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1.
Article in English | MEDLINE | ID: mdl-20208152

ABSTRACT

The trace-element oxyanion molybdate, which is required for the growth of many bacterial and archaeal species, is transported into the cell by an ATP-binding cassette (ABC) transporter superfamily uptake system called ModABC. ModABC consists of the ModA periplasmic solute-binding protein, the integral membrane-transport protein ModB and the ATP-binding and hydrolysis cassette protein ModC. In this study, X-ray crystal structures of ModA from the archaeon Methanosarcina acetivorans (MaModA) have been determined in the apoprotein conformation at 1.95 and 1.69 A resolution and in the molybdate-bound conformation at 2.25 and 2.45 A resolution. The overall domain structure of MaModA is similar to other ModA proteins in that it has a bilobal structure in which two mixed alpha/beta domains are linked by a hinge region. The apo MaModA is the first unliganded archaeal ModA structure to be determined: it exhibits a deep cleft between the two domains and confirms that upon binding ligand one domain is rotated towards the other by a hinge-bending motion, which is consistent with the 'Venus flytrap' model seen for bacterial-type periplasmic binding proteins. In contrast to the bacterial ModA structures, which have tetrahedral coordination of their metal substrates, molybdate-bound MaModA employs octahedral coordination of its substrate like other archaeal ModA proteins.


Subject(s)
ATP-Binding Cassette Transporters/chemistry , Apoproteins/chemistry , Archaeal Proteins/chemistry , Methanosarcina/chemistry , ATP-Binding Cassette Transporters/metabolism , Amino Acid Sequence , Apoproteins/metabolism , Archaeal Proteins/metabolism , Binding Sites , Crystallography, X-Ray , Ligands , Methanosarcina/metabolism , Models, Molecular , Molecular Sequence Data , Protein Binding , Protein Structure, Secondary , Protein Structure, Tertiary , Sequence Alignment , Substrate Specificity
2.
Structure ; 21(4): 627-37, 2013 Apr 02.
Article in English | MEDLINE | ID: mdl-23523424

ABSTRACT

The Mycobacterium tuberculosis (Mtb) genome encodes approximately 90 toxin-antitoxin protein complexes, including three RelBE family members, which are believed to play a major role in bacterial fitness and pathogenicity. We have determined the crystal structures of Mtb RelBE-2 and RelBE-3, and the structures reveal homologous heterotetramers. Our structures suggest RelE-2, and by extension the closely related RelE-1, use a different catalytic mechanism than RelE-3, because our analysis of the RelE-2 structure predicts additional amino acid residues that are likely to be functionally significant and are missing from analogous positions in the RelE-3 structure. Toxicity assays corroborate our structural findings; overexpression of RelE-3, whose active site is more similar to Escherichia coli YoeB, has limited consequences on bacterial growth, whereas RelE-1 and RelE-2 overexpression results in acute toxicity. Moreover, RelE-2 overexpression results in an elongated cell phenotype in Mycobacterium smegmatis and protects M. tuberculosis against antibiotics, suggesting a different functional role for RelE-2.


Subject(s)
Antitoxins/chemistry , Bacterial Toxins/chemistry , Models, Molecular , Multiprotein Complexes/chemistry , Mycobacterium tuberculosis/chemistry , Protein Conformation , Amino Acid Sequence/genetics , Antitoxins/genetics , Bacterial Toxins/genetics , Catalysis , Cell Shape/genetics , Cluster Analysis , Crystallography, X-Ray , Molecular Sequence Data , Multiprotein Complexes/genetics , Mycobacterium smegmatis , Mycobacterium tuberculosis/genetics , Phylogeny , Proteomics/methods , Sequence Alignment , Species Specificity
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