Volume 17, Issue 2, Pages 211-222 (February 2009) Insights into the Architecture of the Replicative Helicase from the Structure of an Archaeal MCM Homolog  Brian Bae, Yi-Hsing Chen, Alessandro Costa, Silvia Onesti, Joseph S. Brunzelle, Yuyen Lin, Isaac K.O. Cann, Satish K. Nair  Structure  Volume 17, Issue 2, Pages 211-222 (February 2009) DOI: 10.1016/j.str.2008.11.010 Copyright © 2009 Elsevier Ltd Terms and Conditions

Figure 1 Multiple Sequence Alignments of Archaeal and Eukaryotic MCM Structure-based multiple sequence alignment of Methanopyrus kandleri MCM2 (GenBank accession code 20094556), Methanopyrus kandleri MCM1 (20094401), Methanothermobacter thermautotrophicus str. Delta H MCM (15679758), Sulfolobus solfataricus MCM (6015702), Homo sapiens MCM4 (33469919), and MCM2 (33356547). Strictly conserved, highly conserved, and semiconserved residues are colored in magenta, yellow, and cyan, respectively. Secondary structure elements are derived from the structure of MkaMCM2, and dashed lines represent disordered regions. Sequence motifs that are characteristic of MCM proteins are denoted below the aligned sequences. Residues that are deleted in the MkaMCM2 sequence or have not been modeled in the structure are drawn as thin line. Secondary-structure elements within the amino-terminal domain are named based on the notation of Fletcher et al., 2003. Structure 2009 17, 211-222DOI: (10.1016/j.str.2008.11.010) Copyright © 2009 Elsevier Ltd Terms and Conditions

Figure 2 Structure of the Full-Length M. kandleri MCM2 Monomer (A) Ribbon diagram of the overall structure of full-length MkaMCM2 illustrating the structural domains. The amino-terminal helical domain (light blue), OB fold domain (pink), and the carboxy-terminal AAA+ ATPase module (red) are shown, with the two β-hairpins of the AAA+ module highlighted in yellow and green. The PS1BH and H2 insert define a distinct clade of AAA+ domains that include the MCM proteins. Note that the secondary structure elements are numbered for consistency with the structure of the MthMCM amino-terminal domain. The nonsequential numbering of the strands in the OB-fold domain reflects the fact that the zinc-binding extension is deleted in the sequence of MkaMCM2. (B) Close-up view of the β-hairpins that are thought to respond to DNA binding and nucleotide hydrolysis. Note that the H2 insert is positioned adjacent to β strands β4 and β7 in the OB-fold domain. These strands form the base of the zinc-binding module (domain B) found in MthMCM. Interaction between these β strands of the OB fold and the PS1BH and H2 insertions provides a mean of cross-talk between the catalytic AAA+ module and the amino-terminal domain. The position of a highly conserved tryptophan residue (Trp-363 in MkaMCM2) that has been shown to respond to ATPase and helicase activities (Jenkinson and Chong, 2006) is situated in the H2 β-hairpin insert. Structure 2009 17, 211-222DOI: (10.1016/j.str.2008.11.010) Copyright © 2009 Elsevier Ltd Terms and Conditions

Figure 3 Cryo-EM Density Map of the MCM Hexamer (A and C) Surface rendering of the cryo-EM density map (displayed at 2σ) viewed from the top and the side, respectively. The side view clearly shows the lateral holes, which are disrupted by an isthmus of electron density. (B and D) Fitting of the AAA+ domain from MkaMCM2 (red) and the N-terminal domain from MthMCM (green, Fletcher et al., 2003). The AAA+ domain of MkaMCM2 matches well the dome-shaped electron density. (E) A view showing the fitting of two hybrid monomers (including the N-terminal domain of MthMCM and the AAA+ domain of MkaMCM2). The N-terminal domain of one subunit (highlighted in green) communicates with the AAA+ domain of the next-neighboring subunit (in red) through the interaction between the PS1BH of the AAA+ domain (shown in yellow) and the β7-β8 loop of the N-terminal domain of an adjacent subunit (in orange) consistent with biochemical studies on MthMCM (Sakakibara et al., 2008). (F) A close up of the same interaction. Structure 2009 17, 211-222DOI: (10.1016/j.str.2008.11.010) Copyright © 2009 Elsevier Ltd Terms and Conditions

Figure 4 Unique Trans-Acting Residues Characterize the MCM AAA+ Domain (A) Model of the MCM hexamer derived as per Figure 3B showing the location of the composite active site formed between two AAA+ domains (colored in blue and red). (B) A close up view of the composite active site showing residues that have been demonstrated to be critical for ATP hydrolysis. The composite active site in MCM has unique features that distinguish the AAA+ modules of these proteins from classical AAA+ ATPases such as DnaA. (C) Within the active site of DnaA (Erzberger et al., 2006), the Walker A and B motifs, sensor I and sensor II helix, are situated in one molecule as cis-acting elements, but the critical arginine finger is a trans-acting residue that is contributed from a neighboring subunit. (D) In contrast, the sensor II helix in MCM functions in trans and is contributed by a neighboring subunit. (E–H) This arrangement of trans-acting elements is similar to that found in viral superfamily III helicases, such as the papillomavirus E1 helicase (Enemark and Joshua-Tor, 2006) (E). An additional trans-site has been identified by biochemical analysis of SsoMCM (Moreau et al., 2007) and consists of a motif containing a highly conserved acidic residue at the base of PS1BH. Comparison of the structure of the SV40 large T antigen bound to (F) ATP and (G) ADP demonstrates that this acidic residue orients the arginine finger in response to the nature of the nucleotide (Gai et al., 2004). Likewise, a similar, highly conserved acidic residue is located as a trans-element in MCM (H) where it might similarly affect intersubunit association in response to nucleotide hydrolysis. Structure 2009 17, 211-222DOI: (10.1016/j.str.2008.11.010) Copyright © 2009 Elsevier Ltd Terms and Conditions