Crystal structure of a thwarted mismatch glycosylase DNA repair complex

The bacterial mismatch-specific uracil-DNA glycosylase (MUG) and eukaryotic thymine-DNA glycosylase (TDG) enzymes form a homologous family of DNA glycosylases that initiate base-excision repair of G:U/T mismatches. Despite low sequence homology, the MUG/TDG enzymes are structurally related to the uracil-DNA glycosylase enzymes, but have a very different mechanism for substrate recognition. We have now determined the crystal structure of the Escherichia coli MUG enzyme complexed with an oligonucleotide containing a non-hydrolysable deoxyuridine analogue mismatched with guanine, providing the first structure of an intact substrate-nucleotide productively bound to a hydrolytic DNA glycosylase. The structure of this complex explains the preference for G:U over G:T mispairs, and reveals an essentially non-specific pyrimidine-binding pocket that allows MUG/TDG enzymes to excise the alkylated base, 3, N(4)-ethenocytosine. Together with structures for the free enzyme and for an abasic-DNA product complex, the MUG-substrate analogue complex reveals the conformational changes accompanying the catalytic cycle of substrate binding, base excision and product release.     

Crystal structure of a MAT alpha 2 homeodomain-operator complex suggests a general model for homeodomain-DNA interactions.

The MAT alpha 2 homeodomain regulates the expression of cell type-specific genes in yeast. We have determined the 2.7 A resolution crystal structure of the alpha 2 homeodomain bound to a biologically relevant DNA sequence. The DNA in this complex is contacted primarily by the third of three alpha-helices, with additional contacts coming from an N-terminal arm. Comparison of the yeast alpha 2 and the Drosophila engrailed homeodomain-DNA complexes shows that the protein fold is highly conserved, despite a 3-residue insertion in alpha 2 and only 27% sequence identity between the two homeodomains. Moreover, the orientation of the recognition helix on the DNA is also conserved. This docking arrangement is maintained by side chain contacts with the DNA--primarily the sugar-phosphate backbone--that are identical in alpha 2 and engrailed. Since these residues are conserved among all homeodomains, we propose that the contacts with the DNA are also conserved and suggest a general model for homeodomain-DNA interactions.     

Dynamical allosterism in the mechanism of action of DNA mismatch repair protein MutS

The multidomain protein Thermus aquaticus MutS and its prokaryotic and eukaryotic homologs recognize DNA replication errors and initiate mismatch repair. MutS actions are fueled by ATP binding and hydrolysis, which modulate its interactions with DNA and other proteins in the mismatch-repair pathway. The DNA binding and ATPase activities are allosterically coupled over a distance of ∼70 Å, and the molecular mechanism of coupling has not been clarified. To address this problem, all-atom molecular dynamics simulations of ∼150 ns including explicit solvent were performed on two key complexes—ATP-bound and ATP-free MutS⋅DNA(+T bulge). We used principal component analysis in fluctuation space to assess ATP ligand-induced changes in MutS structure and dynamics. The molecular dynamics-calculated ensembles of thermally accessible structures showed markedly small differences between the two complexes. However, analysis of the covariance of dynamical fluctuations revealed a number of potentially significant interresidue and interdomain couplings. Moreover, principal component analysis revealed clusters of correlated atomic fluctuations linking the DNA and nucleotide binding sites, especially in the ATP-bound MutS⋅DNA(+T) complex. These results support the idea that allosterism between the nucleotide and DNA binding sites in MutS can occur via ligand-induced changes in motion, i.e., dynamical allosterism.     

Stoichiometry of MutS and MutL at unrepaired mismatches in vivo suggests a mechanism of repair

Mismatch repair (MMR) is an evolutionarily conserved DNA repair system, which corrects mismatched bases arising during DNA replication. MutS recognizes and binds base pair mismatches, while the MutL protein interacts with MutS-mismatch complex and triggers MutH endonuclease activity at a distal-strand discrimination site on the DNA. The mechanism of communication between these two distal sites on the DNA is not known. We used functional fluorescent MMR proteins, MutS and MutL, in order to investigate the formation of the fluorescent MMR protein complexes on mismatches in real-time in growing Escherichia coli cells. We found that MutS and MutL proteins co-localize on unrepaired mismatches to form fluorescent foci. MutL foci were, on average, 2.7 times more intense than the MutS foci co-localized on individual mismatches. A steric block on the DNA provided by the MutHE56A mutant protein, which binds to but does not cut the DNA at the strand discrimination site, decreased MutL foci fluorescence 3-fold. This indicates that MutL accumulates from the mismatch site toward strand discrimination site along the DNA. Our results corroborate the hypothesis postulating that MutL accumulation assures the coordination of the MMR activities between the mismatch and the strand discrimination site.     

Altering the conserved nucleotide binding motif in the Salmonella typhimurium MutS mismatch repair protein affects both its ATPase and mismatch binding activities.

The Salmonella typhimurium and Escherichia coli MutS protein is one of several methyl-directed mismatch repair proteins that act together to correct replication errors. MutS is homologous to the Streptococcus pneumoniae HexA mismatch repair protein and to the Duc1 and Rep1 proteins of human and mouse. Homology between the deduced amino acid sequence of both MutS and HexA, and the type A nucleotide binding site consensus sequence, suggested that ATP binding and hydrolysis play a role in their mismatch repair functions. We found that MutS does indeed weakly hydrolyze ATP to ADP and Pi, with a Km of 6 microM and kcat of 0.26. To show that this activity is intrinsic to MutS, we made a site-directed mutation, which resulted in the invariant lysine of the nucleotide binding consensus sequence being changed to an alanine. The mutant MutS allele was unable to complement a mutS::Tn10 mutation in vivo, and was dominant over wild type when present in high copy number. The purified mutant protein had reduced ATPase activity, with the Km affected more severely than the kcat. Like the wild type MutS protein, the mutant protein is able to bind heteroduplex DNA specifically, but the mutant protein does so with a reduced affinity.     

Oligomerization of a MutS mismatch repair protein from Thermus aquaticus.

The MutS DNA mismatch protein recognizes heteroduplex DNAs containing mispaired or unpaired bases. We have examined the oligomerization of a MutS protein from Thermus aquaticus that binds to heteroduplex DNAs at elevated temperatures. Analytical gel filtration, cross-linking of MutS protein with disuccinimidyl suberate, light scattering, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry establish that the Taq protein is largely a dimer in free solution. Analytical equilibrium sedimentation showed that the oligomerization of Taq MutS involves a dimer-tetramer equilibrium in which dimer predominates at concentrations below 10 microM. The DeltaG(0)(2-4) for the dimer to tetramer transition is approximately -6.9 +/- 0.1 kcal/mol of tetramer. Analytical gel filtration of native complexes and gel mobility shift assays of an maltose-binding protein-MutS fusion protein bound to a short, 37-base pair heteroduplex DNA reveal that the protein binds to DNA as a dimer with no change in oligomerization upon DNA binding.     

Formation of a DNA mismatch repair complex mediated by ATP

The mismatch repair proteins, MutS and MutL, interact in a DNA mismatch and ATP-dependent manner to activate downstream events in repair. Here, we assess the role of ATP binding and hydrolysis in mismatch recognition by MutS and the formation of a ternary complex involving MutS and MutL bound to a mismatched DNA. We show that ATP reduces the affinity of MutS for mismatched DNA and that the modulation of DNA binding affinity by nucleotide is even more pronounced for MutS E694A, a protein that binds ATP but is defective for ATP hydrolysis. Despite the ATP hydrolysis defect, E694A, like WT MutS, undergoes rapid, ATP-dependent dissociation from a DNA mismatch. Furthermore, MutS E694A retains the ability to interact with MutL on mismatched DNA. The recruitment of MutL to a mismatched DNA by MutS is also observed for two mutant MutL proteins, E29A, defective for ATP hydrolysis, and R266A, defective for DNA binding. These results suggest that ATP binding in the absence of hydrolysis is sufficient to trigger formation of a MutS sliding clamp. However, recruitment of MutL results in the formation of a dynamic ternary complex that we propose is the intermediate that signals subsequent repair steps requiring ATP hydrolysis.     

The structure of bovine F1-ATPase inhibited by ADP and beryllium fluoride

The structure of bovine F1-ATPase inhibited with ADP and beryllium fluoride at 2.0 angstroms resolution contains two ADP.BeF3- complexes mimicking ATP, bound in the catalytic sites of the beta(TP) and beta(DP) subunits. Except for a 1 angstrom shift in the guanidinium of alphaArg373, the conformations of catalytic side chains are very similar in both sites. However, the ordered water molecule that carries out nucleophilic attack on the gamma-phosphate of ATP during hydrolysis is 2.6 angstroms from the beryllium in the beta(DP) subunit and 3.8 angstroms away in the beta(TP) subunit, strongly indicating that the beta(DP) subunit is the catalytically active conformation. In the structure of F1-ATPase with five bound ADP molecules (three in alpha-subunits, one each in the beta(TP) and beta(DP) subunits), which has also been determined, the conformation of alphaArg373 suggests that it senses the presence (or absence) of the gamma-phosphate of ATP. Two catalytic schemes are discussed concerning the various structures of bovine F1-ATPase.     

Comparative sequence analysis suggests a conserved gating mechanism for TRP channels

The transient receptor potential (TRP) channel superfamily plays a central role in transducing diverse sensory stimuli in eukaryotes. Although dissimilar in sequence and domain organization, all known TRP channels act as polymodal cellular sensors and form tetrameric assemblies similar to those of their distant relatives, the voltage-gated potassium (Kv) channels. Here, we investigated the related questions of whether the allosteric mechanism underlying polymodal gating is common to all TRP channels, and how this mechanism differs from that underpinning Kv channel voltage sensitivity. To provide insight into these questions, we performed comparative sequence analysis on large, comprehensive ensembles of TRP and Kv channel sequences, contextualizing the patterns of conservation and correlation observed in the TRP channel sequences in light of the well-studied Kv channels. We report sequence features that are specific to TRP channels and, based on insight from recent TRPV1 structures, we suggest a model of TRP channel gating that differs substantially from the one mediating voltage sensitivity in Kv channels. The common mechanism underlying polymodal gating involves the displacement of a defect in the H-bond network of S6 that changes the orientation of the pore-lining residues at the hydrophobic gate.     

The coordinated functions of the E. coli MutS and MutL proteins in mismatch repair

The Escherichia coli MutS and MutL proteins have been conserved throughout evolution, although their combined functions in mismatch repair (MMR) are poorly understood. We have used biochemical and genetic studies to ascertain a physiologically relevant mechanism for MMR. The MutS protein functions as a regional lesion sensor. ADP-bound MutS specifically recognizes a mismatch. Repetitive rounds of mismatch-provoked ADP-->ATP exchange results in the loading of multiple MutS hydrolysis-independent sliding clamps onto the adjoining duplex DNA. MutL can only associate with ATP-bound MutS sliding clamps. Interaction of the MutS-MutL sliding clamp complex with MutH triggers ATP binding by MutL that enhances the endonuclease activity of MutH. Additionally, MutL promotes ATP binding-independent turnover of idle MutS sliding clamps. These results support a model of MMR that relies on two dynamic and redundant ATP-regulated molecular switches.     

Crystal structure of the yeast His6 enzyme suggests a reaction mechanism

The Saccharomyces cerevisiae His6 gene codes for the enzyme phosphoribosyl-5-amino-1-phosphoribosyl-4-imidazolecarboxamide isomerase, catalyzing the fourth step in histidine biosynthesis. To get an insight into the structure and function of this enzyme, we determined its X-ray structure at a resolution of 1.30 A using the anomalous diffraction signal of the protein's sulphur atoms at 1.77 A wavelength. His6 folds in an (alpha/beta)8 barrel similar to HisA, which performs the same function in bacteria and archaea. We found a citrate molecule from the buffer bound in a pocket near the expected position of the active site and used it to model the open form of the substrate (phosphoribulosyl moiety), which is a reaction intermediate. This model enables us to identify catalytic residues and to propose a reaction mechanism where two aspartates act as acid/base catalysts: Asp134 as a proton donor for ring opening, and Asp9 as a proton acceptor and donor during enolization of the aminoaldose. Asp9 is conserved in yeast His6 and bacterial or archaeal HisA sequences, and Asp134 has equivalents in both HisA and TrpF, but they occur at a different position in the protein sequence.     

Replisome structure suggests mechanism for continuous fork progression and post-replication repair

What happens to DNA replication when it encounters a damaged or nicked DNA template has been under investigation for five decades. Initially it was thought that DNA polymerase, and thus the replication-fork progression, would stall at road blocks. After the discovery of replication-fork helicase and replication re-initiation factors by the 1990s, it became clear that the replisome can “skip” impasses and finish replication with single-stranded gaps and double-strand breaks in the product DNA. But the mechanism for continuous fork progression after encountering roadblocks is entangled with translesion synthesis, replication fork reversal and recombination repair. The recently determined structure of the bacteriophage T7 replisome offers the first glimpse of how helicase, primase, leading-and lagging-strand DNA polymerases are organized around a DNA replication fork. The tightly coupled leading-strand polymerase and lagging-strand helicase provides a scaffold to consolidate data accumulated over the past five decades and offers a fresh perspective on how the replisome may skip lesions and complete discontinuous DNA synthesis. Comparison of the independently evolved bacterial and eukaryotic replisomes suggests that repair of discontinuous DNA synthesis occurs post replication in both.     

Crystal structure of MutS2 endonuclease domain and the mechanism of homologous recombination suppression

DNA recombination events need to be strictly regulated, because an increase in the recombinational frequency causes unfavorable alteration of genetic information. Recent studies revealed the existence of a novel anti-recombination enzyme, MutS2. However, the mechanism by which MutS2 inhibits homologous recombination has been unknown. Previously, we found that Thermus thermophilus MutS2 (ttMutS2) harbors an endonuclease activity and that this activity is confined to the C-terminal domain, whose amino acid sequence is widely conserved in a variety of proteins with unknown function from almost all organisms ranging from bacteria to man. In this study, we determined the crystal structure of the ttMutS2 endonuclease domain at 1.7-angstroms resolution, which resembles the structure of the DNase I-like catalytic domain of Escherichia coli RNase E, a sequence-nonspecific endonuclease. The N-terminal domain of ttMutS2, however, recognized branched DNA structures, including the Holliday junction and D-loop structure, a primary intermediate in homologous recombination. The full-length of ttMutS2 digested the branched DNA structures at the junction. These results indicate that ttMutS2 suppresses homologous recombination through a novel mechanism involving resolution of early intermediates.     

ATP hydrolysis-dependent formation of a dynamic ternary nucleoprotein complex with MutS and MutL.

Functional interactions of Escherichia coli MutS and MutL in mismatch repair are dependent on ATP. In this study, we show that MutS and MutL associate with immobilised DNA in a manner dependent on ATP hydrolysis and with an ATP concentration near the solution K m of the ATPase of MutS. After removal of MutS, MutL and ATP, much of the protein in this ternary complex is not stably associated, with MutL leaving the complex more rapidly than MutS. The rapid dissociation reveals a dynamic interaction with concurrent rapid association and dissociation of proteins from the DNA. Analysis by surface plasmon resonance showed that the DNA interacting with dynamically bound protein was more resistant to nuclease digestion than the DNA in MutS-DNA complexes. Non-hydrolysable analogs of ATP inhibit the formation of this dynamic complex, but permit formation of a second type of ternary complex with MutS and MutL stably bound to the immobilised DNA.     

Composite active site of an ABC ATPase: MutS uses ATP to verify mismatch recognition and authorize DNA repair

The MutS protein initiates DNA mismatch repair by recognizing mispaired and unpaired bases embedded in duplex DNA and activating endo- and exonucleases to remove the mismatch. Members of the MutS family also possess a conserved ATPase activity that belongs to the ATP binding cassette (ABC) superfamily. Here we report the crystal structure of a ternary complex of MutS-DNA-ADP and assays of initiation of mismatch repair in conjunction with perturbation of the composite ATPase active site by mutagenesis. These studies indicate that MutS has to bind both ATP and the mismatch DNA simultaneously in order to activate the other mismatch repair proteins. We propose that the MutS ATPase activity plays a proofreading role in DNA mismatch repair, verification of mismatch recognition, and authorization of repair.     

Structures of Escherichia coli DNA mismatch repair enzyme MutS in complex with different mismatches: a common recognition mode for diverse substrates

We have refined a series of isomorphous crystal structures of the Escherichia coli DNA mismatch repair enzyme MutS in complex with G:T, A:A, C:A and G:G mismatches and also with a single unpaired thymidine. In all these structures, the DNA is kinked by ~60° upon protein binding. Two residues widely conserved in the MutS family are involved in mismatch recognition. The phenylalanine, Phe 36, is seen stacking on one of the mismatched bases. The same base is also seen forming a hydrogen bond to the glutamate Glu 38. This hydrogen bond involves the N7 if the base stacking on Phe 36 is a purine and the N3 if it is a pyrimidine (thymine). Thus, MutS uses a common binding mode to recognize a wide range of mismatches.     

Mutations in the signature motif in MutS affect ATP-induced clamp formation and mismatch repair

MutS protein dimer recognizes and co-ordinates repair of DNA mismatches. Mismatch recognition by the N-terminal mismatch recognition domain and subsequent downstream signalling by MutS appear coupled to the C-terminal ATP catalytic site, Walker box, through nucleotide-mediated conformational transitions. Details of this co-ordination are not understood. The focus of this study is a conserved loop in Escherichia coli MutS that is predicted to mediate cross-talk between the two ATP catalytic sites in MutS homodimer. Mutagenesis was employed to assess the role of this loop in regulating MutS function. All mutants displayed mismatch repair defects in vivo . Biochemical characterization further revealed defects in ATP binding, ATP hydrolysis as well as effective mismatch recognition. The kinetics of initial burst of ATP hydrolysis was similar to wild type but the magnitude of the burst was reduced for the mutants. Given its proximity to the ATP bound in the opposing monomer in the crystal and its potential analogy with signature motif of ABC transporters, the results strongly suggest that the loop co-ordinates ATP binding/hydrolysis in trans by the two catalytic sites. Importantly, our data reveal that the loop plays a direct role in co-ordinating conformational changes involved in long-range communication between Walker box and mismatch recognition domains.     

Mitochondrial ATP synthasome: three-dimensional structure by electron microscopy of the ATP synthase in complex formation with carriers for Pi and ADP/ATP

The terminal steps involved in making ATP in mitochondria require an ATP synthase (F(0)F(1)) comprised of two motors, a phosphate carrier (PIC), and an adenine nucleotide carrier (ANC). Under mild conditions, these entities sub-fractionate as an ATP synthase/PIC/ANC complex or "ATP synthasome" (Ko, Y.H., Delannoy, M, Hullihen, J., Chiu, W., and Pedersen, P.L. (2003) J. Biol. Chem. 278, 12305-12309). As a first step toward obtaining three-dimensional information about this large complex or "metabolon" and the locations of PIC and ANC therein, we dispersed ATP synthasomes into single complexes and visualized negatively stained images by electron microscopy (EM) that showed clearly the classical headpiece, central stalk, and basepiece. Parallel immuno-EM studies revealed the presence of PIC and ANC located non-centrally in the basepiece, and other studies implicated an ATP synthase/PIC/ANC stoichiometry near 1:1:1. Single ATP synthasome images (7506) were boxed, and, using EMAN software, a three-dimensional model was obtained at a resolution of 23 A. Significantly, the basepiece is oblong and contains two domains, the larger of which connects to the central stalk, whereas the smaller appears as an extension. Docking studies with known structures together with the immuno-EM studies suggest that PIC or ANC may be located in the smaller domain, whereas the other transporter resides nearby in the larger domain. Collectively, these finding support a mechanism in which the entry of the substrates ADP and P(i) into mitochondria, the synthesis of ATP on F(1), and the release and exit of ATP are very localized and highly coordinated events.     

Crystal structure of the S-adenosylmethionine synthetase ternary complex: a novel catalytic mechanism of S-adenosylmethionine synthesis from ATP and Met

S-Adenosylmethionine synthetase (MAT) catalyzes formation of S-adenosylmethionine (SAM) from ATP and l-methionine (Met) and hydrolysis of tripolyphosphate to PP(i) and P(i). Escherichia coli MAT (eMAT) has been crystallized with the ATP analogue AMPPNP and Met, and the crystal structure has been determined at 2.5 A resolution. eMAT is a dimer of dimers and has a 222 symmetry. Each active site contains the products SAM and PPNP. A modeling study indicates that the substrates (AMPPNP and Met) can bind at the same sites as the products, and only a small conformation change of the ribose ring is needed for conversion of the substrates to the products. On the basis of the ternary complex structure and a modeling study, a novel catalytic mechanism of SAM formation is proposed. In the mechanism, neutral His14 acts as an acid to cleave the C5'-O5' bond of ATP while simultaneously a change in the ribose ring conformation from C4'-exo to C3'-endo occurs, and the S of Met makes a nucleophilic attack on the C5' to form SAM. All essential amino acid residues for substrate binding found in eMAT are conserved in the rat liver enzyme, indicating that the bacterial and mammalian enzymes have the same catalytic mechanism. However, a catalytic mechanism proposed recently by González et al. based on the structures of three ternary complexes of rat liver MAT [González, B., Pajares, M. A., Hermoso, J. A., Guillerm, D., Guillerm, G., and Sanz-Aparicio. J. (2003) J. Mol. Biol. 331, 407] is substantially different from our mechanism.