Crystal structure of the chi:psi subassembly of the Escherichia coli DNA polymerase clamp-loader complex

Jacqueline M. Gulbis, Steven L. Kazmirski, Jeff Finkelstein, Zvi Kelman, Mike O'Donnell, and John Kuriyan

Eur. J. Biochem. 271, 439-449 (2004) (local copy)

Abstract / Figures from the paper / Coordinates


The chi (χ) and psi (ψ) subunits of Escherichia coli DNA polymerase III form a heterodimer that is associated with the ATP-dependent clamp-loader machinery. In E. coli, the χ:ψ heterodimer serves as a bridge between the clamp-loader complex and the single-stranded DNA-binding protein. We determined the crystal structure of the χ:ψ heterodimer at 2.1 resolution. Although neither χ (147 residues) nor ψ (137 residues) bind to nucleotides, the fold of each protein is similar to the folds of mononucleotide-(χ) or dinucleotide-(ψ) binding proteins, without marked similarity to the structures of the clamp-loader subunits. Genes encoding χ and ψ proteins are found to be readily identifiable in several bacterial genomes and sequence alignments showed that residues at the χ:ψ interface are highly conserved in both proteins, suggesting that the heterodimeric interaction is of functional significance. The conservation of surface-exposed residues is restricted to the interfacial region and to just two other regions in the χ:ψ complex. One of the conserved regions was found to be located on χ, distal to the ψ interaction region, and we identified this as the binding site for a C-terminal segment of the single-stranded DNA-binding protein. The other region of sequence conservation is localized to an N-terminal segment of ψ (26 residues) that is disordered in the crystal structure. We speculate that ψ is linked to the clamp-loader complex by this flexible, but conserved, N-terminal segment, and that the χ:ψ unit is linked to the single-stranded DNA-binding protein via the distal surface of χ. The base of the clamp-loader complex has an open C-shaped structure, and the shape of the χ:ψ complex is suggestive of a loose docking within the crevice formed by the open faces of the δ and δ' subunits of the clamp-loader.

Illustrations from the paper.

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Fig. 1. Structure of the χ:ψ heterodimer
(A) Ribbon diagram of the χ:ψ heterodimer crystal structure. ψ is colored cyan and sits on top of the χ subunit. χ is colored green except for the stretch of residues that reside in the ψ binding site which have been colored red. (B) An enlarged view of the contiguous loop region of χ and how it interacts within the cleft of ψ. This loop region has high sequence similarity with a DNA-dependent DNA polymerase from the bacteriophage PRD1. (C) A rotated view of the surface of ψ is shown. ψ has been rotate to show the cleft between α1 and α4 that makes up the χ binding surface. In green are residues 61-66 of χ. The side chain of Phe 64 of χ inserts itself in to a conserved hydrophobic pocket consisting of Val 57, Leu 121, Trp 122 and Ile 125. (D) A schematic diagram of the χ:ψ heterodimer.

Fig. 2. Structural comparisons of χ and ψ with other proteins
(A) A side-by-side comparison of ψ with the mismatch specific DNA uracyl glycosylase MUG [45]. Similar structural features are colored yellow. (B) A side-by-side comparison of χ DEAD box helicase PcrA [46].

Fig. 3. Sequence conservation in χ and ψ
The conservation score using the BLOSUM62 substitution matrix, (see Materials and Methods) for each residue in χ and ψ was calculated for the 12 pairs of sequences shown in Table 2. The surfaces of χ and ψ shown in this figure are colored according to this conservation score. To the right, the binding surfaces of both proteins are shown. Both binding surfaces have been conserved in each protein. On ψ, little surface conservation is observed outside of the χ binding surface. In χ , a large amount of surface area is conserved distal to the ψ binding site. This area is proposed to bind to single stranded DNA binding protein (SSB).

Fig. 4. Potential χ:SSB interaction
A region of χ with high sequence conservation is shown (middle). This surface is suggested to bind to the negatively charged C-terminus tail of SSB. Absolutely conserved and positively charged residues located within this region are shown on the left in a ribbon diagram in the same orientation. A schematic drawing of the inferred interaction between χ and the C-terminus consensus sequence of SSB is shown on the left.

Fig. 5. Conservation of sequences in the N-terminal segment of ψ
An alignment of the first 26 residues of ψ, from the list of sequences given in Table 2, is shown. The alignment is colored according to the degree of sequence conservation. These 26 residues are disordered in the crystal structure of the χ:ψ complex yet a high amount of conservation is observed. It is proposed that the this linker binds to the clamp loader complex tethering the χ:ψ heterodimer to the complex.

Fig. 6. Potential clamp loader: ψ interaction
(A) Two views of the E. coli clamp loader complex are shown [17]. An exposed hydrophobic region of the γ subunit that is highly conserved but is not involved in nucleotide binding or inter-subunit interactions is indicated as a potential binding site for the N-terminal disordered region of ψ. (B) On the left are space filling structures of the E. coli clamp loader and χ:ψ heterodimer colored by different subunits: δ (magenta), δ' (orange),γ1 (green), γ2 (red), γ3 (blue), ψ (cyan), and χ (dark green). A schematic diagram showing a possible mode of interaction between the χ:ψ heterodimer and the clamp-loader complex is shown on the right. The χ:ψ heterodimer is believed to sit in the gap between δ and δ' while the N-terminus of ψ interacts with the proposed binding region of γ inside the C-terminal collar of the clamp loader complex.


1EM8: Crystal Structure Of χ and ψ Subunit Heterodimer From DNA Pol III