PDBsum entry 2z4s

DNA binding protein

PDB id: 2z4s

Contents

Protein chain

240 a.a. *

Ligands

ADP

Metals

_MG

* Residue conservation analysis

PDB id:

2z4s

Name:

DNA binding protein

Title:

Crystal structure of domain iii from the thermotoga maritima replication initiation protein dnaa

Structure:

Chromosomal replication initiator protein dnaa. Chain: a. Engineered: yes

Source:

Thermotoga maritima. Organism_taxid: 2336. Gene: dnaa. Expressed in: escherichia coli. Expression_system_taxid: 562.

Resolution:

3.00Å R-factor: 0.233 R-free: 0.291

Authors:

N.Fujikawa,S.Ozaki,W.Kagawa,S.-Y.Park,T.Katayama,H.Kurumizaka, S.Yokoyama,Riken Structural Genomics/proteomics Initiative (Rsgi)

Key ref:

S.Ozaki et al. (2008). A common mechanism for the ATP-DnaA-dependent formation of open complexes at the replication origin. J Biol Chem, 283, 8351-8362. PubMed id: 18216012 DOI: 10.1074/jbc.M708684200

Date:

25-Jun-07 Release date: 19-Feb-08

Protein chain

P46798  (DNAA_THEMA) -  Chromosomal replication initiator protein DnaA from Thermotoga maritima (strain ATCC 43589 / DSM 3109 / JCM 10099 / NBRC 100826 / MSB8)

Seq: 440 a.a.

Struc: 240 a.a.

DOI no: 10.1074/jbc.M708684200

J Biol Chem 283:8351-8362 (2008)

PubMed id: 18216012

A common mechanism for the ATP-DnaA-dependent formation of open complexes at the replication origin.

S.Ozaki, H.Kawakami, K.Nakamura, N.Fujikawa, W.Kagawa, S.Y.Park, S.Yokoyama, H.Kurumizaka, T.Katayama.

ABSTRACT

Initiation of chromosomal replication and its cell cycle-coordinated regulation bear crucial and fundamental mechanisms in most cellular organisms. Escherichia coli DnaA protein forms a homomultimeric complex with the replication origin (oriC). ATP-DnaA multimers unwind the duplex within the oriC unwinding element (DUE). In this study, structural analyses suggested that several residues exposed in the central pore of the putative structure of DnaA multimers could be important for unwinding. Using mutation analyses, we found that, of these candidate residues, DnaA Val-211 and Arg-245 are prerequisites for initiation in vivo and in vitro. Whereas DnaA V211A and R245A proteins retained normal affinities for ATP/ADP and DNA and activity for the ATP-specific conformational change of the initiation complex in vitro, oriC complexes of these mutant proteins were inactive in DUE unwinding and in binding to the single-stranded DUE. Unlike oriC complexes including ADP-DnaA or the mutant DnaA, ATP-DnaA-oriC complexes specifically bound the upper strand of single-stranded DUE. Specific T-rich sequences within the strand were required for binding. The corresponding conserved residues of the DnaA ortholog in Thermotoga maritima, an ancient eubacterium, were also required for DUE unwinding, consistent with the idea that the mechanism and regulation for DUE unwinding can be evolutionarily conserved. These findings provide novel insights into mechanisms for pore-mediated origin unwinding, ATP/ADP-dependent regulation, and helicase loading of the initiation complex.

Selected figure(s)

Figure 1.
FIGURE 1. Structural models of DnaA oligomers and tmaDnaA mutants. A, crystal structure of tmaDnaA AAA^+ domain. The crystal structure of tmaDnaA AAA^+ domain bound to ADP was solved by molecular replacement. The A. aeolicus DnaA AAA^+ domain structure (22) was used as a search model. The subdomains IIIa and IIIb within AAA^+ domain are depicted as a ribbon model in different colors. B, structure comparison. Structures of AAA^+ domain of tmaDnaA and A. aeolicus DnaA, colored purple and cyan, respectively, were superimposed for subdomain IIIa. Structures are shown in a frame model. C, a ring model structure of tmaDnaA. A hexameric ring model was constructed using the determined structure of tmaDnaA AAA^+ domain. Each protomer is colored differently. The amino acid residues of Val-176, Met-179, Lys-180, Lys-209, and Gly-211 are exposed on the pore surface of the ring and are colored in red. For i–vi, see panel F. D, a spiral filament model structure of tmaDnaA. A spiral filament model was constructed using the structure of tmaDnaA AAA^+ domain, based on the spiral filament structure of the A. aeolicus DnaA AAA^+ domain (37). The model filament is shown viewed down the helical axis (top), as well as perpendicularly to the axis (bottom). Each protomer is colored differently. The amino acid residues described above are also exposed on the pore surface of the spiral filament and are colored red. E, the pore surfaces of the tmaDnaA ring and spiral filament. Three consecutive molecules of tmaDnaA within the ring (C) and spiral (D) models are shown. The amino acid residues described above and analyzed in this study are colored red and indicated. F, amino acid sequences, including the putative pore region of representative DnaA orthologs. i–vi correspond to E. coli DnaA residues of Val-211, Leu-214, Gln-215, Lys-223, Lys-243, and Arg-245, respectively. Residues corresponding to these are highlighted in yellow for hydrophobic residues or in purple for basic residues. The secondary structures of A. aeolicus DnaA (helix 3- 6) are also shown (22). Ecoli, Escherichia coli; Thema, Thermotoga maritima; Aquae, Aquifex aeolicus; Helph, Helicobacter pylori; Bacsu, Bacillus subtilis; Myctu, Mycobacterium tuberculosis; Chlmu, and Chlamydia muridarum. G, tma-oriC unwinding assay. The indicated amounts of wild-type (WT) or mutant tmaDnaA proteins were incubated at 48 °C for 10 min in the presence of pOZ14 (200 fmol), which bears the tma-oriC, followed by digestion with P1 nuclease and AlwNI.

Figure 10.
FIGURE 10. A model for open complex formation. A, a model of DnaA complexes and ATP-dependent conformational change. Light blue, ADP; light pink, ATP; yellow, H-motif; blue, B-motif; and red, arginine finger. B, possible mixed complexes. The initiation activity of the mixed complexes is indicated by ± (moderate) or + (active). Mixed complexes of wild-type DnaA with mutant DnaA of the B-motif (i), H-motif (ii), or arginine finger (iii) are shown. Light gray color indicates mutant DnaA protomers.

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2008, 283, 8351-8362) copyright 2008.

Figures were selected by the author.