Atomic model of the 5‐methylcytosine‐specific restriction enzyme McrA reveals an atypical zinc finger and structural similarity to ββαMe endonucleases (2000)

Sir, Using a novel ffas algorithm for supersensitive sequence comparison and fold recognition, we have identified (Rychlewski et al., 2000, Protein Sci9: 232–241) a domain in the 5-methylcytosine-specific restriction enzyme (ENase) McrA from Escherichia coli that is common to the ββα-Me superfamily of nucleases (Kuhlmann et al., 1999, FEBS Lett463: 1–2). We carried out extensive threading of the McrA sequence onto the known protein structures and detected a high compatibility with conformation assumed by the DNase domain of colicins E7 (Ko et al., 1999, Structure7: 91–102) and E9 (Kleanthous et al., 1999, Nature Struct Biol6: 243–52), although it displays no significant similarity to the sequences of these proteins. The model presented reveals that McrA shares a putative zinc finger with homologous group II intron maturases and the more remotely related coliphage T4 endonuclease VII (Raaijmakers et al., 1999, EMBO J18: 1447–1458), despite the fact that this structure is absent from colicins. This is the first robust structure prediction of an ENase ever performed. It strongly suggests that ENases do not all share the same three-dimensional fold and, as a functional class of proteins, they should be regarded as products of convergent evolution. Comparative analysis of recently published structures of colicins E9 and E7, homing nuclease I-PpoI, coliphage T4 endonuclease VII and a non-specific endonuclease from Serratia marcescens identified a distinct superfamily of proteins dubbed ‘ββα-Me’ for the three secondary structure elements and the metal ion that make up the invariant core embedded in strikingly variable peripheral structures (Friedhof et al., 1999, FEBS Lett443: 209–214 and Nature Struct Biol6: 112–113; Kuhlmann et al., 1999, FEBS Lett463: 1–2). Such eroded homology is also characteristic of the PD-(D/E)XK superfamily of DNases, which includes all structurally characterized type II ENases – commercial enzymes indispensable in recombinant DNA technology and molecular medicine (Kovall and Matthews, 1999, Curr Opin Chem Biol3: 578–583). Based on comparison of crystal structures, one of us has recently demonstrated (Bujnicki, 2000, J Mol Evol50: 39–44) that it is possible to infer the evolutionary history of ENases, despite the notion that their sequences are ‘too divergent’ for phylogenetic inference (for instance, see Jeltsch et al., 1995, Gene160: 7–16). Now, applying a recently developed state-of-the-art ffas algorithm for protein structure prediction (Rychlewski et al., 2000, Protein Sci9: 232–241), we launched a large-scale analysis, attempting to demonstrate that it is also possible to predict the structures of ENases with high confidence. Here, we present the first structural model obtained. McrA (for modified cytosine restriction) was originally identified as an activity of E. coli K-12, named RglA, that restricts non-glucosylated, 5-hydroxymethylcytosine-containing T-even phages (Revel, 1967, Virology31: 668–701), and was also subsequently demonstrated to target 5-methylcytosine (m5C), preferably at Cm5CGG sites (Raleigh and Wilson, 1986, Proc Natl Acad Sci USA83: 9070–9074). Together with non-related modification-directed restriction systems McrBC and Mrr encoded by the immigration control region (ICR; Raleigh, 1992, Mol Microbiol6: 1079–1086), the activity of McrA encumbered early attempts at cloning of DNA with foreign, for instance eukaryotic, methylation patterns. Starting from the McrA sequence, we attempted to build a multiple alignment to obtain as much evolutionary information related to the protein structure as possible. We conducted several cycles of psiblast (Altschul et al., 1997, Nucleic Acids Res25: 3389–3402) searches of the non-redundant database and preliminary genomic data (obtained from http://www.ncbi.nlm.nih.gov/), database threading using ffas (Rychlewski et al., 2000, Protein Sci9: 232–241), homology modelling using modeller (Sali and Blundell, 1993, J Mol Biol234: 779–815) and manual refinement. The final alignment revealed significant homology of the entire McrA sequence with α-sen and other group II intron maturases, whereas the structurally characterized proteins from the ββα-Me family only matched its C-terminal domain (Fig. 1). Interestingly, McrA and maturases seem to share a putative Zn binding site with T4 endonuclease VII (ffas Z-score only 3.95), despite this site being absent from the structurally more compatible colicins (Z-score 9.63). The conserved core of McrA (accession no. g96185) aligned with Podospora anserina mitochondrial maturase α-sen (g1334533) and structurally characterized ββα-Me representatives: colicin E7(PDB# 7cei), colicin E9(1bxi), T4endonuclease VII (1en7), S. marcescens nuclease (1qae) and endonuclease I-PpoI (1ipp). The consensus secondary structure is shown at the bottom. Highly conserved residues are shown in black boxes; moderately conserved in bold. The histidines predicted to co-ordinate the Mg2+ ion, essential for the phosphodiester bond cleavage to occur, are indicated by ‘’□‘’. The cysteines predicted to make up a zinc finger are indicated by an asterisk (*). To obtain further insight into this intriguing ‘mosaic’ similarity, we performed homology modelling of the C-terminal conserved region of McrA using a modified version of the ‘multiple’ approach of Jaroszewski et al. (1998, J Mol Model4: 294–309), with the structures of all homologues as modelling templates. The final model revealed a compact ββα-Me-like domain harbouring a putative active site and suggested that residues from one of the helices might be involved in specific recognition of the m5C-containing target sequence (Fig. 2). Modelling of McrA also revealed that the putative zinc finger does not directly participate in forming the active site, but rather stabilizes the structural scaffold and brings together the catalytic histidines, most probably involved in co-ordinating a Mg2+ ion. The model of McrA catalytic domain (residues 159–272) manually docked to a double-stranded DNA fragment. Both ions are predicted to be tetrahedrally co-ordinated: Zn2+ by four cysteines (C-207, C-210, C-248 and C-251) and Mg2+ by three histidines (H-228, H-252 and H-256) and the oxygen atom of the DNA phosphate backbone respectively. H-229 seems to interact directly with the backbone phosphate groups. The N-terminal tip of the α-helix comprising residues 183-ERLQKVYVRDPMVK-196 is nested in the major groove of the DNA, where the methyl or hydroxymethyl groups of modified cytosines might be exposed. However, the relative position of the McrA cleavage site with respect to the target sequence is not known or whether the N-terminal region, which could not be modelled, might also exert specific DNA binding. Therefore, the precise prediction of specificity determinants must await further experimental study. These predictions are amenable to experimental verification and should assist in the rational design of mutagenesis studies. Experiments guided by our model might explain the role of additional metal binding site and alter the DNA–binding interface of McrA, thereby modifying its substrate specificity. The experience gained from this work will also help to improve the multistep structure prediction protocol, including automation of the most tedious steps.