The protein footprinting data achieved by mass spectrometry and the subsequent model of the Pms1-NTD/DNA complex correlates well with what is already known about their interaction. The limited proteolysis data has shown that the Pms1-NTD bound to DNA is slower than in the unbound form (). While this may be partially attributed to non-specific interactions between the enzyme and DNA precluding proteolysis, it suggests that the Pms1-NTD is in a conformation more stable to proteolysis when in the DNA-bound form. Additionally, proteolysis is slower for protein bound to dsDNA than protein bound to ssDNA indicating that the Pms1-NTD has a higher affinity for dsDNA than ssDNA, which is consistent with previous studies [64
]. Several residues exhibited increased proteolysis. While these residues are not considered potential DNA binding sites, they may become more exposed as a result of the DNA-bound conformation to allow potential interactions with other MMR proteins.
Residues Arg188 and Lys190 both exhibited protection from proteolysis in the presence of DNA. They are conserved in LN40, and in PMS2 from human and mouse, implying biological relevance. Along with Lys197 and Arg198, they reside on a bent α helix (), which is reminiscent of α helices involved in the structures of other DNA binding proteins with the ability to fit well within the major groove of DNA [65
]. It should be noted that mutation rates measured for the K190E mutant did not exhibit a mutator phenotype [18
]; therefore, Lys190, which is clearly protected from proteolysis in the data presented here, is not required for DNA binding. For these reasons, Arg188 and Lys190 were considered important in generating a model for the interaction of the Pms1-NTD with DNA.
Two peptides exhibited complete protection when the Pms1-NTD was bound to dsDNA: R5 and K30. The R5 peptide is generated by cleavage at Arg188 and Arg198. Because cleavage occurs at Arg188 in the presence of DNA, albeit significantly reduced as observed for R4 ( and ), Arg198 must be directly involved in DNA binding or is highly protected from proteolysis when DNA is bound. Arg198 is located on an α helix in the X-ray structure () and is conserved in the PMS2 human and mouse homologs indicating its significance to protein function. The K197E/R198E/K229E mutant exhibited increased susceptibility to proteases in the presence of DNA suggesting a reduced affinity as a result of one or more of these mutations. Arana et al.
] have shown that Lys197 and Arg198 are both important for DNA binding in experiments where K197E and R198E mutants abolished DNA binding in vitro
while the K229E mutant does not display a strong mutator phenotype. Strong mutator phenotypes were observed for Lys218, Arg243 and Lys244 mutants without corresponding reductions in DNA affinity. According to the model presented here, Lys218, Lys229, Arg243, Lys244 and Arg311 are all distant from the DNA-binding site and would not be expected to affect DNA binding. Arana et al.
also identified a surface consisting of positively-charged residues spanning from Lys197/Arg198 to Lys244 that may potentially interact with DNA. Our model agrees with the basic region containing Lys197 and Arg198, but instead places the DNA-binding region along an adjacent positively charged groove, implicating the aforementioned groove as a potential binding surface for other MMR proteins.
The K30 peptide is generated by cleavage at Lys364 and Lys380. Cleavage occurs at Lys380 because K31 is readily formed, which implies that Lys364 is bound to the DNA or shielded from cleavage due to a subsequent conformational change. Lys364 is conserved in human and mouse Pms2 as well as human PMS2 and E.coli LN40, and is located in a loop (). Because loops are flexible, it may move allowing Lys364 to interact directly with DNA. Lys364 forms a salt bridge with Glu61 and Asp64 in the modeled structure; therefore, it is also likely that DNA binding disrupts the salt bridge and that the presence of DNA prevents access of the protease to the backbone. Lys364 is taken to be involved in DNA binding, whether through direct contact or protection by the DNA itself, and was used to guide DNA docking in generating the model for the DNA-binding site of the Pms1-NTD.
Oxidative surface mapping data demonstrated similar protection from oxidation with either DNA or ATP as a ligand. This is attributed to electrostatic interactions of AMP-PNP with the DNA-binding site and vice versa, as well as a similar stabilization of the Pms1-NTD to local unfolding during γ-irradiation. Perhaps in the absence of DNA, ATP binding generates an allosteric effect that better presents the binding site to DNA during MMR. Peptide T38 was uniquely protected upon Pms1-NTD binding to AMP-PNP. Phe313 is only ~10% accessible while Tyr315 is completely buried; therefore, in the absence of ligand, local unfolding of the structure may allow access by the hydroxyl radical. Furthermore, the base of the ATP binding site involves the α-helix comprised of residues 55-69 () that constricts the motion of the α helix comprised of residues 195-214 when nucleotide is bound, which in turn protects the structure proximal to Phe313 and Tyr315 from local unfolding. Protection of T38 is attributed to stability of the structure upon AMP-PNP binding.
In the presence of dsDNA, only T39 is uniquely protected from oxidation with statistical significance. There is no significant protection in the presence of AMP-PNP, nor is there significant protection in the presence of both ligands, suggesting that the conformation differs somewhat in the presence of both ligands. Protection from oxidation may result from interaction with DNA instead of, or in addition to, stabilization of the structure. Tyr323, the site of oxidation on T39, is mapped on the crystal structure at the base of the positively-charged groove that also encompasses Arg188, Lys190, Arg198 and Lys364. The diameter of this groove in the crystal structure is approximately 20 Å, making it suitable for accommodating dsDNA.
The electrostatic surface potential diagram of the Pms1-NTD model () shows the positively charged groove where DNA binding occurs according to our model. Likewise, homodimerization of E. coli
LN40 in the presence of ADP-PNP forms a highly positive-charged surface potential where ssDNA may bind [20
] (). An overlay of the Pms1-NTD/DNA model with the model of the LN40/ADP-PNP complex shows the double-stranded DNA passing through the same groove thought to be the binding site for ssDNA in E. coli
MutL (). An R266E mutation in this putative DNA binding groove of LN40 () abolishes DNA binding, providing further evidence for a DNA-binding site [20
]. When a homologous mutation, K328E, was made in the Pms1-NTD, there was little reduction in DNA binding whereas its MutLα binding partner, Mlh1, lost its ability to bind DNA when the homologous R274E mutation was introduced [16
]. Our model suggests that DNA binding via a positively-charged groove has been conserved between E. coli
and eukaryotic homologs of MutL although the basic residues of contact may differ.
Comparison of models of the LN40/ADP-PNP and the Pms1-NTD/DNA complexes
Understanding the role of Pms1 is important to human health because mutations in the homologous human PMS2
gene are associated with hereditary non-polyposis colon cancer (HNPCC), which results in a propensity toward colon cancer, but is also associated with endometrial and ovarian tumors [4
]. Most of the PMS2
mutations that are associated with cancer are deletion, frameshift and nonsense mutations that result in truncation of the protein and prevent ATP binding, DNA binding and heterodimer formation with Mlh1. Four nucleotide substitutions have been reported that correlate with colon or endometrial cancer [66
]. Two of the substitutions, which generate the protein variants I18V and R20Q, are located 5′ to the sequence that defines the ATP-binding motif and probably disrupt nucleotide binding. The third substitution results in a silent mutation in the protein product, yet is associated with cancer indicating that the mutation may affect transcription. The fourth substitution translates into an A182T mutant in PMS2 and is associated with endometrial cancer. The homologous version of this mutation on the Pms1-NTD would occur on the same α-helix () immediately downstream of Lys197 and Arg198, and, therefore, within the proposed DNA-binding site. This implies that mutations in the DNA binding site of Pms1 or its homologs could affect its ability to perform MMR with serious consequences to the organism.
The model presented here defines the DNA binding interface along a positively-charged groove of the Pms1-NTD. Our data, as well as previous functional studies, indicate that Lys197 and Arg198 are directly involved while Arg188, Lys190, Tyr323 and Lys 364 define the DNA-binding surface without directly participating in DNA binding. In addition, this study has shown how the complementary techniques of limited proteolysis and oxidative surface mapping coupled with mass spectrometry can be used to obtain structural information from a large and dynamic complex. This model of the Pms1-NTD/DNA complex will provide further insight into the structure-function relationship of Pms1 in mismatch repair.