Allostery is conformation regulation by propagating a signal from one site to another distal site. This study focuses on the long-range communication in DNA mismatch repair proteins MutS and its homologs where intramolecular signaling has to travel over 70 Å to couple lesion detection to ATPase activity and eventual downstream repair. Using dynamic network analysis based on extensive molecular dynamics simulations, multiple preserved communication pathways were identified that would allow such long-range signaling. The pathways appear to depend on the nucleotides bound to the ATPase domain as well as the type of DNA substrate consistent with previously proposed functional cycles of mismatch recognition and repair initiation by MutS and homologs. A mechanism is proposed where pathways are switched without major conformational rearrangements allowing for efficient long-range signaling and allostery. We are proposing a new model for how long-range allosteric communication may be accomplished via switching of pre-existing pathway as a result of only minor structural perturbations. The systems studied here are the bacterial mismatch repair enzyme MutS and its eukaryotic homologs where we identified strong communication pathways connecting distant functional domains. The functionally-related exchange of nucleotides in a distant ATPase domain appears to be able to switch between those pathways providing a new paradigm for how long-range allostery may be accomplished in large biomolecular assemblies. Allostery is a fundamental part of many if not most biological processes. It is classically defined as the induced regulation at one site by an event at another distal site. Venerable models for allostery, such as the MWC (Monod-Wyman-Changeux)  and KNF It is difficult to obtain detailed insight into allostery from experiments, especially for larger and more complex systems, because NMR spectroscopy is generally limited to small and soluble proteins that can be easily labeled and expressed in large quantities. On the other hand, crystallography is not well-suited for studying allosteric effects due to its inherent dynamic nature.Computational approaches such as statistical coupling analysis (SCA) , normal MMR is responsible for correcting errors that escape immediate proofreading during DNA replication and the mechanism is widely conserved from prokaryotic to eukaryotic organisms. MMR alone can increase the accuracy of DNA replication by 20–400 fold . While sThe crystal structures of prokaryotic MutS and its eukaryotic homologs, complexed with mismatched DNA heteroduplexes, feature a similar overall Θ shape , 24–26. Previous studies have examined MutS and eukaryotic analogs via molecular dynamics simulations , 39–43, Thermus aquaticus MutS in a recent study [A number of very similar MutS and MSH crystal structures are available with different nucleotide bound states and different mismatches or IDLs. The structural variations that can be discerned primarily focus on the MBD, ATPase and clamp domains and involve mostly local side-chain displacements rather than larger conformational changes of the main chain. For example, the MutS crystal structures 1E3M (with a single ADP)  and 1W7Ant study , althougnt study .A coupling is strongest in ADP-None, while the strongest positive MBD(I)-connector(II)B coupling is observed in None-ADP. Also in the case of ADP-None, the MBD and connector domains of subunit A are strongly negatively coupled with the lever and clamp domains of subunit B. The two clamp domains are positively coupled in cases of ATP-ADP and None-None, which are stronger than the others. The positive coupling between the two ATPase domains is strongest in ATP-ADP. Similar direct correlations between MutS domains have also been observed in other work based on MD simulations of Thermus aquaticus MutS [Average dynamical cross-correlation matrices (DCCM) were calculated from the MD simulations. cus MutS . HoweverNext, we turned to dynamical network analysis to allow for a more dynamic model of allostery where direct correlations between distant sites are not required. In this approach, pathways connecting residue pairs along the shortest path with the highest pairwise local correlations based on the converged DCCMs from 200 ns MD sampling are determined. We focused our analysis on the functionally most relevant signal propagation between the MBD, ATPase, and clamp domains using specific key residues as anchor points . A firstMapping of the resulting pathways onto the MutS structure is shown in Tables Three major types of MBD-ATPase pathways were identified see : The firFocusing then on states where either ADP or ATP is bound in the A (mismatch-bound) domain of the MutS dimer, we find two paths with significantly lower weights: path ② for ADP-None and path ③ for the ATP-ADP state. These paths are visualized in more detail in Communication along path ② appears to involve many alternate routes, rather than a single well-defined path. A broad ensemble of possible paths is a consequence of considering just slightly suboptimal paths see  as foundIt is readily apparent that path ③ has only very little overlap with path ② except for a few residues near the start (F36) and end (K620) points. This suggests that simply exchanging ADP for ATP in the A site may be sufficient to switch between entirely different pathways. In order to understand the structural basis in more detail we compared the detailed conformations of the pathway residues in the simulations with the ADP-None and ATP-ADP states. As shown in Q626, which is almost perfectly conserved along with R625 , is therA large number of mutations in the human MutS homolog MSH6-MSH2 are known to be associated with cancer phenotypes . Most muFour out of 16 non-frameshift mutations in MSH6 that map onto MutS coincide with one of the 97 pathway residues shown in To further test whether mutations hypothesized to impact communication between the MBD and ATPase domains we examined the E169P and L240D mutants in the presence of ADP-None. As shown in Path ② is a plausible communication route that could be taken in the presence of ADP in the A site to communicate mismatch recognition to the ATPase site. Mismatch recognition is experimentally known to be followed by exchange of ADP for ATP. Our analysis suggests that once such an exchange takes place, the communication along path ② is lost and instead communication along the lever (path ③) would be preferred. In generating path ③, we required that a connection is made between the MBD and ATPase domains. However, path ③ largely overlaps with communication to the clamp (see below) suggesting that exchange from ADP to ATP may in fact switch communication between the MBD and the ATPase to communication between ATPase and the clamp domain and/or changes in the lever and core domain. We hypothesize that the latter could also place MutS into a MutL-binding conformation, the next functional step in the MutS mismatch repair cycle.We identified two major pathways connecting the ATPase domain with the clamp, one along chain A, the other along chain B. All of the paths would connect through the lever along the long kinked helix (helices α20 and α21) as suggested by the crystal structures , 25. We To test the proposed importance of 558, we carried out simulations of L558R mutants in either chain A or B. Results are shown in Tables The ADP-None combination  is believed to be the mismatch scanning configuration of MutS . We woulA-ATPaseB pathways obtained from our computational analysis can be roughly classified into three types  and the main result of this work is the identification of such paths in a nucleotide-dependent manner. Integrating previous biochemical data with such a dynamic allosteric model allowed us to arrive at the mechanism depicted in A strongly couples with the connector and ATPase domains in the ADP-None state via the proposed pathway ② that consists of a broad ensemble of individual paths with a few bottlenecks at domain boundaries. This communication is then proposed to result in exchange of ADP for ATP in the ATPaseA domain upon mismatch recognition [A domains because an optimal or suboptimal path via the connector domain is either absent altogether (ATP-ADP) or present with less favorable weights or low minimum pairwise correlations  that suggest inefficient coupling. At the same time, the ATPaseA -ATPaseB communication would engage the Walker B motif of chain B when ATP is present in the A site. We further hypothesize that ADP or ATP binding to the B site would follow, leading to the ATP-ADP state. Since the lifetime of the ATP-None state is believed to be short [A domain connects to the lever instead of the connector. Because the connector primarily connects with the MBD domain while the lever domain provides a route to the clamp domain, we hypothesize that in the ATP-ADP state communication from the ATPaseA domain would be switched from the MBD domain to the clamp. At the same time, strong coupling between the ATPaseA -ATPaseB domains via the signature loop could disrupt the strong ATPaseB-clamp connection present in the ADP-None state and allow release of MutS from the mismatch site.In the absence of DNA, the ADP-ADP state is presumed to be dominant . The ADPognition , 35. In be short  this wouThe mechanism above postulates communication routes within the context of a dynamic allosteric mechanism that could be tested further experimentally, e.g. via mutations of pathway residues. Based on the dynamics sampled in the underlying simulations we are able to propose a structural basis for how pathways are switched in the presence of different nucleotides, however, the model is still lacking a clear mechanism for how ADP would be exchanged for ATP following mismatch recognition, for how the clamp domains would respond to signaling resulting from nucleotide exchange as proposed here, and what role MutL binding plays in this process. We speculate that the altered correlated dynamics induces subtle shifts in the overall conformational landscape which would favor ADP-ATP exchange and lead to clamp opening. To address this idea in more detail, significant additional simulations are required to probe the DNA binding process and clamp dynamics leading to the sliding clamp conformation in excess of the scope of the present work. Such a model could also conceptually integrate recent conformational landscape-based ideas of allostery with the communication-focused analysis presented here into a complete model for allostery in a large, complex system such as MutS where simpler concepts of conformational selection or induced-fit may not be able to adequately describe the allosteric mechanism.MutS can recognize a broad range of lesions, mismatches and IDLs, but MSHs have differentiated substrate specificities. MutSα (MSH2-MSH6) primarily recognizes mismatches and single base IDLs, whereas MutSβ (MSH2-MSH3) recognizes DNA loops composed of 2–13 bases. Based on previous simulations of MutSα and MutSβ with native and swapped substrates and no DNA at all , we alsoA domains (and subsequent initiation of repair) would depend on both the nature of the DNA substrate and the nucleotides bound in the ATPase domains.In the MSH complexes we identified pathways analogous to paths ①, ②, and ③ in MutS see  suggestiE.coli MutS and MSHs where several long-range signaling steps are essential for initiating DNA repair following mismatch recognition. Using dynamic network analysis based on extensive molecular dynamics simulations we developed a model consisting of a number of communication pathways that depend on strong local pairwise residue dynamical coupling where signaling would be expected to progress stochastically along those paths. In this model, different combinations of ATPase-bound nucleotides would result in switching between different pathways to implement the functional cycle of MutS without significant conformational rearrangements. A signaling mechanism based on pre-existing pathways that are switched on or off by different nucleotides and/or different DNA substrates is consistent with previous crystallographic and simulation studies that show surprisingly little structural variations in mismatch-bound MutS and homologs. The benefit of such a mechanism could be energetic economy, especially when considering the very long range over which the pathways appear to operate. Experimental validation of the hypotheses presented here could involve mutations of key residues, but it will also be interesting to see whether similar mechanisms are at play in other large enzymes. However, further computational studies will also be necessary to develop a more complete mechanistic understanding of how exactly signaling along the proposed pathways would promote and depend on nucleotide exchange and how it would lead to sliding clamp formation and complex formation with MutL.Long-range signaling and allostery is a key mechanistic component of many large biomolecular complexes. Here, we present a detailed analysis of E.coli MutS protein bound to a G:T mismatch DNA (PDB ID: 1W7A) [E. coli MutS system to test the mechanistic hypotheses developed in this study: E169P (ADP-None), L240D (ADP-None), and Q626A (ATP-ADP) in chain A as well as L558R in either chain A or B (ATP-ADP). These simulations were simulated using the same protocol as the previous simulations of the wild-type systems (see below).MD simulations of the D: 1W7A)  were preD: 1W7A) . Each ATapo structures, where the DNA heteroduplex was removed (MutSα/Apo and MutSβ/Apo), and for MutSα and MutSβ where the respective substrates were swapped (MutSα/IDL-4L and MutSβ/G:T) [MD simulations of human MutSα and MutSβ were started from the crystal structure 2O8B  and 3THXtSβ/G:T) .In total, 15 previous simulations and five new simulations , each foNetworkView plugin of VMD [ijw = −log(|ijC|), where Cij are the elements of the correlation matrix calculated as Allosteric networks within the proteins were identified using the n of VMD , 66. Thesoftware . The lensoftware . The numsoftware . The nodAll pathways were determined between residues in the MBD (located within 10 Å of the mismatch site) and residues in the ATPase domain (located within 10 Å of a bound nucleotide) or between residues in the clamp domain (within 10 Å of DNA) and residues in the ATPase domain (within 10 Å of a bound nucleotide). The residue pairs with the shortest optimal path were finally selected as representative residues . SuboptiS1 Table(DOCX)Click here for additional data file.S2 TableSelected residues used as anchor points in network analysis in MutS and eukaryotic homologs.(DOCX)Click here for additional data file.S3 TableCritical inter-domain edges in MutS with major edges shown in bold(DOCX)Click here for additional data file.S4 TableCancer-associated non-frameshift/non-mistranslation mutations in MSH2(DOCX)Click here for additional data file.S5 TableCancer-associated non-frameshift/non-mistranslation mutations in MSH6(DOCX)Click here for additional data file.S1 FigDynamical cross-correlation matrices for Ca atoms for E. coli MutS simulated systems as a functions of nucleotides bound to ATPase domains using 50, 100, 150, and 200 ns of the simulation trajectories (from left to right).(TIF)Click here for additional data file.S2 FigCommunities of the MutS protein in all MutS systems . Different communities were colored differently.(TIF)Click here for additional data file.S3 FigA pathways for additional NTP states.MBD-ATPase(TIF)Click here for additional data file.S4 FigA–clamp pathways for additional NTP states.ATPase(TIF)Click here for additional data file.S5 FigB–clamp pathways for additional NTP states.ATPase(TIF)Click here for additional data file.S6 FigA -ATPaseB pathways for additional NTP states.ATPase(TIF)Click here for additional data file.S7 FigThe conserved motifs in NBD domain.(TIF)Click here for additional data file.S8 FigMBD-ATPase pathways in MutSα and MutSβ with native DNA substrates as in (TIF)Click here for additional data file.S9 FigATPase-clamp pathways in MutSα and MutSβ with native DNA substrates as in (TIF)Click here for additional data file.