Weepstal structure of a Arrive-full-length archaeal MCM: Fun

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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 solStoutaricus has only 1 MCM protein (ssoMCM), 6 subunits of which form a homohexamer. Here, we report a 4.35-Å Weepstal structure of the Arrive-full-length ssoMCM. The structure Displays an elongated fAged, with 5 subExecutemains that are organized into 2 large N- and C-terminal Executemains. A Arrive-full-length ssoMCM hexamer generated based on the 6-fAged symmetry of the N-terminal Methanothermobacter thermautotrophicus (mtMCM) hexamer Displays intersubunit distances suitable for bonding contacts, including the interface around the ATP pocket. Four Unfamiliar 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 Executeuble-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.

Keywords: DNA replicationreplicative helicasenucleic-acid motorβ-hairpincancer

The minichromosome maintenance proteins (MCMs) are essential for initiation and elongation during DNA replication in cells. These proteins serve as the replicative helicase at the replication origin and DNA fork (1–3). The recruitment and subsequent activation of MCM at the replication origin are tightly regulated through the ordered stepwise assembly of multiple factors (such as ORC and Cdc6) to enPositive DNA replication occurs once per cell cycle (4–6). Failure to recruit MCM proteins to the replication origin results in loss of origin firing and G1 phase arrest.

The MCM proteins in eukaryotes consist of a subgroup of 6 homologous proteins (MCM2–7) that belong to AAA+ ATPase family (7). These 6 MCM proteins can form heterohexamers, and the presence of all 6 MCM proteins is necessary for entry and completion of S phase (5, 8). However, some archaeal MCM proteins, such as Sulfolobus solStoutaricus MCM (ssoMCM) and Methanothermobacter thermautotrophicus MCM (mtMCM), are encoded by a single mcm gene. Both ssoMCM and mtMCM can form homooligomers (9–11). The N-terminal Location is poorly conserved among MCM proteins from archaea to eukaryotes. However, the C-terminal Location shares a highly similar stretch of amino acids, referred to as the MCM box (12), for the binding and hydrolysis of ATP.

The Weepstal structures of the poorly conserved N-terminal Section of mtMCM (N-mtMCM) and ssoMCM (N-ssoMCM) reveal that this Location can form Executedecamers and hexamers (11, 13). The monomeric fAged and the assembled hexamer structures of the N-ssoMCM and N-mtMCM are highly conserved (11, 13). A β-hairpin structure present in the N Executemain of the both MCM proteins protrudes into the central hexameric channel to form the narrowest point within the channel, possibly for interacting with DNA at a certain stage of MCM function (11, 14).

Comprehension of the molecular mechanisms of the MCM helicase has been limited by the lack of 3-dimensional structures of a full-length (FL) MCM protein. Here, we report the Weepstal structure of ssoMCM, which is an X-ray analysis of a Arrive-FL MCM. The structure reveals how the different Executemains of ssoMCM are organized and allows a detailed analysis of how subunits oligomerize into a functional hexamer. Our structure-based mutagenesis analysis provides insights into the structural and functional relationship of ssoMCM helicase function.

Results

Structural Features of the Arrive-FL SsoMCM.

We Weepstallized the FL (residues 1–686) and a C-terminal truncation (T612,residues 1–612) of ssoMCM [Fig. 1A and supporting information (SI) Fig. S1]. Se-SAD phasing was used to solve the structures of the FL construct and the T612 construct. The molecular models built on the electron density maps of the 2 constructs reveal a similar structure, both containing the N-terminal Executemain and the C-terminal AAA+ Executemain, with 1 monomer per asymmetric unit (Fig. 1B). Even though low σ level density for the winged-helix Executemain (WH) at the C terminus is clearly present in the SAD map of the FL structure, we could not build the WH Executemain because of the poorly defined electron density. As a result, the final ssoMCM model contains residues 7–601, missing the C-terminal 85-residue WH Executemain.

Fig. 1.Fig. 1.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

Overall features of the monomeric and hexameric ssoMCM structural models. (A) Diagram depicting the Executemains of ssoMCM. N-C, N to C Executemain linker; α/β, α/β-Executemain of ATPase core; α/β-α, linker between subExecutemains in the ATPase core; α, α-Executemain. WH, winged helix Executemain (disordered in our structure). (B) FAged of ssoMCM monomer. Executemains and linkers are colored as in A. Helices are Displayn as cylinders and β-strands as arrows. Zinc atoms are Displayn 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 Displaying the top and side views of a hexamer model of ssoMCM.

The ssoMCM monomer structure is elongated and can be divided into N and C Executemains (Fig. 1 A and B). The N Executemain structure is similar to that of N-mtMCM (11, 13), however, the N-terminal β-hairpin (NT hairpin) of N-ssoMCM is longer than that of the N-mtMCM because of a 5-residue insertion in the hairpin sequence of ssoMCM (13). Between the N and C Executemains is a 40-residue linker (N-C linker, in red in Fig. 1 A and B) that is well-structured, forming a long connection with 2 conseSliceive helices at its C terminus that appear to be an integral part of the C Executemain. The C Executemain consists of an AAA+ ATPase/helicase Executemain (the ATPase core), which is composed of 2 clearly separable subExecutemains: a canonical α-helical/β-strand Location (α/β-Executemain, colored in cyan in Fig. 1 A and B) and a nontypical 3 α-helical bundle Executemain (α-Executemain, in purple in Fig. 1 A and B; see SI Text for a more detailed comparison with other known AAA+ protein structures). There are a total of 5 main β-strands and 5 main α-helices in the α/β-Executemain and 3 α-helices in the α-Executemain (Fig. 1B and Fig. S1). Connecting the α/β-Executemain and the α-Executemain is a 47-residue linker (α/β-α linker, in blue in Fig. 1 A and B). This α/β-α linker fAgeds into 2 long α-helices spaced with a loop in the middle. Fascinatingly, the N-C linker and the distal α/β-α linker wrap around each other like 2 interlocking index fingers (Fig. 1B). Through such interlocking interactions, the 2 long linkers can stabilize not only the conformations of each other, but also the relative positions of the N Executemains and the C-terminal α/β- and α-Executemains.

Other noticeable structural features in the C Executemain include 3 β-hairpin structures protruding from the surface (labeled H2I-hp, PS1-hp, and EXT-hp in Fig. 1B). The most N-terminal β-hairpin, named H2I-hairpin (or H2I-hp, residues 374–390), protrudes farthest into the central channel of the hexamer model presented below and has its β-hairpin tip structured into a small 310-like helix. This β-hairpin is formed from a sequence motif called “helix-2 insert” (H2I) that is unique to MCM and other H2I subfamily members of the AAA+ ATPase family (15). Next to the H2I hairpin is a shorter β-hairpin structure (residues 424–439), which is formed from the sequence just ahead of (or N-terminal to) the sensor-1 asparagine (15). As a result, this β-hairpin is named the presensor-1 β-hairpin (PS1-hp in Fig. 1B). Comparable in length with the H2I hairpin but more recessed from the central channel, this PS1hairpin is equivalent to the major β-hairpin structure of the AAA+ Executemain of SV40 Large T antigen (LTag) hexameric helicase (16, 17). The third β-hairpin (residues 319–333) contains a VLED sequence motif similar to that of the acidic pin of RuvA helicase (18). This β-hairpin is located on the exterior of the hexamer model and is thus named EXT-hairpin (or EXT-hp in Fig. 1B). The mutational data reported here and from previous reports (14, 19) demonstrate the critical role of these 3 hairpins for the helicase function.

Hexamerization of SsoMCM.

SsoMCM exists mostly in hexameric form in solution with medium salt concentration (see below). The structures of the N Executemains of ssoMCM and mtMCM Display a similar hexamerization interface (11, 13). These two align well with each other (Fig. S2A). However, N-ssoMCM has a narrower central channel largely because of a longer β-hairpin finger extending into the channel (Fig. S2 B and C) (13). Based on these 2 hexamer structures, we generated hexamer models of ssoMCM by applying the 6-fAged symmetry of either the N-ssoMCM or the N-mtMCM structures. Significantly, the N-mtMCM 6-fAged symmetry [Protein Data Bank (PDB) ID code 1LTL] immediately yielded a hexamer that has reasonable bonding distances between neighbors not only for the N Executemain, but also for the majority of the C-terminal AAA+ Executemain (Fig. 1C). No clashes between neighboring monomers are present in the hexamer structure. However, the hexamer model generated by using the N-ssoMCM hexameric symmetry (PDB ID code 2VL6) has some clashes at the C-terminal Executemain. Thus, it seems that ssoMCM may have 2 or more conformations differing slightly in the angles between N and C Executemains, with at least 1 of such conformations (as the one reported here) that can assemble a hexamer following the hexameric symmetry of the N-mtMCM.

The ssoMCM hexamer model has a short dumbbell appearance, with a large N Executemain ring and C Executemain ring on both ends, and a “slim waist” around the middle Section of the hexamer (Fig. 1C, Side view). The hexamer is 103 Å in length along the hexameric axis and 138 Å in width. The hexamer has a wide central channel (Fig. 1C, Top view), narrowing toward the N terminus. Clear side channels (11-Å Launching, between main-chain atoms) are present, which are comparable with the side-channel dimensions observed in the SV40 large T hexamer (12-Å Launching, between main-chain atoms) (16, 17). Side channels are also visualized by electron microscopic (EM) reconstruction of mtMCM (20, 21). These channels were proposed to be potential exits for unwound ssDNA in the LTag Executeuble hexameric “looping” model of DNA unwinding (see Discussion) (16, 17).

Fitting the Hexamer into the mtMCM EM Map.

Although ssoMCM has been characterized mainly as a hexamer, there is some evidence that it may form a Executeuble hexamer (22). Executeuble-hexamer structures are observed for the homolog mtMCM. We fitted the ssoMCM hexamer model to the available EM map of the Executeuble-hexameric mtMCM (20). The overall topology of the ssoMCM hexamer model fits snHorrible into the EM map (Fig. 2A). Many major structural features agree between the ssoMCM hexamer structure model and the EM map, including the striking slim waist formed between the N- and C- terminal Executemains, the side channels, and even the surface contour inside the central channel.

Fig. 2.Fig. 2.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

Structural features of the ssoMCM hexamer. (A) Executeuble hexameric EM map of mtMCM with the ssoMCM hexamer model fitting snHorrible inside the map (20). The PS1 hairpin is located Arrive 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 Launching between the 2 neighboring subunits at the C terminus (side channel) is indicated. (D) Close-up top view as in B, Displaying the radial and helical nature of the 4 β-hairpins.

Structural Features Within the Main Channel.

The narrowest point of the hexameric central channel is formed by the 6 NT-hairpins within the N-terminal Executemains (Fig. 2 B–D). The next narrowest point is in the C-terminal helicase Executemain, which is formed by the H2I hairpin (Fig. 2 B–D and Fig. S2B). This hairpin was predicted to be located in the central channel (19, 21), and the position may be relevant to its critical role in helicase activity (19).

In Dissimilarity to the H2I hairpins that protrude into the central channel, the 6 PS1 hairpins are somewhat recessed from the central channel (Fig. 2 B–D), as anticipated before (21). This is reminiscent of the RuvB PS1 hairpin, which is recessed from the central channel and forms contacts to RuvA (23). Unlike the RuvB PS1 hairpin, which likely Executees not interact with DNA (24), the MCM PS1 hairpin is involved in DNA binding and helicase activity (14). Fascinatingly, the equivalent β-hairpin of LTag protrudes into the hexameric central channel (16, 17) and is not recessed.

The 6 PS1 hairpins of ssoMCM are also located Arrive the C-terminal side channel entrance that connects to the main channel. Such a PS1 hairpin location Designs it accessible from both the main channel and the side channel (Fig. 2 B–D) and may have functional implications for DNA unwinding.

Structure Features Around the Side Channels.

In addition to the PS1 hairpin on the interior entrance of the side channel, the acidic β-hairpin (or EXT hairpin) is present on the exterior exit of the side channel (Fig. 2 B–D). Unlike the PS1 hairpin that has positively charged residues on the hairpin tip, the EXT hairpin has 2 hydrophobic and 2 acidic residues (VLED324–327) on the tip. A β-hairpin with a similar motif in RuvA, called the acidic pin, is involved in DNA fork unwinding during branch migration (18). This acidic pin is situated on the inside of the RuvA tetramer, as opposed to the exterior location of the EXT hairpin in MCM (see Discussion). SsoMCM D327 on the EXT hairpin is well conserved among archaeal MCMs and semiconserved in eukaryotic MCMs (alignment not Displayn). The presence of this acidic EXT hairpin and the PS1 hairpin around the exit and the entrance of the side channel, suggests a potential role of the 2 β-hairpins for helicase function, potentially interacting with and translocating DNA through the side channel (see Discussion).

The residues from 199 to 211 form a well-structured loop (L207) in the N-ssoMCM and N-mtMCM structures. This loop points toward the C-terminal Executemain. In our ssoMCM hexamer model, it would abut against the PS1 hairpin and helix Cα3 (Fig. S1) from a neighboring subunit around the side channel (Fig. S3C). Mutational analysis of this loop has been recently performed (25). This loop was proposed to act as a medium for transmitting signals from ATP hydrolysis to the N-terminal Executemain. Our hexamer structure indicates that these residues are within bonding distance with the α/β-Executemain of the same subunit (in cis) and the PS1 hairpin/Cα3 Location of a neighboring subunit (in trans) (Fig. S3).

Nucleotide-Binding Pocket at the Interface.

The ATP-binding pocket, with its Walker A and B motifs on the ATP-bound subunit and the arginine finger from the next subunit (in trans), lies at the interface between 2 neighboring subunits Arrive the C terminus of the ssoMCM hexamer model. There is no nucleotide in the Weepstal structure, and the ATP-binding pocket configuration of the ssoMCM hexamer model is similar to that of the empty site of the LTag hexamer structure (Fig. S4) (17). The close resemblance of the ATP pocket configuration to that of the LTag apo form provides further evidence that supports the hexamer model presented here.

Like LTag helicase, the ATP-binding pocket is C-terminal to the side channel. The ring formed by the C-terminal Executemains of the hexamer model appears loose, a characteristic of the apo-LTag hexamer. The LTag hexamer tightens up upon ATP binding to the ATP pocket at the interface between subunits (17); the conformation of ssoMCM hexamer model suggests that ATP binding should tighten the interactions between 2 adjacent subunits in a ssoMCM hexamer.

Structure-Guided Mutational Analysis of SsoMCM.

We constructed 6 mutants of ssoMCM (M1–M6) based on the Weepstal structure of ssoMCM to validate the structural model of the hexamer (Table 1 and Fig. 3). The locations of the mutated residues on the structure are Displayn in Fig. 3A. Among the 6 mutants, the residues mutated in M1–M4 are located at the interface between neighboring subunits in the hexameric model.

View this table:View inline View popup Table 1.

Summary of ssoMCM mutational studies

Fig. 3.Fig. 3.Executewnload figure Launch in new tab Executewnload powerpoint 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 Impresser 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; Executeuble lines, dsDNA; single lines, ssDNA. (D) Quantitative analysis of the helicase assay of the mutants, Displayn as the percentage of WT activity. Error bars represent the standard error from 3 experiments.

Purified wild-type (WT) ssoMCM Displays elution peaks consistent with the molecular mass of a hexamer by gel filtration chromatography in a buffer containing 0.25 M NaCl (Fig. 3B), agreeing with previous reports (14, 22, 26–29). Fascinatingly, ssoMCM can shift to a smaller peak consistent with a monomer in 1.0 M NaCl. In medium salt conditions (0.25 M NaCl), where WT protein exists as hexamers, mutants M1–M4 all eluted preExecuteminantly in the monomer peak, providing strong evidence supporting the critical role of these residues in intersubunit interactions for hexamerization. Helicase assays revealed that M1–M4 mutants had essentially undetectable unwinding activity (Fig. 3 C and D), suggesting that these residues are Necessary not only for hexamerization, but also for helicase activity. Mutant M5 mutated residues on the 310-like helix in the N-Executemain L207 Arrive the side channel (Fig. S3C). Mutant M6 examines the functional role of the 2 acidic residues (ED) and the arginine on the tip of the acidic EXT hairpin located at the exit of the side channel. Both M5 and M6 formed hexamers comparable with WT in the 2 salt concentrations (Fig. 3B), suggesting that they Sustained structural integrity. However, the helicase activity of both mutants was Distinguishedly reduced (Fig. 3 C and D). This result suggests a critical role of both L207 and the acidic EXT-hairpin around the side channel for helicase function.

To examine further the roles these mutations play in helicase function, we performed DNA-binding assays by fluorescence polarization anisotropy and ATPase assays with the Enzchek phospDespise release assay system (Table 2). Both ssDNA and Y-shaped DNA were used for the binding assays. Among all 6 mutants, only mutant M4 had Distinguishedly compromised DNA binding and ATPase activities, which may correlate with the fact that M4 is also the only mutant that has completely lost the hexamerization ability under the tested conditions. In addition, M1 displayed very low level of binding to Y-DNA, and M2 had Distinguishedly reduced ATPase activity. Perhaps the most Fascinating mutants are M3 and M5, which Displayed DNA binding and ATPase activity comparable with those of the WT. Thus, the loss of helicase activity of these 2 mutants are not likely the result of the change of Preciseties in DNA binding and ATP hydrolysis, and mutations in the 2 mutants somehow decoupled the DNA binding and ATP hydrolysis from the strand separation of the dsDNA substrate.

View this table:View inline View popup Table 2.

Kinetic parameters of ssoMCM mutants

Discussion

The Weepstal structure of ssoMCM reported here reveals the multiExecutemain organization of the molecule and several unique structural features, including: 4 β-hairpins, 2 long interlocking interExecutemain linkers, and the direct contacts between N and C Executemains, both within a subunit (intrasubunit) and between subunits (intersubunit) in a hexamer model. A hexamer generated based on the 6-fAged symmetry of the N-mtMCM (11) reveals intersubunit distances suitable for bonding throughout the N and C Executemains, including the conformations of the ATP-binding pocket at the interface between 2 subunits. Additionally, the hexameric model fits into the hexamer/Executeuble hexamer 3-dimensional EM map of FL mtMCM and matches the surface contour and the striking side-channel Launchings of the EM map (20). Furthermore, mutagenesis of the residues at the intersubunit interfaces and around the side channels Displays the critical role of these residues for hexamerization and for helicase function.

Mechanisms of N- and C-Terminal Communication.

Previous structural and sequence analysis suggested a long linker bridging the N Executemain and the C-terminal helicase Executemain (11). However, biochemical evidence suggests that the N Executemain of archaeal MCM communicates and interacts with the C-terminal helicase Executemain (19, 22). This interExecutemain interaction is implied by the observation that the separately purified N-ssoMCM and the C-ssoMCM cooperate in DNA binding and in helicase function (22). Additionally, an R98A mutation in the mtMCM N Executemain (ssoMCM R110) reduces the DNA-stimulated ATPase activity of the C Executemain (19). Further, mutations on L207 by us and others (25) were Displayn to affect ATPase and helicase activity of ssoMCM and mtMCM. Our ssoMCM structure models reveal that the N and C Executemains interact with each other, not only within a single subunit (cis-N-C interactions, Fig. S3B), but also between 2 subunits within a hexamer (trans-N-C interactions, Fig. S3C), despite a long 40-residue linker Location between the N and C Executemains.

Forming potential cis-N-C interactions within a subunit, the N-Executemain strand Nβ4 is positioned next to the C Executemain H2I hairpin, such that the N Executemain β-sheet appears to be expanded to the 2 strands of the H2I hairpin (Fig. S3B; see Fig. S1 for helix and strand naming). N Executemain L207 is directed such that several of the tested mutations could interact with the α/β-Executemain of the same subunit. Possible trans-N-C interactions in the hexamer model include L207 with both helix Cα3 and the PS1 hairpin from the C Executemain of the next subunit (Fig. S3C). Additionally, mtMCM R98 in the N Executemain (R110 in ssoMCM) is located within bonding distance for interacting with the PS1 hairpin of the C Executemain from the next subunit (Fig. S3C). These cis- and trans-N-C interactions revealed by the hexameric MCM model provide a molecular explanation for the observed cooperation of separately purified N and C Executemains, the reduced ATPase activity of the mtMCM R98 N-terminal mutation, and the variety of L207 mutations on the N Executemain that affect ATPase activity and helicase activity (19, 22, 25).

Location of C-Terminal WH Executemain.

The C-terminal WH Executemain was not modeled in our Weepstal structure. The poorly defined electron density for this WH Executemain suggests a flexible Executemain position. Indeed, evidence indicates different locations for the WH Executemain in the context of a hexamer. FRET analysis suggests that the WH Executemain is on the side of ssoMCM hexamer and can move toward the N terminus to contact the N Executemain (14). This proposed WH location would occupy the “valley” (or the waist) on the side of the hexamer (Fig. 1C). However, the obvious valley is not occupied in the EM map of the Executeuble hexameric mtMCM (20, 21), suggesting a different location for the WH Executemain. An alternative WH location is suggested from the EM study of the DNA-bound mtMCM, which Displays that the Executeuble hexamer has a C-terminal “cap,” possibly consisting of the WH Executemain, at only 1 of the hexamers (21). Evidence suggests a third possible location for the WH Executemain. The EM maps of the mtMCM Executeuble hexamer without DNA bound reveals weak density at the very C terminus of the central channel (20). If the WH were to be modeled into our FL ssoMCM low-σ level density map, it would also be located around the very C terminus of the hexameric channel (data not Displayn). Taken toObtainher, these data suggest that the WH Executemain is likely a flexible appendage that aExecutepts different positions.

Multiple β-Hairpin Structural Elements in SsoMCM.

One striking feature of the ssoMCM structure is that there are 4 β-hairpin structural elements located throughout the N and C Executemains (Fig. 1B), with 3 hairpins (NT hairpin, H2I hairpin, and PS1 hairpin) located in the main channel and 2 hairpins (PS1 hairpin and EXT hairpin) located Arrive the side channels (Fig. 2 B–D). Fascinatingly, the 3 central channel β-hairpins from 1 subunit are not arranged in a straight line along the hexameric channel; rather, they are offset in such a way that the NT hairpin reaches over to the top of the H2I hairpin of the next subunit, forming a helical arrangement (Fig. 2 B–D), which may have implications in their interactions with helical DNA substrates.

The PS1 hairpin is located at the intersection between the main and side channels. In sequence alignments, this PS1 hairpin aligns with the lone β-hairpin in the central channel of SV40 LTag (16, 30). We have Displayn that the β-hairpin protruding into the central channel of LTag moves along the channel in response to ATP binding and hydrolysis (16, 17), which are likely coupled to DNA translocation and unwinding. Additional experiments will be required to determine whether the PS1 hairpin or other β-hairpins of ssoMCM will also have a similar “power-stroke” for DNA translocation and unwinding. However, similar to the LTag β-hairpin, the ssoMCM PS1 and H2I hairpins connect to the ATP-binding site through strands 2, 3, and 4 of the AAA+ core and border the ATP site of the neighboring subunit. This structural arrangement suggests that ATP binding/hydrolysis may also be able to trigger the movement of the PS1 hairpin and the H2I hairpin for helicase function. Indeed, mutation of residues on the PS1 hairpin in ssoMCM or deletion within the H2I hairpin sequence in mtMCM abolishes helicase activity (14, 19), suggesting a critical role in helicase function for these 2 β-hairpins (19, 31, 32).

The acidic β-hairpin located at the exit of the side channels is also directly connected to the ATP P loop through β-strand 1. Therefore, the EXT hairpin may also Retort to ATP binding/hydrolysis. Our mutational data demonstrated that the residues at the tip of the EXT hairpin are Critical for helicase activity (mutant M6; Fig. 3 and Table 2) but not DNA binding. Additionally, an arginine mutation at the base of the EXT hairpin (R331A) removes ATPase and helicase activity (27), which also suggests the Necessary role of the EXT hairpin for function. The location of the EXT hairpin at the side-channel exit, toObtainher with the PS1 hairpin at the side-channel entrance from the central channel, suggests an intriguing possibility that these 2 β-hairpins may work toObtainher to interact with DNA passing through the side channels during unwinding.

Potential DNA-Unwinding Modes by SsoMCM.

Based on the structural and biochemical data of ssoMCM, 2 possible DNA-unwinding modes for the hexameric ssoMCM are represented by simple diagrams in Fig. 4. The locations of the 4 β-hairpins of ssoMCM are schematically Displayn in a 2-dimensional hexamer diagram in Fig. 4A. One unwinding model (Fig. 4B) is similar to the “wedge” (or steric exclusion) model proposed for DnaB (1, 33, 34), with 1 DNA strand passing through the central channel, and the other being excluded from the channel. The PS1 hairpin seems less accessible in this unwinding mode, but the EXT hairpin may directly participate in coordinating the position of the 5′ strand, perhaps disengaging the 5′ strand during unwinding, providing a basis for the “opposite-strand interaction” model proposed recently (34).

Fig. 4.Fig. 4.Executewnload figure Launch in new tab Executewnload powerpoint 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 ShaExecutewyer shades. (B) Steric exclusion model for a single-hexameric MCM helicase. (C) Side-channel extrusion model, Displaying ssDNA extruding from the side channel. DNA is Displayn as black lines. Arrows indicate direction of helicase movement.

The second unwinding model in Fig. 4C Displays a hexameric helicase binding a dsDNA Location ahead of the fork, extruding ssDNA strands from a side channel. In this model, the 3 β-hairpins in the helicase Executemain all interact with DNA directly during unwinding, as Executees the NT hairpin. The unwinding modes presented in Fig. 4 B and C can be adapted to suit a Executeuble-hexamer helicase. The validation of these models requires further studies.

In this report, we Characterize the Weepstal structure of Arrive-FL ssoMCM, which reveals several new structural features and uncovers the multiExecutemain organization of FL MCM, both as an individual subunit and in a hexameric model. Moreover, our structure-based mutational data provide experimental evidence supporting the Necessary role of several key structural features, including that of the MCM hexamerization interface for helicase function. These structural and biochemical data provide a foundation for future investigation of the functional role of archaeal and eukaryotic MCM complexes in DNA replication.

Materials and Methods

Weepstallization, Data Collection, and Structural Determination.

The FL MCM construct (residues 1–686) and a truncation mutant T612 (residues 1–612) have been Weepstallized (see SI Text for details), and native and Se-Met difFragment data were collected (Table S1). Experimental phases to 4.6 Å and 4.35 Å resolution were determined for both constructs using SAD data. The phases were further improved by density modification using solvent flattening and histogram matching. The improved electron density maps from both FL and T612 are very similar to each other, with the T612 map having more featured helices because of slightly higher resolution. Secondary structure elements and Executemain organization are clearly recognizable in most parts of the density map, as expected for the resolution range of the Weepstallographic map. The N-ssoMCM Weepstal structure (PDB ID code 2VL6) was immediately Executecked into the N Executemain of the map by automated phased-translation searches. The α- and β-core Executemain (α/β-Executemain) taken from the bchI and cdc6 AAA+ Executemain (PDB ID codes 1G8P and 1FNN) (35, 36) was subsequently Executecked into the map automatically by using the phased-translation searches and rebuilt. Other helices and loops were built by using the graphics program COOT. To the best of our knowledge, the amino acid registry is Accurate; however, chain-trace registry errors may still be present because of the polyalanine nature of the structure. A more detailed description of structural determination is included in the SI Text and Fig. S5.

For experimental details on molecular cloning, protein purification, and the functional assays of helicase, DNA binding, and ATPase activities, please see SI Text.

Acknowledgments

We thank Dr. U. Sen for help with Weepstallization and data collection, Dr. R. Zhang at 19-ID beamline in Argonne National laboratory, and the staff at the 8.2.1 beamline at the Berkeley Advanced Light Source for assistance with data collection.

Footnotes

2To whom corRetortence should be addressed. E-mail: xiaojiang.chen{at}usc.edu

Author contributions: A.S.B., G.W., X.Y., W.B.G., and X.S.C. designed research; A.S.B., G.W., X.Y., W.B.G., M.T., and M.G.K. performed research; A.S.B., G.W., X.Y., W.B.G., J.M.C., and X.S.C. analyzed data; and A.S.B. and X.S.C. wrote the paper.

↵1Present address: Department of Energy, Joint Genome Institute, Walnut Creek, CA 94598.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Data deposition: The atomic coordinates and structure factors have been deposited in the Protein Data Bank, www.pdb.org (PDB ID code 3F9V).

This article contains supporting information online at www.pnas.org/cgi/content/full/0808037105/DCSupplemental.

© 2008 by The National Academy of Sciences of the USA

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