzymes. Mutational analysis of several residues in a highly conserved surface cavity of hUCE revealed that they are essential for function. The bacterial enzyme (BACOVA_00430) has ∼1% of the catalytic activity of hUCE toward the substrate GlcNAc-P-mannose, the precursor of the Man-6-P lysosomal targeting signal. GlcNAc-1-P is a poor substrate for both enzymes. We conclude that, for at least a subset of proteins in this family, DUF2233 functions as a phosphodiester glycosidase.     

Comparative analysis of GT14/GT14-like gene family in Arabidopsis, Oryza, Populus, Sorghum and Vitis

Glycosyltransferase family14 (GT14) belongs to the glycosyltransferase (GT) superfamily that plays important roles in the biosynthesis of cell walls, the most abundant source of cellulosic biomass for bioethanol production. It has been hypothesized that DUF266 proteins are a new class of GTs related to GT14. In this study, we identified 62 GT14 and 106 DUF266 genes (named GT14-like herein) in Arabidopsis, Oryza, Populus, Sorghum and Vitis. Our phylogenetic analysis separated GT14 and GT14-like genes into two distinct clades, which were further divided into eight and five groups, respectively. Similarities in protein domain, 3D structure and gene expression were uncovered between the two phylogenetic clades, supporting the hypothesis that GT14 and GT14-like genes belong to one family. Therefore, we proposed a new family name, GT14/GT14-like family that combines both subfamilies. Variation in gene expression and protein subcellular localization within the GT14-like subfamily were greater than those within the GT14 subfamily. One-half of the Arabidopsis and Populus GT14/GT14-like genes were found to be preferentially expressed in stem/xylem, indicating that they are likely involved in cell wall biosynthesis. This study provided new insights into the evolution and functional diversification of the GT14/GT14-like family genes.     

Crystal structure of JHP933 from Helicobacter pylori J99 shows two‐domain architecture with a DUF1814 family nucleotidyltransferase domain and a helical bundle domain

The jhp0933 gene in the plasticity region of Helicobacter pylori J99 encodes a hypothetical protein (JHP933), which may play some roles in pathogenesis. Here, we have determined the crystal structure of JHP933 at 2.17 Å. It represents the first crystal structure of the DUF1814 protein family. JHP933 consists of two domains: an N‐terminal domain of the nucleotidyltransferase (NTase) fold and a C‐terminal helix bundle domain. A highly positively charged surface patch exists adjacent to the putative NTP binding site. Structural similarity of JHP933 to known NTases is very remote, suggesting that it may function as a novel NTase. Proteins 2014; 82:2275–2281. © 2014 Wiley Periodicals, Inc.