Structure and regulatory role of the C-terminal winged helix domain of the archaeal minichromosome maintenance complex

Abstract

The minichromosome maintenance complex (MCM) represents the replicative DNA helicase both in eukaryotes and archaea. Here, we describe the solution structure of the C-terminal domains of the archaeal MCMs of Sulfolobus solfataricus (Sso) and Methanothermobacter thermautotrophicus (Mth). Those domains consist of a structurally conserved truncated winged helix (WH) domain lacking the two typical ‘wings’ of canonical WH domains. A less conserved N-terminal extension links this WH module to the MCM AAA+ domain forming the ATPase center. In the Sso MCM this linker contains a short α-helical element. Using Sso MCM mutants, including chimeric constructs containing Mth C-terminal domain elements, we show that the ATPase and helicase activity of the Sso MCM is significantly modulated by the short α-helical linker element and by N-terminal residues of the first α-helix of the truncated WH module. Finally, based on our structural and functional data, we present a docking-derived model of the Sso MCM, which implies an allosteric control of the ATPase center by the C-terminal domain.

Mechanisms and regulation of DNA replication initiation in eukaryotes

“…Insofar as the MCM CTD, solution structures have shown this region to adopt a canonical winged-helix (WH) domain (Figure 4A) (Wei et al, 2010, Liu et al, 2012a, Wiedemann et al, 2015), which based on the weak or absent CTD density in structures of many full-length MCM proteins, appears flexibly tethered to the AAA+ core. Although the position of this domain with respect to the hexamer was at first unclear, recent structural and biochemical work demonstrates that the WH domain sits distal to the NTD (Li et al, 2015, Wiedemann et al, 2015, Yuan et al, 2016).…”

“…Although the position of this domain with respect to the hexamer was at first unclear, recent structural and biochemical work demonstrates that the WH domain sits distal to the NTD (Li et al, 2015, Wiedemann et al, 2015, Yuan et al, 2016). The S. solfataricus MCM WH domain can bind single-stranded DNA weakly (Pucci et al, 2007); however, what role this activity plays in vivo , and whether the WH domains of the eukaryotic MCMs can also bind DNA, are open questions.…”

“…The S. solfataricus MCM WH domain can bind single-stranded DNA weakly (Pucci et al, 2007); however, what role this activity plays in vivo , and whether the WH domains of the eukaryotic MCMs can also bind DNA, are open questions. The majority of data reported thus far suggests that, at least in archaea, the MCM WH domain functions to allosterically modulate the ATP hydrolysis rate of the AAA+ domain (Jenkinson and Chong, 2006, Barry et al, 2007, Wiedemann et al, 2015). In addition, this region can serve as a protein/protein interaction site, with the WH domains of eukaryotic MCM3 and MCM6 having been shown to engage Cdc6 and Cdt1, respectively (You and Masai, 2008, Liu et al, 2012a, Frigola et al, 2013).…”

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

“…Because this is the only other region that shows significant deviation between the hexameric model and our HDX data, we propose that the difference is due to the flexible C-terminal helix-turn-helix (HTH) domain interacting at this site. The HTH domain is absent from all available x-ray structural data and is typically removed for crystallization trials because of its flexibility; however, a recent NMR structure of the HTH domain alone has been determined and docked onto the nearly full-length x-ray structure at the ␣6 -␣8 helices (35). Unlike other data supporting this hypothesis, our HDX experiments include all residues of SsoMCM and can therefore directly identify protected regions unobserved in other structurally modified experiments.…”

“…The detection of deuteration changes on the exterior surface of SsoMCM could highlight an initial engaged DNA binding state for the helicase that is a precursor to loading. External regions of reduced deuteration occur at the extreme C terminus (residues 590 -599, ␣8-helix) near where the ssDNA-dsDNA junction is proposed to bind (33) and where the HTH domain is proposed to interact (35). The HTH domain itself shows no significant changes upon DNA binding.…”