Structural analysis of the Sulfolobus solfataricus MCM protein N-terminal domain

Abstract

The Mini-Chromosome Maintenance (MCM) proteins are candidates of replicative DNA helicase in eukarya and archaea. Here we report a 2.8 A crystal structure of the N-terminal domain (residues 1-268) of the Sulfolobus solfataricus MCM (Sso MCM) protein. The structure reveals single-hexameric ring-like architecture, at variance from the protein of Methanothermobacter thermoautotrophicus (Mth). Moreover, the central channel in Sso MCM seems significantly narrower than the Mth counterpart, which appears to more favorably accommodate single-stranded DNA than double-stranded DNA, as supported by DNA-binding assays. Structural analysis also highlights the essential role played by the zinc-binding domain in the interaction with nucleic acids and allows us to speculate that the Sso MCM N-ter domain may function as a molecular clamp to grasp the single-stranded DNA passing through the central channel. On this basis possible DNA unwinding mechanisms are discussed.

Figure 1.

Overall and subunit structure of the Sso MCM N-terminal domain. Top (A) and side (B) view of the hexamer forming a single-layered ring. The six subunits are painted with different colors. (C) Ribbon model of the subunit structure showing the three distinct domains (A, B and C) rendered in yellow, green and cyan, respectively. The zinc atoms are denoted by magenta spheres.

Figure 2.

The central cavity and the β-hairpin loop encircling the narrowest space. (A) The accessible surface showing the shape and dimension of the cavity interior of the Sso structure, and (B), the same representation of the crystal structure of Mth MCM N-ter (17). For clarity, two subunits were taken away from the front. (C) Electron density (2Fo–Fc) map contoured at 1.0 σ level around the peptide region from residues 241 to 251. The protein atoms are represented in stick model.

Figure 3.

Structural comparison between the Sso and Mth MCM N-terminal domains. (A) Structure-based sequence alignment. (B) Superimposition of two structures: Sso MCM colored in blue, Mth MCM in green, zinc atoms are represented by magenta spheres. The three regions producing a large rmsd are labeled with numbers. C and D, The zinc-finger motives in Sso (C) and Mth (D) MCM.

Figure 4.

DNA-binding activity of Sso MCM and its truncated forms on blunt dsDNA. Representative EMSAs were carried out with increasing concentrations of each indicated protein using a radio-labeled 56-bp DNA as a ligand. Control mixtures without protein were run on the lanes indicated by B. Plots of the shifted DNA versus the free protein concentration (as a monomer) are shown. Experiments were performed in triplicate and the results averaged. Curves represent best fits to the data points. The error bars on the graphs are the standard error of the mean.