Nucleic Acids Research, 2010, Vol. 38, No. 5 1733 Figure 9. Lipophilic surface representation of the KlcA136 protein. Two views, rotated by 180 about the y-axis, of a MOLCAD-generated lipophilic surface rendition of the protein. Regions of high lipophilicity or hydrophobicity are coloured brown and regions of high hydrophilicity are coloured blue. Additionally, the lipophilic surface rendition generated by MOLCAD reveals two regions of spatially contiguous hydrophobic side-chains on one face of the molecule, Figure 9 (left-hand panel) and another more prominent hydrophobic region on the opposite face of the protein, Figure 9 (right-hand panel). Among these, F21, L22, W54, F83, L87, A128 and I141 are largely conserved. Some of the aromatic side-chains are involved in aromatic– aromatic stacking interactions e.g., F64 with F21, Y25, F26; Y57 with Y65, F127; Y39 with W54 and W41 with F99. Comparisons between the 43 ArdB and KlcA protein sequences showed that they have highly conserved a-helices and b-strands, while loops vary in length and amino acid composition. Figure 10 shows a subset of the sequences comprised of the proteins listed in Table 1 and the archetypal KlcA from plasmid RK2 (20) and archetypal ArdB from pKM101 (11). Mapping sequence conservation (Supplementary Figure S5) onto the surface of KlcA136 shows only three well-conserved patches, comprising ﬁrstly H81 and D142, secondly L87, S88 and E133 and lastly E35, Y49, G51, A52 and W54, Figure 11. Only the ﬁrst of these three patches projects out from the surface, the others are on concave regions of the surface. Discussion In this study, we have solved the atomic resolution struc- ture of KlcA136, an anti-restriction protein belonging to the ArdB family and showed its anti-restriction function against Type I RM systems. We have also demonstrated the same anti-restriction function for ﬁve other examples of the ArdB/KlcA family from a range of organisms. A particularly striking result is the absence of any inhi- bition of a Type I RM enzyme in vitro. The EcoKI RM enzyme was able to bind and cleave unmodiﬁed DNA in the presence of either ArdB or KlcA proteins in contrast to the result in vivo in which the enzyme failed to operate against invading phage DNA in the presence of these anti-restriction proteins. Thus the ArdB/KlcA proteins must operate in a diﬀerent manner to the DNA mimics. Several protein-based anti-restriction strategies aimed at negating the activity of Type I RM systems have been identiﬁed in addition to DNA mimicry (2), namely, hydro- lysis of the essential S-adenosyl-L-methionine cofactor (77), proteolysis of the HsdR subunit required for DNA cleavage (30,32,71), enhancement of the Mtase activity via the action of the lambda ral gene to allow protection of unmethylated DNA targets (78) and interference with the subunit assembly process forming the complete RM enzyme (26). Our data and that of Belogurov et al. (11), who demonstrated that ardB did not stimulate DNA mod- iﬁcation by the EcoKI Type I RM system using a ral-like mechanism, show that these mechanisms are not applica- ble to the ArdB/KlcA proteins. Thus the mechanism of anti-restriction by ArdB and KlcA remains unclear at present. However, the variable quaternary structure observed using size exclusion chromatography may suggest a role for large scale conformational changes in both tertiary and quaternary structure in ArdB and KlcA as being involved in anti-restriction. The structure of KlcA136 is the ﬁrst ArdB/KlcA struc- ture reported and adopts a novel fold. Although three distinct regions of charge are apparent from the electrostatic surface representation, only a handful of charged residues are conserved among the 43 non-redundant related sequences of the ArdB/KlcA group. Other anti-restriction proteins, such as ocr from