PMC:2682197 / 22049-27899 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/2682197","sourcedb":"PMC","sourceid":"2682197","source_url":"http://www.ncbi.nlm.nih.gov/pmc/2682197","text":"The crystal structure of M. tuberculosis Rv2623: Dimer assembly and ATP-binding capacity\nTo examine the biochemical mechanisms responsible for Rv2623 function, we determined the crystal structure of wild-type Rv2623 at a resolution of 2.9 Å. The structure reveals a compact, 2-fold symmetric dimer. Each monomer is composed of tandem USP domains [residues 6–154 (domain 1), 155–294 (domain2)] that share 26% sequence identity and significant structural homology (residues 6–154 and 155–294 comprise domains 1 and 2, respectively; interdomain rms = 2.04 Å for 140 equivalent Cα's). Individual domains, which consist of a twisted, five-stranded, parallel β sheet flanked by four α helices, unite through an antiparallel, cross-strand (β5–β10) interaction that produces a central dyad axis between β5/β10 and a continuous, ten-stranded, mixed β sheet in the complete monomer. Each domain possesses a pair of conserved βαβ motifs (domain 1: β1-L1-α1- β2, β4-L2-α4-β5; domain 2: β6-L3-α5-β7, β9-L4-α8-β10) that encompass four loops (designated L1–L4) responsible for ATP recognition (Figure 6A and C). A “U-shaped” ATP molecule that lies within a cleft near the monomer surface is stabilized by 1) a cluster of hydrophobic residues (I14, V41, H42, V116/132/261/277/281, L136, A175) that forge the adenine/ribose-binding scaffold, 2) a pair of conserved L1/L3 aspartates (D15-L1/D167-L3), and 3) small phosphoryl/ribosyl-binding residues within the G-2X-G-9X-G (S/T) motifs that comprise L2/L4 (G120/265/267/268 and S131/276) (Figures 6A,C and 7A). Dimerization of Rv2623 occurs along a 2-fold axis orthogonal to the intramonomer dyad and juxtaposes ATP binding pockets from opposing monomers (Figure 6B).\n10.1371/journal.ppat.1000460.g006 Figure 6 Structure and phylogeny of Rv2623 from M. tuberculosis.\n(A,B) A ribbon representation of the Rv2623 monomer (A) and dimer (B) with bound ATP (sticks) and Mg2+ (chocolate spheres). The three, mutually perpendicular pseudo-two-fold axes of the dimer are represented by lines with double arrows (along x, y) and a central ellipse (along z). The atoms of the bound ATP are colored cyan (carbon), red (oxygen), blue (nitrogen), and orange (phosphorus) in (A) and (B). (C) A structure-based sequence alignment of Rv2623, the N631 subfamily consensus, Methanococcus jannaschii protein 0577 (MJ0577), and domains 1 and 2 of Rv2623. Invariant residues in the alignment (\u003e85% conserved in N631) are shaded in bold red and similarities are boxed in blue but left unshaded. Regions with consensus ATP binding motifs comprising L1/L2 (domain 1) and L3/L4 (domain 2) are colored dark violet and smudge, respectively. The positions of the mutated amino acids (D15, G117) are indicated in green. The structure-based sequence alignment was produced using ESPript and the structural representations were produced using PyMOL.\n10.1371/journal.ppat.1000460.g007 Figure 7 Design and stability of ATP-binding–deficient Rv2623 mutants.\n(A) A ribbon and stick representation of the mutation sites within the ATP binding pocket of domain 1. Mg2+ is shown as a green sphere; dotted lines indicate hydrogen-bonding contacts; atoms that constitute ATP are colored cyan (carbon), red (oxygen), blue (nitrogen), and orange (phosphorus). (B) The ATP-binding capacity of mutant Rv2623 was compared to that of wild type protein following nucleotide extraction and HPLC. Data presented are derived from analysis of three independent protein preparations. ATP binding capacity is expressed as: [(the amount of ATP bound in mutant)/(the amount of ATP bound by wild type Rv2623) * 100]. (C) Thermal denaturation curves of two individual protein preparations of Rv2623WT (WT-1, WT-2) as compared to Rv2623G117A (G117A) and Rv2623D15E (D15E). The data is expressed as the negative first-derivative of the fluorescence intensity as a function of temperature. Phylogenomic analysis places Rv2623 in a Uniprot/TrEMBL family (Q5YVE7) of 370 tandem-domain USPs, and a 113-member subfamily (N631) that consists almost exclusively of actinobacterial representatives (Text S1). Structure-based sequence alignments of both Rv2623 domains with the N631 consensus suggest that domain 2, which exhibits significantly higher conservation than domain 1 across global and ATP-binding subfamily consensus sequences, represents the ancestral domain among ATP-binding USPs with tandem-type architectures. Interestingly, the domain fold and interdomain organization observed for Rv2623 is broadly conserved: these features are shared among single domain USP structures, both monomeric and dimeric, that are presently represented within the PDB. As this manuscript was under preparation, a second, lower resolution (3.2 Å) crystal form of Rv2623 (PDB ID 2JAX) was released for public access. This structure is nearly identical to the present model as demonstrated by superposition over the ATP ligands and the monomeric and dimeric forms (rmsds are 0.57 and 0.81 for 258 and 517 matched CA's, respectively). The differences localize primarily to flexible loop regions (residues 44–58, 150–159) that, while disordered in 2JAX, are partially stabilized in the present structure by local crystal contacts.\nTo gain insight into the ATP-binding mode(s) exhibited by Rv2623, the structural features of the ATP-binding pocket of domains 1/2 were compared to the monomer fold of the representative ATP-binding USP, MJ0577 (PDBID 1MJH) [26]. Overlay of these structures reveals very considerable similarity for the residues that form the binding pockets and the associated ATP molecules, for which the triphosphoryl moieties assume virtually indistinguishable conformations. Relatively subtle structural and phylogenetic differences that exist between the ATP-binding pockets might nevertheless confer divergent binding and/or regulatory properties to the tandem domains.","divisions":[{"label":"Title","span":{"begin":0,"end":88}},{"label":"Figure caption","span":{"begin":1700,"end":2853}},{"label":"Title","span":{"begin":1744,"end":1799}},{"label":"Figure caption","span":{"begin":2852,"end":3865}},{"label":"Title","span":{"begin":2896,"end":2957}}],"tracks":[{"project":"2_test","denotations":[{"id":"19478878-9860944-97945831","span":{"begin":5416,"end":5418},"obj":"9860944"}],"attributes":[{"subj":"19478878-9860944-97945831","pred":"source","obj":"2_test"}]},{"project":"bionlp-st-id-2011-training","denotations":[{"id":"T243","span":{"begin":25,"end":40},"obj":"Organism"},{"id":"T244","span":{"begin":41,"end":47},"obj":"Protein"},{"id":"T245","span":{"begin":68,"end":71},"obj":"Chemical"},{"id":"T246","span":{"begin":143,"end":149},"obj":"Protein"},{"id":"T247","span":{"begin":209,"end":215},"obj":"Protein"},{"id":"T248","span":{"begin":1062,"end":1065},"obj":"Chemical"},{"id":"T249","span":{"begin":1110,"end":1113},"obj":"Chemical"},{"id":"T250","span":{"begin":1559,"end":1565},"obj":"Protein"},{"id":"T251","span":{"begin":1644,"end":1647},"obj":"Chemical"},{"id":"T252","span":{"begin":3895,"end":3901},"obj":"Protein"},{"id":"T253","span":{"begin":4122,"end":4128},"obj":"Protein"},{"id":"T254","span":{"begin":4265,"end":4268},"obj":"Chemical"},{"id":"T255","span":{"begin":4346,"end":4349},"obj":"Chemical"},{"id":"T256","span":{"begin":4468,"end":4474},"obj":"Protein"},{"id":"T257","span":{"begin":4727,"end":4733},"obj":"Protein"},{"id":"T258","span":{"begin":4878,"end":4881},"obj":"Chemical"},{"id":"T259","span":{"begin":5216,"end":5219},"obj":"Chemical"},{"id":"T260","span":{"begin":5249,"end":5255},"obj":"Protein"},{"id":"T261","span":{"begin":5288,"end":5291},"obj":"Chemical"},{"id":"T262","span":{"begin":5378,"end":5381},"obj":"Chemical"},{"id":"T263","span":{"begin":5395,"end":5401},"obj":"Protein"},{"id":"T264","span":{"begin":5552,"end":5555},"obj":"Chemical"},{"id":"T265","span":{"begin":5735,"end":5738},"obj":"Chemical"}],"attributes":[{"subj":"T243","pred":"source","obj":"bionlp-st-id-2011-training"},{"subj":"T244","pred":"source","obj":"bionlp-st-id-2011-training"},{"subj":"T245","pred":"source","obj":"bionlp-st-id-2011-training"},{"subj":"T246","pred":"source","obj":"bionlp-st-id-2011-training"},{"subj":"T247","pred":"source","obj":"bionlp-st-id-2011-training"},{"subj":"T248","pred":"source","obj":"bionlp-st-id-2011-training"},{"subj":"T249","pred":"source","obj":"bionlp-st-id-2011-training"},{"subj":"T250","pred":"source","obj":"bionlp-st-id-2011-training"},{"subj":"T251","pred":"source","obj":"bionlp-st-id-2011-training"},{"subj":"T252","pred":"source","obj":"bionlp-st-id-2011-training"},{"subj":"T253","pred":"source","obj":"bionlp-st-id-2011-training"},{"subj":"T254","pred":"source","obj":"bionlp-st-id-2011-training"},{"subj":"T255","pred":"source","obj":"bionlp-st-id-2011-training"},{"subj":"T256","pred":"source","obj":"bionlp-st-id-2011-training"},{"subj":"T257","pred":"source","obj":"bionlp-st-id-2011-training"},{"subj":"T258","pred":"source","obj":"bionlp-st-id-2011-training"},{"subj":"T259","pred":"source","obj":"bionlp-st-id-2011-training"},{"subj":"T260","pred":"source","obj":"bionlp-st-id-2011-training"},{"subj":"T261","pred":"source","obj":"bionlp-st-id-2011-training"},{"subj":"T262","pred":"source","obj":"bionlp-st-id-2011-training"},{"subj":"T263","pred":"source","obj":"bionlp-st-id-2011-training"},{"subj":"T264","pred":"source","obj":"bionlp-st-id-2011-training"},{"subj":"T265","pred":"source","obj":"bionlp-st-id-2011-training"}]}],"config":{"attribute types":[{"pred":"source","value type":"selection","values":[{"id":"2_test","color":"#aeec93","default":true},{"id":"bionlp-st-id-2011-training","color":"#ec93c8"}]}]}}