Structural Insight into the DNA-Binding Mode of the Primosomal Proteins PriA, PriB, and DnaT
Figure 4
(a) Amino acid sequence alignment of EcPriB. An alignment consensus of 111 sequenced PriB homologs by the program ConSurf reveals the degree of variability at each position along the primary sequence. In general, the overall amino acid sequences among PriB proteins are not highly conserved, including many residues found important for ssDNA binding by EcPriB, such as Phe42, Trp47, Lys82, Lys84, and Lys89. (b) EcPriB is a homodimer with polypeptide chains of 104 amino acid residues. Each PriB monomer has an OB-fold structure with three flexible β-hairpin loops: L12 (residues 20–24; colored in blue), L23 (residues 37–44; colored in red), and L45 (residues 81–88; colored in purple blue). The ssDNA in the complex is shown in gold. (c) Crystal structure of EcPriB in complex with DNA. The complex structure of EcPriB (Protein Data Bank entry: 2CCZ) shows that a single dT15 ssDNA periodically interacts with two OB folds from two symmetrically related EcPriB dimers in the crystal and that the DNA is sandwiched by PriB dimers via their L45 loops. (d) Possible working model of interaction between two PriB proteins on ssDNA. PriB proteins cooperatively bind to ssDNA in two steps: two PriB proteins independently interact with ssDNA and then interact with each other through His64 on the ssDNA. The ssDNA in the complex is shown in gold. The region in ssDNA that does not directly interact with PriB, proposed in this two-step binding model, is colored in yellow. (e) Proposed models for PriA-DNA-PriB structure. These models are based on these observations: (1) two PriB dimers are complexed with a single dT15; (2) PriA has a highly electropositive ssDNA-binding region in DBD, and the basic DBD in PriA may be involved in complex with PriB; (3) DBD of PriA alone in solution forms a dimer and not a monomer as the full-length PriA.
