The interacting domains of three MutL heterodimers in man: hMLH1 interacts with 36 homologous amino acid residues within hMLH3, hPMS1 and hPMS2

Abstract

In human cells, hMLH1, hMLH3, hPMS1 and hPMS2 are four recognised and distinctive homologues of MutL, an essential component of the bacterial DNA mismatch repair (MMR) system. The hMLH1 protein forms three different heterodimers with one of the other MutL homologues. As a first step towards functional analysis of these molecules, we determined the interacting domains of each heterodimer and tried to understand their common features. Using a yeast two-hybrid assay, we show that these MutL homologues can form heterodimers by interacting with the same amino acid residues of hMLH1, residues 492-742. In contrast, three hMLH1 partners, hMLH3, hPMS1 and hPMS2 contain the 36 homologous amino acid residues that interact strongly with hMLH1. Contrary to the previous studies, these homologous residues reside at the N-terminal regions of three subdomains conserved in MutL homologues in many species. Interestingly, these residues in hPMS2 and hMLH3 may form coiled-coil structures as predicted by the MULTICOIL program. Furthermore, we show that there is competition for the interacting domain in hMLH1 among the three other MutL homologues. Therefore, the quantitative balance of these three MutL heterodimers may be important in their functions.

Figure 1

Domains of hMLH1 interacting with hMLH3, hPMS1 and hPMS2. The β-gal activities in yeast cells containing various lexA DNA BD and VP16 transcriptional AD plasmids. The left figure represents schematic diagrams of the full-length (residues 1–756) and the 15 hMLH1 deletion constructs in AD plasmids. Lane hMLH3, hPMS1 or hPMS2 indicates the β-gal activities of yeast cells that contain the full-length hMLH3, hPMS1 or hPMS2 BD plasmid and the corresponding truncated hMLH1 AD plasmid. The mean and SD of three independent transformants are shown. The mean β-gal activities in yeast cells containing only hMLH3, hPMS1 or hPMS2 BD plasmid are 0.3, 0.1 or 0.1 U, respectively.

Figure 2

Summary of the two-hybrid assay, GST-IVTT and immunoprecipitation study between the full-length hMLH1 and various hPMS2 deletion constructs. These three assays were performed as described in Materials and Methods. The left figure represents schematic diagrams of the full-length (residues 1–862) and the twenty-six hPMS2 deletion constructs. The mean and SD of β-gal activities in three independent yeast transformants containing various hPMS2 deletion constructs in the BD plasmid and the full-length hMLH1 cDNA in the AD plasmid are indicated in the two-hybrid lane. The values in parentheses represent the mean β-gal activity in the yeast cell containing only the corresponding BD plasmid. Results of the GST-IVTT assay are presented as the mean and SD of three separate experiments and are indicated as relative values when the binding ability of the full-length hPMS2 to GST-hMLH1 is 100. Lane IP indicates the presence (+) or absence (–) of the immunoprecipitated band. ND, not detected.

Figure 3

Determination of the hMLH1-interactive domain of hPMS2 at residues 612–674 using the immunoprecipitation study (A) and the GST-IVTT assay (B). (A) Extracts of HEC-1-A cells transfected with pFLAG plasmids containing various hPMS2 deletion constructs were subjected to immunoprecipitation analyses using the anti-FLAG M2 monoclonal antibody. Immunoprecipitated proteins were analysed by the use of anti-hMLH1 monoclonal antibody. Lane HEC-1-A illustrates 20 µg of the cell extract that was subjected to immunoblotting without prior immunoprecipitation as the control. The arrow indicates the position of the hMLH1 protein. The minimal interaction domain of hPMS2 with hMLH1 lies between residues 612 and 674. (B) ³⁵S-labeled full-length and deletion mutant proteins of IVTT-hPMS2 were added to glutathione beads that had been pre-treated with either GST alone (–) or GST-hMLH1 (+). Samples were resolved on 8% (top left), 15% (top right) or 10–20% (bottom) SDS-PAGE and examined by BAS-1500 and LAS-1000 (Fuji Film). Again the minimal interacting domain of hPMS2 with hMLH1 resides at residues 612–674.

Figure 4

36 homologous amino acid residues conserved in hPMS2, hPMS1 and hMLH3. The proportions of identity or similarity with residues 612–674 of hPMS2 are presented. Lowercase letters indicate positions of the heptad repeat in the predicted coiled-coil motif. Bold letters in the consensus sequence correspond to identical amino acids. X, non-conserved amino acids. As for hMLH3, four homologous regions were identified, however, only residues 860–895, the highest homologous region, can interact with hMLH1.

Figure 5

Domain of hPMS1 interacting with hMLH1. Results of the two-hybrid assay in yeast cells that contain the corresponding hPMS1 constructs (left figure) in the BD plasmid and the full-length hMLH1 cDNA in the AD plasmid. Parentheses indicate the mean β-gal activities in the yeast cells containing only the corresponding BD plasmid. Each β-gal activity is the mean and SD of three independent transformants. The minimal interacting domain of hPMS1 with hMLH1 resides at residues 693–728 as established in Figure 4.

Figure 6

Domains of hMLH3 interacting with hMLH1. Results of the two-hybrid assay in yeast cells that contain the corresponding hMLH3 constructs (left figure) in the BD plasmid and the full-length hMLH1 cDNA in the AD plasmid. Parentheses indicate the mean β-gal activities in the yeast cells containing only the corresponding BD plasmid. Each β-gal activity is the mean and SD of three independent transformants. The domain of hMLH3 interacting with hMLH1 resides at residues 860–895, as established in Figure 4. There are two additional domains of hMLH3 interacting with hMLH1 at residues 1–244 and 1399–1453.

Figure 7

The structural relationship between each interacting domain and three subdomains in four MutL homologues: hMLH1, hPMS2, hPMS1 and hMLH3. The 36 homologous amino acid residues showing strong interacting activities were all located in the N-terminal regions of three MutL subdomains.

Figure 8

Competitive inhibition of hPMS2 binding by hPMS1 or hMLH3 when complexed with GST-hMLH1. The GST-hMLH1 and 2 µl of ³⁵S-labeled IVTT-hPMS2 were incubated with indicated amounts of unlabeled IVTT-luciferase, IVTT-hPMS1 or IVTT-hMLH3. Then hPMS2 complexed with GST-hMLH1 was resolved by 10–20% SDS–PAGE and examined by BAS-1500. The arrow indicates the position of hPMS2 protein. Both hPMS1 and hMLH3 compete with hPMS2 for the binding to hMLH1.