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

Sir, Using a novel FFAS algorithm for supersensitive sequence comparison and fold recognition, we have identified (Rychlewski et al., 2000, Protein Sci 9: 232± 241) a domain in the 5-methylcytosine-specific restriction enzyme (ENase) McrA from Escherichia coli that is common to the bba-Me superfamily of nucleases (Kuhlmann et al., 1999, FEBS Lett 463: 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, Structure 7: 91±102) and E9 (Kleanthous et al., 1999, Nature Struct Biol 6: 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 J 18: 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 `bba-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 Lett 443: 209±214 and Nature Struct Biol 6: 112±113; Kuhlmann et al., 1999, FEBS Lett 463: 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 Biol 3: 578±583). Based on comparison of crystal structures, one of us has recently demonstrated (Bujnicki, 2000, J Mol Evol 50: 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, Gene 160: 7±16). Now, applying a recently developed state-of-the-art FFAS algorithm for protein structure prediction (Rychlewski et al., 2000, Protein Sci 9: 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-hydroxymethylcytosinecontaining T-even phages (Revel, 1967, Virology 31: 668±701), and was also subsequently demonstrated to target 5-methylcytosine (mC), preferably at CmCGG sites (Raleigh and Wilson, 1986, Proc Natl Acad Sci USA 83: 9070±9074). Together with non-related modificationdirected restriction systems McrBC and Mrr encoded by the immigration control region (ICR; Raleigh, 1992, Mol Microbiol 6: 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 PSI-BLAST (Altschul et al., 1997, Nucleic Acids Res 25: 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 Sci 9: 232±241), homology modelling using MODELLER (Sali and Blundell, 1993, J Mol Biol 234: 779±815) and manual refinement. The final alignment revealed significant homology of the entire McrA sequence with a-sen and other group II intron maturases, whereas the structurally characterized proteins from the bba-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). To obtain further insight into this intriguing `mosaic' similarity, we performed homology modelling of the Cterminal conserved region of McrA using a modified version of the `multiple' approach of Jaroszewski et al. (1998, J Mol Model 4: 294±309), with the structures of all