Dataset Information

The domain structure of Helicobacter pylori DnaB helicase: the N-terminal domain can be dispensable for helicase activity whereas the extreme C-terminal region is essential for its function.

ABSTRACT: Hexameric DnaB type replicative helicases are essential for DNA strand unwinding along with the direction of replication fork movement. These helicases in general contain an amino terminal domain and a carboxy terminal domain separated by a linker region. Due to the lack of crystal structure of a full-length DnaB like helicase, the domain structure and function of these types of helicases are not clear. We have reported recently that Helicobacter pylori DnaB helicase is a replicative helicase in vitro and it can bypass Escherichia coli DnaC activity in vivo. Using biochemical, biophysical and genetic complementation assays, here we show that though the N-terminal region of HpDnaB is required for conformational changes between C6 and C3 rotational symmetry, it is not essential for in vitro helicase activity and in vivo function of the protein. Instead, an extreme carboxy terminal region and an adjacent unique 34 amino acid insertion region were found to be essential for HpDnaB activity suggesting that these regions are important for proper folding and oligomerization of this protein. These results confer great potential in understanding the domain structures of DnaB type helicases and their related function.

SUBMITTER: Nitharwal RG

PROVIDER: S-EPMC1888833 | biostudies-other | 2007

REPOSITORIES: biostudies-other

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The domain structure of Helicobacter pylori DnaB helicase: the N-terminal domain can be dispensable for helicase activity whereas the extreme C-terminal region is essential for its function.

Nitharwal Ram Gopal RG Paul Subhankar S Dar Ashraf A Choudhury Nirupam Roy NR Soni Rajesh K RK Prusty Dhaneswar D Sinha Sukrat S Kashav Tara T Mukhopadhyay Gauranga G Chaudhuri Tapan Kumar TK Gourinath Samudrala S Dhar Suman Kumar SK

Nucleic acids research 20070411 9

Hexameric DnaB type replicative helicases are essential for DNA strand unwinding along with the direction of replication fork movement. These helicases in general contain an amino terminal domain and a carboxy terminal domain separated by a linker region. Due to the lack of crystal structure of a full-length DnaB like helicase, the domain structure and function of these types of helicases are not clear. We have reported recently that Helicobacter pylori DnaB helicase is a replicative helicase in ...[more]

PMID: 17430964

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Project description:Helicobacter pylori is a gram-negative bacterium that colonizes the human stomach and contributes to the development of gastric cancer and peptic ulcer disease. VacA, a toxin secreted by H. pylori, is comprised of two domains, designated p33 and p55. Analysis of the crystal structure of the p55 domain indicated that its structure is predominantly a right-handed parallel beta-helix, which is a characteristic of autotransporter passenger domains. Substitution mutations of specific amino acids within the p33 domain abrogate VacA activity, but thus far, it has been difficult to identify small inactivating mutations within the p55 domain. Therefore, we hypothesized that large portions of the p55 domain might be non-essential for vacuolating toxin activity. To test this hypothesis, we introduced eight deletion mutations (each corresponding to a single coil within a beta-helical segment spanning VacA amino acids 433-628) into the H. pylori chromosomal vacA gene.All eight of the mutant VacA proteins were expressed by the corresponding H. pylori mutant strains and underwent proteolytic processing to yield ~85 kDa passenger domains. Three mutant proteins (VacA Delta484-504, Delta511-536, and Delta517-544) were secreted and induced vacuolation of mammalian cells, which indicated that these beta-helical coils were dispensable for vacuolating toxin activity. One mutant protein (VacA Delta433-461) exhibited reduced vacuolating toxin activity compared to wild-type VacA. Other mutant proteins, including those containing deletions near the carboxy-terminal end of the beta-helical region (amino acids Val559-Asn628), exhibited marked defects in secretion and increased susceptibility to proteolytic cleavage by trypsin, which suggested that these proteins were misfolded.These results indicate that within the beta-helical segment of the VacA p55 domain, there are regions of plasticity that tolerate alterations without detrimental effects on protein secretion or activity, as well as a carboxy-terminal region in which similar alterations result in protein misfolding and impaired secretion. We propose that non-essential beta-helical coils and a carboxy-terminal beta-helical segment required for proper protein folding and secretion are features shared by numerous autotransporter passenger domains.

Dataset Information

The domain structure of Helicobacter pylori DnaB helicase: the N-terminal domain can be dispensable for helicase activity whereas the extreme C-terminal region is essential for its function.

Publications

The domain structure of Helicobacter pylori DnaB helicase: the N-terminal domain can be dispensable for helicase activity whereas the extreme C-terminal region is essential for its function.

Similar Datasets

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