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-Å 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 β-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.

ATP-dependent conformational dynamics underlie the functional asymmetry of the replicative helicase from a minimalist eukaryote

“…3D reconstructions (at 24 Å resolution) showed that these images correspond to side and top/bottom views, respectively, of a lock washer-shaped hexameric particle ( Conversion of 2D EM projections into 3D structures by singleparticle reconstitution results in ambiguous chirality (27). Docking studies using the crystal structure of an archaeal MCM monomer (28) initially showed that six subunits could be reasonably fitted into both left-and right-handed EcuMCM2-7 reconstructions. However, cross-correlation values somewhat disfavored the right-handed conformation (Fig.…”

DNA Interactions Probed by Hydrogen-Deuterium Exchange (HDX) Fourier Transform Ion Cyclotron Resonance Mass Spectrometry Confirm External Binding Sites on the Minichromosomal Maintenance (MCM) Helicase

“…Examination of the apo-SsoMCM data reveals significant correlation with solvent-exposed regions within a previously developed hexameric model (6-fold symmetry) of the monomeric SsoMCM crystal structure (Protein Data Bank code 3F9V) ( Fig. 2) (14). There is one other nearly full-length crystal structure of a hexameric archaeal MCM that consists of a hybrid of S. solfataricus (residues 7-269) and Pyrococcus furiosus (residues 257-361 and 729 -965) (Protein Data bank code 4POG) (15).…”

“…Our HDX data seem to indicate a tighter compaction between subunits in this area than represented in the model. Given that the hexameric structure for SsoMCM is modeled based on 6-fold symmetry from a monomeric crystal structure (14), precise intersubunit contacts containing specific hydrophobic patches may be poorly represented by the model. Analysis of a helical filamentous SsoMCM structure (34) shows a tighter compaction in this area (region ii) between subunits (data not shown), but it is arranged in a much larger oligomeric structure and shifted slightly between subunits, which is not consistent with our hexameric solution state.…”

“…Using HDX-MS, we are able to probe interfacial residues within the SsoMCM hexamer from the monomeric x-ray structure (14) and define a more global interaction path of the excluded strand with the exterior surface, thus validating the SEW model as a bona fide unwinding mechanism.…”

“…Archaeal MCM helicases from Sulfolobus solfataricus (SsoMCM) and Methanothermobacter thermoautotrophicus serve as simplified models for understanding mechanisms of ATP hydrolysis, DNA binding, and unwinding for more complex eukaryotic hexameric DNA replication helicases (11). Crystal structures of archaeal MCMs with the N-terminal domain (NTD) alone (12,13) as well as nearly full-length structures with both the NTD and AAA ϩ domain (14,15) have been solved and show similar symmetry and subunit arrangements. It is interesting to note that an EM structure of M. thermoautotrophicus MCMs has identified a binding site for DNA on the external surface of the helicase, possibly implicated as a prebound engaged state prior to loading and encircling onto DNA at replication origins (16).…”