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To extend the structural coverage of proteins with unknown functions, we targeted a novel protein family (Pfam accession number PF08807, DUF1798) for which we proposed and determined the structures of two representative members. The MW1337R gene of Staphylococcus aureus subsp. aureus Rosenbach (Wood 46) encodes a protein with a molecular weight of 13.8 kDa (residues 1-116) and a calculated isoelectric point of 5.15. The lin2004 gene of the nonspore-forming bacterium Listeria innocua Clip11262 encodes a protein with a molecular weight of 14.6 kDa (residues 1-121) and a calculated isoelectric point of 5.45. MW1337R and lin2004, as well as their homologs, which, so far, have been found only in Bacillus, Staphylococcus, Listeria, and related genera (Geobacillus, Exiguobacterium, and Oceanobacillus), have unknown functions and are annotated as hypothetical proteins. The genomic contexts of MW1337R and lin2004 are similar and conserved in related species. In prokaryotic genomes, most often, functionally interacting proteins are coded by genes, which are colocated in conserved operons. Proteins from the same operon as MW1337R and lin2004 either have unknown functions (i.e., belong to DUF1273, Pfam accession number PF06908) or are similar to ypsB from Bacillus subtilis. The function of ypsB is unclear, although it has a strong similaritymore » to the N-terminal region of DivIVA, which was characterized as a bifunctional protein with distinct roles during vegetative growth and sporulation. In addition, members of the DUF1273 family display distant sequence similarity with the DprA/Smf protein, which acts downstream of the DNA uptake machinery, possibly in conjunction with RecA. The RecA activities in Bacillus subtilis are modulated by RecU Holliday-junction resolvase. In all analyzed cases, the gene coding for RecU is in the vicinity of MW1337R, lin2004, or their orthologs, but on a different operon located in the complementary DNA strand. Here, we report the crystal structures of MW1337R and lin2004, which were determined using the semiautomated, high-throughput pipeline of the Joint Center for Structural Genomics (JCSG), part of the National Institute of General Medical Sciences Protein Structure Initiative.« less

TyrA is a member of the dye-decolorizing peroxidase (DyP) family, a new family of heme-dependent peroxidase recently identified in fungi and bacteria. Here, we report the crystal structure of TyrA in complex with iron protoporphyrin (IX) at 2.3 A. TyrA is a dimer, with each monomer exhibiting a two-domain, alpha/beta ferredoxin-like fold. Both domains contribute to the heme-binding site. Co-crystallization in the presence of an excess of iron protoporphyrin (IX) chloride allowed for the unambiguous location of the active site and the specific residues involved in heme binding. The structure reveals a Fe-His-Asp triad essential for heme positioning, as well as a novel conformation of one of the heme propionate moieties compared to plant peroxidases. Structural comparison to the canonical DyP family member, DyP from Thanatephorus cucumeris (Dec 1), demonstrates conservation of this novel heme conformation, as well as residues important for heme binding. Structural comparisons with representative members from all classes of the plant, bacterial, and fungal peroxidase superfamily demonstrate that TyrA, and by extension the DyP family, adopts a fold different from all other structurally characterized heme peroxidases. We propose that a new superfamily be added to the peroxidase classification scheme to encompass the DyP family of heme peroxidases.

The crystal structure of an RmlC-type cupin with zinc acetate bound at the putative active site reveals significant differences from a previous structure without any bound ligand. The functional implications of the ligand-induced conformational changes are discussed., In the plant pathogen Xanthomonas campestris pv. campestris, the product of the tcmJ gene, XcTcmJ, encodes a protein belonging to the RmlC family of cupins. XcTcmJ was crystallized in a monoclinic space group (C2) in the presence of zinc acetate and the structure was determined to 1.6 Å resolution. Previously, the apo structure has been reported in the absence of any bound metal ion [Chin et al. (2006 ▶), Proteins, 65, 1046–1050]. The most significant difference between the apo structure and the structure of XcTcmJ described here is a reorganization of the binding site for zinc acetate, which was most likely acquired from the crystallization solution. This site is located in the conserved metal ion-binding domain at the putative active site of XcTcmJ. In addition, an acetate was also bound within coordination distance of the zinc. In order to accommodate this binding, rearrangement of a conserved histidine ligand is required as well as several nearby residues within and around the putative active site. These observations indicate that binding of zinc serves a functional role in this cupin protein.

The crystal structure of the first representative of the Pfam PF07336 (DUF1470) family reveals a two-domain organization that contains a new fold, termed the ABATE domain, at the N-terminus and a treble-clef zinc finger that is likely to bind DNA at the C-terminus., The crystal structure of Jann_2411 from Jannaschia sp. strain CCS1, a member of the Pfam PF07336 family classified as a domain of unknown function (DUF1470), was solved to a resolution of 1.45 Å by multiple-wavelength anomalous dispersion (MAD). This protein is the first structural representative of the DUF1470 Pfam family. Structural analysis revealed a two-domain organization, with the N-terminal domain presenting a new fold called the ABATE domain that may bind an as yet unknown ligand. The C-terminal domain forms a treble-clef zinc finger that is likely to be involved in DNA binding. Analysis of the Jann_2411 protein and the broader ABATE-domain family suggests a role as stress-induced transcriptional regulators.

Structures of the first representatives of PF06684 (DUF1185) reveal a Bacillus chorismate mutase-like fold with a potential role in amino-acid synthesis., The crystal structures of BB2672 and SPO0826 were determined to resolutions of 1.7 and 2.1 Å by single-wavelength anomalous dispersion and multiple-wavelength anomalous dispersion, respectively, using the semi-automated high-throughput pipeline of the Joint Center for Structural Genomics (JCSG) as part of the NIGMS Protein Structure Initiative (PSI). These proteins are the first structural representatives of the PF06684 (DUF1185) Pfam family. Structural analysis revealed that both structures adopt a variant of the Bacillus chorismate mutase fold (BCM). The biological unit of both proteins is a hexamer and analysis of homologs indicates that the oligomer interface residues are highly conserved. The conformation of the critical regions for oligomerization appears to be dependent on pH or salt concentration, suggesting that this protein might be subject to environmental regulation. Structural similarities to BCM and genome-context analysis suggest a function in amino-acid synthesis.

Crystal structures of two homologous peptidases from cyanobacteria Anabaena variabilis and Nostoc punctiforme at 1.05 A and 1.60 A resolution represent the first structures of a large class of cell-wall, cysteine peptidases that contain an N-terminal bacterial SH3-like domain (SH3b) and a C-terminal NlpC/P60 cysteine peptidase domain. The NlpC/P60 domain is a primitive, papain-like peptidase in the CA clan of cysteine peptidases with a Cys126/His176/His188 catalytic triad and a conserved catalytic core. We deduced from structure and sequence analysis, and then experimentally, that that these two proteins act as γ-D-glutamyl-L-diamino acid endopeptidases (EC 3.4.22.-). The active site is located near the interface between the SH3b and NlpC/P60 domains, where the SH3b domain may help define substrate specificity, instead of functioning as a targeting domain, so that only muropeptides with an N-terminal L-alanine can bind to the active site.