Crystal structure of a near-full-length archaeal MCM: functional insights for an AAA+ hexameric helicase

Abstract

The minichromosome maintenance protein (MCM) complex is an essential replicative helicase for DNA replication in Archaea and Eukaryotes. Whereas the eukaryotic complex consists of 6 homologous proteins (MCM2-7), the archaeon Sulfolobus solfataricus has only 1 MCM protein (ssoMCM), 6 subunits of which form a homohexamer. Here, we report a 4.35-A crystal structure of the near-full-length ssoMCM. The structure shows an elongated fold, with 5 subdomains that are organized into 2 large N- and C-terminal domains. A near-full-length ssoMCM hexamer generated based on the 6-fold symmetry of the N-terminal Methanothermobacter thermautotrophicus (mtMCM) hexamer shows intersubunit distances suitable for bonding contacts, including the interface around the ATP pocket. Four unusual beta-hairpins of each subunit are located inside the central channel or around the side channels in the hexamer. Additionally, the hexamer fits well into the double-hexamer EM map of mtMCM. Our mutational analysis of residues at the intersubunit interfaces and around the side channels demonstrates their critical roles for hexamerization and helicase function. These structural and biochemical results provide a basis for future study of the helicase mechanisms of the archaeal and eukaryotic MCM complexes in DNA replication.

Fig. 1.

Overall features of the monomeric and hexameric ssoMCM structural models. (A) Diagram depicting the domains of ssoMCM. N-C, N to C domain linker; α/β, α/β-domain of ATPase core; α/β-α, linker between subdomains in the ATPase core; α, α-domain. WH, winged helix domain (disordered in our structure). (B) Fold of ssoMCM monomer. Domains and linkers are colored as in A. Helices are shown as cylinders and β-strands as arrows. Zinc atoms are shown as red spheres. β-Hairpins are labeled as follows: NT-hp, N-terminal hairpin; H2I-hp, helix-2 insert hairpin; PS1-hp, presensor 1 hairpin; EXT-hp, external hairpin. (C) Ribbon diagram showing the top and side views of a hexamer model of ssoMCM.

Fig. 2.

Structural features of the ssoMCM hexamer. (A) Double hexameric EM map of mtMCM with the ssoMCM hexamer model fitting snugly inside the map (20). The PS1 hairpin is located near the side channels of the EM map (indicated by an arrow). (B) Side and top views of the ssoMCM hexamer model. Subunits are labeled a–f. Two subunits in the front are removed in the side view to reveal the interior. The 4 β-hairpins located inside the central and side channels are colored. (C) Close-up view of subunits a and b in the back side of the hexamer in B (side view). β-Hairpins are labeled as in Fig. 1B. The opening between the 2 neighboring subunits at the C terminus (side channel) is indicated. (D) Close-up top view as in B, showing the radial and helical nature of the 4 β-hairpins.

Fig. 3.

Structure-based mutagenesis and functional analysis of the mutants (also see Tables 1 and 2). (A) Location of mutations on the ssoMCM monomer structure. (B) Superose-6 size exclusion FPLC analysis of ssoMCM mutants in 0.25 M (blue line) and 1.0 M (pink line) NaCl. The molecular marker positions are indicated. The calculated molecular mass of ssoMCM monomer is 77 kDa, hexamer 462 kDa. (C) Representative helicase assay. B, Boiled dsDNA; UB, unboiled dsDNA; double lines, dsDNA; single lines, ssDNA. (D) Quantitative analysis of the helicase assay of the mutants, shown as the percentage of WT activity. Error bars represent the standard error from 3 experiments.

Fig. 4.

Two possible DNA unwinding modes by MCM helicase. (A) Schematic representation of a MCM hexamer helicase. The 4 β-hairpins (NT, H2I, PS1, and EXT hairpins) are represented by short solid bars; the central channel and the side channels are in darker shades. (B) Steric exclusion model for a single-hexameric MCM helicase. (C) Side-channel extrusion model, showing ssDNA extruding from the side channel. DNA is shown as black lines. Arrows indicate direction of helicase movement.