Intersubunit allosteric communication mediated by a conserved loop in the MCM helicase

Abstract

The minichromosome maintenance (MCM) helicase is the presumptive replicative helicase in archaea and eukaryotes. The archaeal homomultimeric MCM has a two-tier structure. One tier contains the AAA+ motor domains of the proteins, and these are the minimal functional helicase domains. The second tier is formed by the N-terminal domains. These domains are not essential for MCM helicase activity but act to enhance the processivity of the helicase. We reveal that a conserved loop facilitates communication between processivity and motor tiers. Interestingly, this allostery seems to be mediated by interactions between, rather than within, individual protomers in the MCM ring.

Fig. 1.

A conserved loop in the N-terminal tier of MCM is required for intersubunit communication. (A) Mutant doping assay using the C-half of both wild-type MCM (residues 267–612) and Walker A lysine–alanine mutant (K346A in the full-length protein) in varying proportions in helicase assays. The red line indicates the nonlinear behavior of the full-length proteins (ref. 8). (B) Structure of the N-terminal 265 residues of SsoMCM [Protein Data Bank (PDB) ID code 2vl6]. The figure was by using PyMOL (http:://pymol.sourceforge.net). The ACL is highlighted in red, and residues E202, E203, and Q208 are shown in brown stick form. The N-terminal DNA-binding β-hairpin is shown in dark blue. A zinc ion is shown as a brown sphere. (C) Sequence line-up of the ACL of seven representative archaeal MCM sequences (Afu, Archaeoglogus fulgidus; Csy, Cenarchaeum symbiosum; Hby, Hyperthermus butylicus; Mth, M. thermautotrophicus; Neq, Nanoarchaeum equitans, Sso, S. solfataricus; Tpe, Thermofilum pendens) together with human MCM2–9. The ACL is indicated in red, and residues corresponding to SsoMCM E202, E203, and Q208 are indicated by brown asterisks. (D) DNA binding to a flayed duplex substrate for wild-type MCM (WT-MCM), ΔACL, and ACL-PM (E202A, E203A, and Q207A) are shown (analogous results for ssDNA and dsDNA are shown in Fig S2). Error bars in this and all other panels are ±SD. (E) DNA helicase activity of WT, ΔACL, and ACL-PM MCM. (F) ATPase rates of the WT, ΔACL, and ACL-PM MCM. Values are picomoles of ATP hydrolyzed per second per nanomole of MCM (as monomer). (G) Mutant doping with ΔACL or Walker A mutant (K346A) into wild-type MCM. The helicase activities of the protein mixtures were measured and plotted relative to the activity of wild-type protein. Simulations (red and cyan lines) were performed as described in ref. .

Fig. 2.

Functional consequences of ACL deletion. (A) Pulldown assays of wild-type (WT) or ΔACL versions of the N-terminal domains of MCM (residues 1–266) on a matrix with immobilized C-half of MCM. After pulldown and washing, proteins were eluted by boiling in SDS/PAGE loading buffer. Proteins were detected after SDS/PAGE by staining with Coomassie brilliant blue. The positions of the various species are indicated. Analogous assays performed in the presence of ADP, ATP, AMP-PNP, and ADP-AlFx are shown in Fig. S5. (B) Helicase assays mediated by 0.5 μM C-half of MCM supplemented with increasing amounts of either wild-type or ΔACL N-half. (C) Helicase assays with full-length proteins with either wild-type, deleted (ΔNβHP) or point-mutated (KR246AA) N-terminal β-hairpin proteins with additional deletion of the ACL (ΔACL) as indicated. (D) Comparison of the relative processivity of wild-type or ΔNβHPΔACL proteins.

Fig. 3.

Nucleotide-modifiable communication between the C-terminal pre-Sensor 1 β-hairpin and the ACL. (A) (Left) Superposition of the MthMCM EM structure (4) with the known structure of MthMCM N-terminal domains (in cyan; the ACL is highlighted in red). The C-terminal domains are modeled based on the structure of a bacterial ATPase (PDB ID code 2R44) that is predicted to have secondary structure architecture similar to MCM (conserved domain database expected value = 1×e⁻⁴). (Right) Enlargement of the selected area. The C-terminal β-hairpin is highlighted in yellow. (B) DEER time trace after background correction shown in black for nucleotide-free MCM and in red MCM in the presence of 10 mM ATP. The best-fit line was found by using a Tikhonov regularization parameter of 1,000 and is shown in red. (C) The distance distributions corresponding to the DEER traces shown in B; black is nucleotide-free MCM, and red is MCM in the presence of 10 mM ATP. (D) Results of cross-linking assays with S206C (Ncys), K430C (Csys) single mutants or S206CK430C (NcysCsys) double mutants. The positions of monomer, dimer, and higher-order species are indicated. Wild-type (cysteine-free protein) is labeled W. M refers to mock-treated (reaction minus BMOE), and X indicates reactions treated with BMOE. (E) Cross-linking assays performed between S206C (Ncys) and K430C (Ccys) in the presence or absence of the indicated nucleotide (at 10 mM).

Fig. 4.

Model for the interplay between C-terminal β-hairpin of one subunit and the ACL of its neighboring subunit in modulation of the position of the neighbor's N-terminal β-hairpin. We propose that, during the cycle of ATP binding and hydrolysis and release of ADP from one subunit, its C-terminal β-hairpin is repositioned, allowing it to contact the ACL of its neighbor and thus reposition the N-terminal β-hairpin of that subunit. (Left) The 34-Å distance indicated in the white subunit would correspond to the ATP-bound version of the protein. (Right) The 30-Å distance between the white subunit ACL and C-terminal β-hairpin and close apposition of the β-hairpin to the ACL of the neighboring subunit would correspond to the nucleotide-free form.