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Originally published In Press as doi:10.1074/jbc.M409307200 on September 14, 2004

J. Biol. Chem., Vol. 279, Issue 47, 49338-49345, November 19, 2004
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Mapping Protein-Protein Interactions between MutL and MutH by Cross-linking*

Luis Giron-Monzon{ddagger}, Laura Manelyte{ddagger}, Robert Ahrends{ddagger}, Dieter Kirsch§, Bernhard Spengler§, and Peter Friedhoff{ddagger}

From the {ddagger}Institut für Biochemie (FB 08), Justus-Liebig-Universität Giessen, D-35392 Giessen, Germany and §Institut für Anorganische und Analytische Chemie (FB 08), Justus-Liebig Universität Giessen, D-35392 Giessen, Germany

Received for publication, August 13, 2004 , and in revised form, September 7, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Strand discrimination in Escherichia coli DNA mismatch repair requires the activation of the endonuclease MutH by MutL. There is evidence that MutH binds to the N-terminal domain of MutL in an ATP-dependent manner; however, the interaction sites and the molecular mechanism of MutH activation have not yet been determined. We used a combination of site-directed mutagenesis and site-specific cross-linking to identify protein interaction sites between the proteins MutH and MutL. Unique cysteine residues were introduced in cysteine-free variants of MutH and MutL. The introduced cysteines were modified with the cross-linking reagent 4-maleimidobenzophenone. Photoactivation resulted in cross-links verified by mass spectrometry of some of the single cysteine variants to their respective Cys-free partner proteins. Moreover, we mapped the site of interaction by cross-linking different combinations of single cysteine MutH and MutL variants with thiol-specific homobifunctional cross-linkers of varying length. These results were used to model the MutH·MutL complex and to explain the ATP dependence of this interaction.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
DNA repair systems are crucial for maintaining a stable genome (1). One of these systems, DNA mismatch repair, is present in most organisms and enhances the fidelity of DNA replication by a factor of up to 1000 by correcting replication errors (2–6). mismatch repair is a complex process that requires the coordination of a number of specialized enzymatic activities involving many different proteins. The paradigm of such a process is the MutHLS system from Escherichia coli (7). Because mismatch repair proteins MutS and MutL are evolutionarily conserved among the three kingdoms of life, it is believed that the basic mechanisms of mismatch repair are similar. In the presence of ATP and a mismatch, MutS recruits MutL and together they activate MutH, which in turn cleaves the newly replicated daughter strand at a transiently unmethylated d(GATC) site. The nick marks the erroneous strand for excision, which is started by the action of DNA helicase II, single-stranded DNA-binding protein, and one of several exonucleases (8). DNA polymerase III, single-stranded DNA-binding protein, and DNA ligase carry out repair synthesis.

One of the interesting questions in DNA mismatch repair is how the different Mut proteins interact in time and space. In the E. coli system, the assembly of the active MutS·MutL complex will activate the strand discrimination endonuclease MutH. Both MutS and MutL are ATPases, and the roles of ATP binding and hydrolysis are just emerging (9–13). The mismatch activated form of this complex will stimulate the latent endonuclease activity of MutH to cleave the transiently unmethylated error-containing daughter strand, thereby marking this strand for removal. We have previously shown that the proper discrimination of the unmethylated strand from the methylated strand can be mapped to a single amino acid residue in MutH, i.e. Tyr212. Biochemical analyses indicated that only MutL, not MutS, interacts directly with MutH (11) and that communication between MutS and MutH was mediated by MutL in an ATP hydrolysis-dependent manner.

In previous studies, we used interference analysis and photochemical cross-linking experiments with single cysteine variants of MutH that had been covalently labeled with either the probes of different sizes or a photochemical cross-linker to identify specific interactions of MutH with MutL (14). These results revealed topographical details of the MutL binding site of MutH located in a region around {alpha}-helix E of MutH.

In this work, we focused on the MutH binding site of MutL. To determine which face of MutL is interacting with MutH, we designed a series of MutL variants containing a single cysteine in topographically distinct regions of the protein. These regions were limited to the N-terminal domain (NTD)1 of MutL, sufficient for MutH activation in vitro (15). Residue selection was guided by a phylogenetic sequence analysis that allowed us to choose positions that are not conserved, thereby maximizing the likelihood of obtaining active single cysteine protein variants suitable for subsequent biochemical studies.

We constructed 13 single cysteine full-length E. coli MutL mutants containing an N-terminal histidine tag for the purpose of attaching photoactivatable or various bifunctional thiol-specific cross-linkers. Photocross-linking experiments were carried out in the presence or absence of the non-hydrolyzable ATP analogue ADPNP with the homodimeric MutL variants and the binary MutL·MutH complex or the ternary MutL·MutH· DNA complex. Our results yielded a detailed map of the MutH binding site of MutL and, together with published results, allowed us to construct a MutL·MutH complex model with implications for the activation of the MutH endonuclease by MutL.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Strains, Plasmids, Enzymes, and Reagents—E. coli K12 strains CC106 ((P90C [ara{Delta}[lac-pro)XIII (F'laciZ proB+])) (16), TX2652 (CC106 mutL::{Omega}4 (BsaAI; Kanr)), and TX2928 (CC106 mutH471::tn5; Kanr) (17) and the pET-15b (Novagen) derived plasmids pTX412 and pTX418 containing the mutS and mutL genes, respectively, under control of the T7 promotor were kindly provided by Dr. M. Winkler (17). Plasmid pMQ402 (His6-MutH), a pBAD18 derivative, was a kind gift from Dr. M. Marinus (18). For the protein expression of MutS and MutL, the E. coli strain HMS174({lambda}DE3) (Novagen) was used. For MutH, the E. coli strain XL1-blue (Stratagene) was used. Pfu DNA polymerase was expressed and purified as described previously (19).

DNA Substrates—Linear heteroduplex substrates were generated by annealing two 484-bp PCR products amplified by Taq-DNA polymerase with a single GATC site at position 210 and a G/C or a A/T base pair at position 385 using plasmids and primers as described previously (20). This procedure results in a mixture of 50% homoduplex substrates (G/C and A/T) and 50% heteroduplex substrates (G/T and A/C). In general, 40–60% of this DNA was cleavable by MutH in a mismatch and MutS-dependent manner.

Site-directed Mutagenesis—pTX418/Cys-free containing the gene for a cysteine-free MutL variant (termed LCys-free) was generated by replacing all seven codons for the native cysteine residues from the pTX418 template using the QuikChange protocol (Stratagene) essentially as described previously (21). The substitutions C61S, C216L, C256F, C276F, C446S, C480S, and C588S are numbered with respect to the E. coli MutL sequence. E. coli XL1-blue MRF' was transformed with the full-length PCR product. Marker positive clones were inoculated and grown overnight in LB medium-containing ampicillin. Plasmid DNA was isolated using the QIAprep Spin Miniprep (Qiagen), and the entire mutL gene was sequenced. Plasmids coding for single cysteine MutL variants were generated from pTX418/Cys-free using the same method described above. Protein variants are labeled as LQ4C, LK52C, and so forth where the superscript indicates the position of the cysteine residues in the MutL protein, i.e. LQ4C, represents the cysteine-free MutL variant with a glutamine to cysteine change at position 4.

The gene coding for the NTD of MutL comprising amino acids 1–331 (MutL1–331) was generated using plasmid pTX418 as a template for an inverse PCR with primers 5'-AAA AAT GCA TTT TGT AGC CAC GCT CAG CAC GC-3' and 5'-AAA AAT GCA TGA GGA TCC GGC TGC TAA CAA AG-3' followed by cutting with NsiI and ligation.

Complementation Mutator Assay—Cells lacking a functional chromosomal mutL gene show a mutator phenotype, which can be analyzed by the frequency of occurrence of Rifampicin (Rif)-resistant clones (18). Single colonies of mutL-deficient TX2652 cells transformed with pET-15b vector alone or with pET-15b containing the wild type or mutant MutL gene were grown overnight at 37 °C in 3 ml of LB medium containing 100 µg/ml Amp. Aliquots of 50 µl from the undiluted culture were plated on LB-agar containing 25 µg/ml Amp and 100 µg/ml Rif. Colonies were counted following overnight incubation at 37 °C.

Protein Purification—Recombinant His6-tagged MutH, MutL, and MutS proteins were purified by nickel-nitrilotriacetic acid chromatography essentially as described previously (14, 17). When necessary, proteins were purified by gel filtration on a Superdex 200 column (Amersham Biosciences). MutH proteins were stored at -20 °C in 10 mM Hepes-KOH, 500 mM KCl, 1 mM EDTA, 1 mM DTT, 50% glycerol, pH 7.9. MutL and MutS proteins were snap-frozen in liquid nitrogen and stored at -70 °C in 10 mM Hepes-KOH, 200 mM KCl, 1 mM EDTA, pH 7.9. Protein concentrations were determined using theoretical extinction coefficients (22).

MutH Endonuclease Assay—MutH endonuclease was assayed on heteroduplex DNA substrate (484 bp) containing a G/T or A/C mismatch at position 385 and a single unmethylated d(GATC) site at position 210. 25 nM heteroduplex DNA was incubated at 37 °C with 500 nM MutH, 2 µM MutL, 1 µM MutS in 10 mM Tris-HCl, pH 7.9, 5 mM MgCl2, 1 mM ATP, 125 mM KCl. At suitable time points, 10-µl aliquots were removed and the reaction was stopped by the addition of 3 µl of 250 mM EDTA, 25% (w/v) sucrose, 1.2% (w/v) SDS, 0.1% (w/v) bromphenol blue, pH 8.0, to 10-µl aliquots. Substrate and product were separated by electrophoresis on 2% agarose gels. Initial rates were determined from the linear portion of a plot of the time course.

Site-specific Photocross-linking—Proteins at 20 µM were incubated with 4-maleimidobenzophenone (MBP, Sigma) in 20 mM Hepes-KOH, pH 6.5, 200 mM KCl for 30 min at room temperature with a 2–100-fold molar excess of the thiol-specific reagent. Reactions were stopped by adding a 5-fold molar excess of DTT over the thiol-specific reagent. Excess reagent was removed using protein-desalting spin columns (Pierce). For photocross-linking, 5 µM single cysteine protein (MutH or MutL) modified with MBP was mixed with an equimolar concentration of the respective interaction partner (wild type or cysteine-free) in 20 µl of 10 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 0.8 mM ADPNP and kept on ice in the dark or irradiated for 10–30 min at 354 nm with a handheld UV lamp at a distance of 5 cm. After the addition of 5 µl of 160 mM Tris-HCl, pH 6.8, 5% (v/v) 2-mercaptoethanol, 2% (w/v) SDS, 40% (v/v) glycerol, the proteins were separated by SDS-PAGE (10% gel) followed by Coomassie Blue staining (PageBlue, Fermentas).

Tryptic Digests and Mass Spectrometry—Coomassie Blue-stained protein bands were excised and treated essentially as described previously (23). After washing, reduction, acetamidation, and dehydration, gel slices were rehydrated in 25 µl of 100 ng/µl trypsin (Promega sequencing grade), 50 mM NH4HCO3, pH 8.0, and incubated for 45 min on ice. The supernatant was removed, and 20 µl of 50 mM NH4HCO3, pH 8.0, was added. Digestion was performed for 10 h at 37 °C. The supernatant containing the tryptic peptides was evaporated to dryness and stored at -20 °C until further use. For mass spectrometry, peptides were dissolved in 10 µl of 0.1% trifluoroacetic acid, 40% ethanol and 1 µl of the mixture was mixed with an equal volume of 10 mg/ml 2.5-dihydroxy benzoic acid in 0.1% (w/v) trifluoroacetic acid, 50% (v/v) acetonitrile. Mass spectra were recorded with the Advanced Laser Desorption Ionization Mass analyzer (ALADIM II) in the reflector mode. After the assignment of monoisotopic peaks, the data were analyzed with ASAP (24) and PAWS (ProteoMetrics).

Thiol-Thiol Cross-linking—The homobifunctional cross-linkers of varying length, 1,8-bismaleimidotetraethyleneglycol (BM[PEO]4) and bismaleimidoethane (BMOE), were obtained from Pierce, and dibromobimane (bBBr) was from Calbiochem (Fig. 1). Stock solutions at 10 mM were made in water, Me2SO, or acetonitrile. The single cysteine MutL and MutH variants were preincubated on ice at 5 µM each for 10 min in 10 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 0.8 mM ADPNP. Samples then were centrifuged for 30 min at 10,000 x g in a tabletop centrifuge at 4 °C. The supernatant was transferred to a new tube and incubated at room temperature (25 °C) with a 100-fold molar excess of cross-linker over thiol groups. The reaction was quenched after 30 s with a 5-fold molar excess of DTT over the cross-linker, and samples were subjected to SDS-PAGE analysis. In the case of bBBr cross-linking, the gels were analyzed under UV light for dibromobimane fluorescence prior to Coomassie Blue staining. All of the gels were analyzed with a video documentation system (Bio-Rad). The intensity of the stained protein bands was quantified using TINA, version 2.07d, software.



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  		FIG. 1.
Thiol-specific cross-linkers used in this study. 4-Maleimidobenzophenone is a heterophotocross-linker with a 8.6–11.4-Å span that is used to modify a single cysteine residue in a protein, which after photoactivation reacts largely nonspecifically with amino acids residues in close proximity (A). The homobifunctional reagents dibromobimane (B), BM[PEO]4 (C), and BMOE (D) cross-link thiol groups selectively. These cross-linkers can span a range of a S–S distance from 3.17 to 6.61, 6.27 to 10.52, and 3.51 to 16.56 Å (17.8 Å according to Pierce), respectively (36).

 
Protein-Protein Docking—All of the models were generated using the algorithms implemented in Chemera 2.0 (www.cqfb.fct.unl.pt/bioin/chemera/) (25). BiGGER was run with two docking partners selecting the MutL dimer (Protein Data Bank code 1b63 [PDB] ) as the target and the MutH protein (2azo [PDB] chain B) as the probe, and the parameters were as follows: resolution 1.0; added radius of 1.0; and angular steps of 15 with a minimal overlap of 100. Ten thousand solutions were obtained by this procedure. After the docking run, models were filtered using a 4-Å cutoff to give 2517 unique clusters. We ranked the models by their agreement with the 4 cross-link-derived distance constraints. The docking models were scored similarly as described previously (24) using Equation 1,

where n is the number of constraints, di is the C{alpha}-C{alpha} distance in the model for the two residues used as the constraint I, and dmax is 16.1 or


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
CONCLUSIONS
 REFERENCES
 
Nucleotide Dependence of Photocross-linking—At low ionic strength, activation of MutH by MutL is independent of MutS and a mismatch on the DNA substrate (26, 27). Under these conditions, binding but not hydrolysis of ATP by MutL is required for the activation of MutH (11, 13, 27). Upon ATP binding, the N-terminal ATPase domain of MutL dimerizes (27), which is a common theme in all of the members of the GHKL family, e.g. gyrase B and Hsp90 (28). MutL1–331 (also named LN40) exists as a monomer in solution in its nucleotide-free or ADP-bound form but dimerizes upon the binding of ATP or ADPNP (27). Moreover, ATP binding and dimerization of the NTD are sufficient for activation of the MutH endonuclease in vitro. Chemical cross-linking with the homobifunctional cross-linker bissulfosuccinimidyl suberate revealed that MutH can interact with the dimeric form of the MutL NTD (29). However, the site of interaction has not yet been determined.

We have previously identified a region in MutH around helix E that binds to MutL (14). The single cysteine variant of MutH (HR172C) labeled with MBP could be photocross-linked to MutL. Consequently, we asked whether the photocross-linking of MutL to MutH is dependent on the dimerization of the MutL NTD. We conducted photocross-linking experiments with MutL and MutH labeled with MBP in the presence or absence of ATP or ADPNP. In addition, we tested the N-terminal fragment of MutL (MutL1–331) for cross-linking to MutH. Only in the presence of ADPNP was photocross-linking with labeled MutH (BP-HR172C) to both MutL and MutL1–331 observed (Fig. 2). This finding indicates that only dimeric MutL can form a complex with MutH and that the MutL NTD is sufficient for the interaction with MutH, in agreement with previous results using a chemical cross-linker (27). We also examined the dependence of the formation of photocross-links at higher ionic strength (125 mM KCl) and in the presence of DNA (data not shown). Cross-links were observed under both conditions, implying that physical interaction is possible under conditions optimal for DNA mismatch repair (13).



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  		FIG. 2.
Nucleotide dependence of MutL·MutH photocross-linking. Photocross-linking of MutL with MutH variant HR172C modified with MBP in the presence and absence of ADPNP. Samples were irradiated at 354 nm as described under "Experimental Procedures" and analyzed by SDS-PAGE. Results for the MutL (full-length protein) (A) and for MutL1–331 (B) are shown. The position of the molecular mass markers (in kDa) are indicated to the left of the gels. Note that, only in the presence of ADPNP, a significant formation of cross-link is observed. H, free MutH; L, free MutL; L-H, cross-linked MutH·MutL complex; L1–331, free MutL1–331; L1–331-H, cross-linked MutL1–331·MutH complex. Proteins bands between the assigned bands of MutL and MutH are due to degraded MutL proteins. Cross-linked bands were assigned after identification of the proteins by tryptic digest and mass spectrometry (data not shown).

 
Single Cysteine Variants of MutL—Our next goal was to determine the MutH interaction site of MutL. Methods for mapping protein interaction sites include cross-linking, protection, interference, and "footprinting" (30). We chose a strategy that in principle allowed us to perform any of these approaches in a site-directed manner using the specific chemistry of cysteine residues. Here, we report our results for the cross-linking method. To perform site-specific labeling of MutL, we needed MutL variants that contained only a single cysteine at selected positions. To this end, we first generated a cysteine-free variant of MutL by site-directed mutagenesis. Replacement of all seven endogenous cysteine residues by alternate amino acid residues was guided by a phylogenetic sequence analysis of the MutL proteins. The cysteine-free variant of MutL (LCys-free) (see "Experimental Procedures") was tested for function in vivo and in vitro and displayed activity comparable with wild type (Table I and Table II).


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  		TABLE I
In vivo analysis to determine the ability of MutL variants to complement a mutL-deficient E. coli strain

 


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  		TABLE II
Mismatch and MutS-dependent stimulation of MutH endonuclease by MutL variants MutH activation on a linear 484-bp heteroduplex substrate containing a single d(GATC) site was assayed as described under "Experimental Procedures." Reactions contained 25 nM heteroduplex, 500 nM MutH, 2 µM MutL, and 1 µM MutS. Incubation was at 37°C, and 10-µl samples were scored for d(GATC) incision as described. Errors shown are mean ± 1 S.D.

 
We next used the phylogenetic sequence analysis of the MutL proteins and the available structural information for E. coli MutL to identify potential surface residues that could serve as targets for replacement with a cysteine residue. Since it had been shown before that the MutL1–331 is sufficient to activate MutH in vitro (27) and that it could be photocross-linked to MBP-modified MutH (BP-HR172C) (see above), we focused on this domain to introduce cysteine residues at thirteen key sites (Fig. 3). Most of these residues with the exception of Lys52 and Gln314 have <60% similarity to MutL proteins from {beta}/{gamma} proteobacteria, and together they cover a large surface of the MutL NTD.



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  		FIG. 3.
Position of cysteine residues in single cysteine variants of MutL. A, the dimeric structure of the MutL1–331 (Protein Data Bank code 1b63 [PDB] ) in the presence of ADPNP is shown. Chains A and B are colored in dark and light gray, respectively. Externally oriented residues selected for mutagenesis to cysteine are indicated. B, same as A but rotated by 90° around the horizontal axis.

 
A prerequisite for successful mapping protein interactions via cross-linking is that the mutations do not affect the function of the protein. In some cases, a mutation directly identifies the protein-protein interface. Such "hot spots" are generally important for complex formation (31, 32). Therefore, we assayed the MutL variants for their in vivo function in mismatch repair. MutL variants were tested for their ability to complement a mutator phenotype in the mutL-deficient E. coli strain TX2652 (17). All of the variants with the exception of LK52C and the MutL1–331 were able to complement a mutL-mutator phenotype at least 10-fold better than the vector control (Table I). This indicates that none of the residues tested with the exception of Lys52 are hot spots for function in mismatch repair and thus for the interaction with MutL. The MutL variants were purified and assayed for their ability to activate MutH in a mismatch-dependent manner by MutS (Table II). All of the variants with the exception of the MutL1–331 retained the ability to mediate communication between MutS and MutH, in good agreement with our in vivo complementation data. The two variants having the slightly conserved residues LK52C and LQ314C exchanged showed reduced activity in vitro. Moreover, we found only a weak correlation between the degree of conservation and the in vitro activity of MutL in the MutH endonuclease activation assay (data not shown).

Photocross-linking of Single Cysteine MutL Variants to MutH—To gain insight into which residues of MutL are in proximity to MutH, photocross-linking experiments were carried out with MBP coupled to cysteine residues in MutL (Fig. 4). MutL variants BP-LN169C and BP-LA251C in the presence of ADPNP and MutH produced a band with low mobility (apparent molecular mass of ~120–150 kDa) in PAGE analysis. This band was only observed in the presence of MutH and ADPNP and contained both MutL and MutH as verified by tryptic digestion and mass spectrometry (see below).



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  		FIG. 4.
Photocross-linking of MutL variants to MutH. Single cysteine MutL variants labeled with MPB (indicated by the amino acid substitution) were subjected to photocross-linking with the partner protein MutH (Cys-free). With 3 of the 13 MutL variants, new bands resulting from a MutL·MutH complex or a MutL·MutL dimer complex appeared in the SDS-PAGE gel. A new band corresponding to a Mr of 120,000 only appeared in the presence of MutH, indicating the formation of a cross-linked MutH·MutL complex as verified by mass spectrometry (see Fig. 5). The band corresponding to a Mr > 200,000 that appeared after UV irradiation was present even in the absence of MutH, indicating the formation of a cross-linked MutL·MutL complex.

 
In the case of BP-LA251C, BP-LH297C, and BP-LQ314C, a high molecular weight complex was observed, even in the absence of MutH. We attributed this to a MutL·MutL photocross-link. This result was expected for BP-LH297C and BP-LQ314C, because the cysteine residues in these variants were within 12 Å to the other subunit. The results for BP-LA251C were unexpected but may be explained by a more compact structure of MutL in solution compared with the crystal, as observed for yeast topoisomerase IIA (33).

We performed tryptic digests and mass spectrometry analysis of the photocross-link products to confirm the presence of MutH and MutL. We tried to identify any cross-linked peptide that could allow us to determine the position of the photocross-link. For BP-HR172C photocross-linked to MutL, we were able to identify tryptic peptides of both MutH and MutL in the photocross-link product. Unfortunately, no cross-linked peptide or missing peptide could be identified since the sequence coverage for MutL peptides was too low (data not shown). In contrast, an analysis of the tryptic digest of BP-LN169C photocross-linked to MutH allowed us to identify two peptides of MutH that were present in the unmodified form of MutH but were missing in the photocross-linked product (Fig. 5), indicating that these peptides may have been cross-linked to MutL. These peptides, comprising amino acids 153–172 and 156–172, were part of the region in MutH that we had previously identified by interference analysis to be the MutL interaction site (14).



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  		FIG. 5.
Mass spectrometry analysis of photocross-links between BP-LN169C and MutH. The MutL variant LN169C modified with MBP was photocross-linked to the cysteine-free MutH variant. A, after separation by SDS-PAGE, the bands corresponding to the MutH (H) and the cross-link (L-H) were cut out and the protein material was digested with trypsin and analyzed by MALDI mass spectrometry. B, MALDI spectra of the products from the tryptic digest of MutH and MutH cross-linked to BP-LN169C. Peaks corresponding to tryptic peptides of MutH are labeled with their monoisotopic mass, whereas peaks corresponding to the tryptic peptide of MutL are labeled with an asterisk. C, an enlargement of the mass spectra shown in B, which highlights the absence of two tryptic peptides of MutH. D, a representation of found sequence matches for MutH after trypsin digestion of the uncross-linked protein and cross-linked protein using PAWS. Note that peptides corresponding to region 156–172 (peptides 2092.17 and 2489.41, respectively) are missing in the mass spectra of the cross-linked MutH protein.

 
Thiol-Thiol Cross-linking of Single Cysteine MutL and MutH Variants—To obtain a more detailed picture of the MutL·MutH complex, we determined the distance between cysteine residues using various length cross-linkers. Different combinations of single cysteine MutH and MutL variants in complex were subjected to thiol-thiol cross-linking using the homobifunctional cysteine-specific cross-linkers BM[PEO]4, BMOE, and bBBr (Fig. 1). Because the structure of the dimeric MutL1–331 is known, distances between the C{alpha} atoms of cysteine residues for the MutL variants could be measured and compared with experimental results of cross-linking with reagents of different length. As the NTD was shown to dimerize only in the presence of ATP or ADPNP (27), we tested for similar nucleotide dependence in the thiol-thiol cross-linking experiments. To minimize the formation of nonspecific cross-links, we used an excess of cross-linker over thiol groups and quenched the reaction by the addition of excess DTT or 2-mercaptoethanol (data not shown). The specificity of the cross-links was assessed by performing experiments with the MutL variants LN131C and LK135C, which have distances between C{alpha} atoms of 26.7 and 48.7 Å, respectively, in the dimeric form of MutL1–331 (Fig. 3). Incubation of MutL variant LN131C, but not LK135C, with the long range cross-linker BM[PEO]4 in the presence of ADPNP resulted in a species with a lower mobility attributed to an L–L dimer (Fig. 6). No cross-links were observed using the shorter cross-linker BMOE or in the absence of ADPNP. Although the 26.7-Å C{alpha}–C{alpha} distance between positions 131 in the MutL1–331 was slightly larger than the 22–23-Å maximum span for C{alpha}–C{alpha} of the cross-linker BM[PEO]4 (using a 2.8-Å C{alpha}–S{gamma} distance for each cysteine and the reported S–S maximum span of 17.8 Å) our cross-linking data are in agreement with the structure of the dimeric MutL1–331. Single cysteine MutH variants incubated with BM[PEO]4 or BMOE did not result in the formation of significant amounts of cross-links (data not shown). These data indicate that, under our assay conditions, the selected cross-linkers can be used to measure the distance of cysteine residues.



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  		FIG. 6.
Cross-linker length and ADPNP dependence. Two single cysteine MutL variants (LN131C and LK135C) were cross-linked with thiol-specific cross-linkers of varying size in the presence or absence of ADPNP. The C{alpha}–C{alpha} atom distance of residues Asn131/Asn131 and Lys135/Lys135 are 26.7 and 48.7 Å, respectively, whereas the maximum span of C{alpha} –C{alpha} atoms bridged by BMOE and BM(PEO)4 is ~16 and 22 Å, respectively. In the case of the variant LN131C, the formation of a cross-linked MutL dimer was possible only in the presence of ADPNP with the longer cross-linker BM(PEO)4.

 
We tested a total of 65 combinations of MutL and MutH variants for thiol-thiol cross-linking with BMOE (Fig. 7). Only four combinations resulted in cross-link formation with a yield of >25%, which is significantly (p < 0.01) higher than the average cross-linking yield of <5%. From this analysis, we conclude that the cysteine residues in the complex formed by HS104C/LL327C, HE156C/LQ314C, HR172C/LN169C, and HR172C/LA251C must be at a distance not larger than the range of the BMOE cross-linker. Neither the MutH variants HS85C and HH115C nor any other MutL variant resulted in a significant formation of thiol-thiol cross-links with any of the MutL variants tested, suggesting that these residues are unlikely to be a part of the MutH·MutL interface. These data are in agreement with our previous finding that the modification of the cysteine residues in MutH variants HS104C,HE156C,HV166C,HL167C, and HR172C with bulky reagents (i.e. mPEG 5000) resulted in reduced activation of MutH endonuclease activity by MutL, whereas this was not observed with HS85C and HH115C (14). To narrow down the distances between different positions in MutH and MutL, we employed the homobifunctional bBBr with an S–S range of only 3.17–6.61 Å. From the combinations that yielded a significant formation of cross-links using BMOE, only HR172C/LA251C and, to a much lesser extent, HR172C/LN169C were able to form a cross-link using bBBr, implying that these residues are in close proximity within the complex (Fig. 8).



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  		FIG. 7.
Quantitative analysis of cross-linking MutH and MutL variant with BMOE. Single cysteine variants of MutH and MutL (indicated by the position of the single cysteine) were incubated at 5 µM with a 100-fold excess of BMOE. The reaction was quenched after 30 s by adding a 5-fold molar excess of DTT over BMOE. Samples were subjected to SDS-PAGE and Coomassie Blue staining. Means ± S.E. are based on three independent experiments.

 



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  		FIG. 8.
Length dependence of thiolthiol cross-linking of single cysteine MutL and MutH variants. Combinations of MutH and MutL variants that gave a significant MutH·MutL complex (L-H) cross-linked with BMOE (Fig. 7) were tested further with bBBr, which spans a smaller range. Only the combination LA251C/HR172C gave a cross-link with this shorter cross-linker. The position of the cysteine residues are indicated in the structure of MutL (top) and MutH (bottom).

 
Model of the MutL·MutH Complex—The program Chemera/BiGGER was used to dock the MutH and MutL proteins and generate a model of the complex (25). The initial 10,000 docking models were then scored with the results from our thiol-thiol cross-linking experiments using BMOE and/or bBBr. The average score of all of the docking models was 23 ± 12 Å. In contrast, the best ten models had an average score of only 2.2 ± 0.4 Å. In these models (Fig. 9), MutH was located between the two subunits of the dimeric MutL, in good agreement with our previous interference analysis (14). Moreover, the models suggested an explanation for the dependence of ATP binding and thus dimerization of MutL for interaction and activation of MutH. The binding site was formed by both subunits of the MutL NTD.



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  		FIG. 9.
Models for complexes comprising MutL and MutH. A, the top ten cluster solutions of the docking process are represented. MutL is shown in ribbons, whereas for MutH only the C{alpha} trace is shown. The subunits of MutL are colored in dark and light gray, respectively. The different MutH solutions are colored from red to green where red represents the best score and green represents the worst score. B, residues that have been implicated in DNA binding or cleavage in MutL and MutH (12, 14) are shown as red and blue spheres, respectively. Note that residues involved in DNA binding/cleavage of MutL and MutH are aligned. Thus, DNA may bind to both proteins at the same time.

 
Previous mutational analyses implicated that the DNA binding site of MutL was located in a deep saddle-shaped groove formed between the two protein subunits, mainly between the transducer domains involving residues Lys159, Arg177, and Arg266 as observed in gyrase B (27, 34). The DNA binding site of MutH has been determined experimentally using a combination of mutational analyses and structure comparisons (14, 15, 18, 34). In several of our top scoring models, the orientation of the two DNA binding sites suggested that both proteins could, in principle, bind to the same DNA molecule in a side by side manner (Fig. 9). Such an interaction with DNA may be important for the activation of the MutH endonuclease by MutL.


   CONCLUSIONS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 CONCLUSIONS
REFERENCES
 
Several studies have shown that MutL can interact both with MutH and MutS in the absence of DNA and that MutL can mediate binding of MutH to MutS, explaining the formation of a ternary complex (11, 26). Although the activation mechanism of MutH mediated by MutL is unknown, it has been suggested that a long-lived MutS·MutL·MutH endonuclease-competent intermediate exists (13). Here, we have characterized the physical interactions between MutL and MutH by cross-linking and have demonstrated that (i) ATP-dependent dimerization of the MutL NTD is required for complex formation between MutL and MutH in the presence and absence of DNA, (ii) the MutL NTD is sufficient for physical interaction with MutH, (iii) the interaction site can be mapped to a region comprising residue positions 169, 251, 314, and 327 of MutL and positions 104, 156, and 172 of MutH, and (iv) a low resolution molecular model of the MutL·MutH complex suggests that both proteins can bind to the same DNA molecule.

Interestingly, the MutH binding site of MutL is in an analogous region to that recently proposed for the client binding site of the structurally related heat shock protein HtpG (35). However, without further experimental data, a more detailed model of the binary and/or ternary complex of MutH, MutL, and DNA remains speculative. Nevertheless, although the mechanism of MutH activation by MutL remains elusive, we are now in a better position to test for complex formation and possible conformational changes involved during activation.

Our model provides insight into several features of the physical and functional interaction of MutL and MutH. It suggests that only in the closed dimeric form of MutL after binding ATP is the interaction site for MutH formed. Furthermore, in principle, both MutH and MutL can bind to DNA simultaneously in a side by side manner. Thus, MutL may either enhance the DNA binding affinity of MutH and/or stabilize the catalytically competent form of MutH. Additional biophysical and biochemical experiments are needed to understand the dynamics of this complex formation and the mechanism of MutH activation.

In conclusion, we have identified a region in MutL that is likely to be the binding site of MutH and determined the spatial distance of different amino acid residues of MutL and MutH by chemical cross-linking. Our results, the first for a complex of mismatch repair proteins, shed light on the structural basis of MutHLS activation. The approach used here is currently being used to analyze the MutL·MutS complex.


   FOOTNOTES
 
* This work was supported by the Deutsche Forschungsgemeinschaft (Fr-1495/3-1) and the Deutsche Akademische Austauschdienst (International Quality Network "Biochemistry of Nucleic Acids"). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

To whom correspondence should be addressed: Institut für Biochemie (FB 08), Justus-Liebig-Universität Giessen, Heinrich-Buff-Ring 58, D-35392 Giessen, Germany. Tel.: 49-641-99-35407; Fax: 49-641-99-35409; E-mail: friedhoff{at}chemie.bio.uni-giessen.de.

1 The abbreviations used are: NTD, N-terminal domain; ADPNP, adenosine 5'-({beta}-{gamma}-imido)triphosphate; bBBr, dibromobimane; BMOE, bismaleimidoethane; DTT, dithiothreitol; BM[PEO]4, 1,11-bismaleimidotetraethyleneglycol; BP-L, MutL protein with 4-maleimidobenzophenone covalently linked to a single cysteine; MBP, 4-maleimidobenzophenone; MALDI, matrix-assisted laser desorption ionization. Back


   ACKNOWLEDGMENTS
 
The expert technical assistance of Ina Steindorf is gratefully acknowledged. We thank Dr. A. Pingoud and Dr. G. Silva for comments and critical reading of the paper.



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 CONCLUSIONS
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