The structure of the KlcA and ArdB proteins reveals a novel fold and antirestriction activity against Type I DNA restriction systems in vivo but not in vitro

Abstract

Plasmids, conjugative transposons and phage frequently encode anti-restriction proteins to enhance their chances of entering a new bacterial host that is highly likely to contain a Type I DNA restriction and modification (RM) system. The RM system usually destroys the invading DNA. Some of the anti-restriction proteins are DNA mimics and bind to the RM enzyme to prevent it binding to DNA. In this article, we characterize ArdB anti-restriction proteins and their close homologues, the KlcA proteins from a range of mobile genetic elements; including an ArdB encoded on a pathogenicity island from uropathogenic Escherichia coli and a KlcA from an IncP-1b plasmid, pBP136 isolated from Bordetella pertussis. We show that all the ArdB and KlcA act as anti-restriction proteins and inhibit the four main families of Type I RM systems in vivo, but fail to block the restriction endonuclease activity of the archetypal Type I RM enzyme, EcoKI, in vitro indicating that the action of ArdB is indirect and very different from that of the DNA mimics. We also present the structure determined by NMR spectroscopy of the pBP136 KlcA protein. The structure shows a novel protein fold and it is clearly not a DNA structural mimic.

Figure 1.

The effect of expressing KlcA₁₃₆ in a 2-AP sensitive strain of E. coli. A single colony of the ClpX⁻ strain NM1041(DE3) harbouring either the KlcA expression construct (pET20b-klcA₁₃₆) or the corresponding vector alone (pET20b) was picked with a loop and spread onto an LB agar plate supplemented with 100 μg/ml carbenicillin, 25 μg/ml kanamycin in the absence (A) or presence (B) of 2-AP (80 μg/ml). The plates were then incubated at 37°C for 14 h.

Figure 2.

An overlay of elution profiles for different proteins from a small volume (25 cm × 0.46 cm diameter) size exclusion chromatography column showing the variable quaternary structure of the four different proteins in 20 mM Tris, 20 mM MES, 0.2 M NaCl, 0.1 mM EDTA, 7 mM β-mercaptoethanol, pH 6.5: ArdB_CFT (black, 4 µM)), ArdB_YAF (blue, 3 µM), ArdB_YFJ (red, 5 µM) and KlcA₁₃₆ (green, 4 µM). The proteins were detected using tryptophan fluorescence emission and are arbitrarily scaled vertically.

Figure 3.

Nuclease assay to test for inhibition of EcoKI by ArdB proteins. Lane 1, undigested DNA; lane 2, EcoKI digested DNA; lane 3, plus ocr; lane 4, plus Orf18 ArdA; lane 5, plus ArdB_YAF; lane 6, plus ArdB_YFJ; lane 7, plus KlcA₁₃₆; lane 8, plus ArdB_CFT; lane M, DNA ladder with sizes in kb. Plasmid DNA used was non-methylated pBRsk1. Ratio of the anti-restriction proteins (monomers) to EcoKI was 20:1. In each case the reaction mixture minus DNA was made up and incubated for 2 min at room temperature. The reaction was then initiated by adding DNA. Digestion was carried out for 8 min at 37°C before quenching at 68°C for 10 min. CC, closed circular plasmid; L, linear plasmid; OC, open circular plasmid.

Figure 4.

Raw ITC data showing heat changes for the interaction of KlcA₁₃₆ with the EcoKI Mtase compared with data for the interaction of ArdA with the Mtase as previously published (13). The traces show from top to bottom: Mtase being titrated with ArdA at 25°C, buffer being titrated with Mtase at 25°C, Mtase being titrated with KlcA₁₃₆ at 25°C and Mtase being titrated with KlcA₁₃₆ at 10°C. It is clear that there is no interaction between the Mtase and KlcA₁₃₆.

Figure 5.

The effect of adding ArdB or KlcA during the assembly of the EcoKI RM enzyme. Where relevant, Mtase and the R subunit were preincubated at 25°C in the presence of a × 20 excess of ArdB or KlcA protein for ∼5 min prior to mixing and addition of DNA. Reactions were performed at 37°C for 8 min. Lane 1, uncut pBRsk1 (3 nM); lane 2, pBRsk1 (3 nM) digested with EcoKI (30 nM); lane 3, pBRsk1 digested with reconstituted nuclease (equivalent to 30 nM EcoKI); lane 4, nuclease reconstituted in the presence of ArdB_YAF (20-fold excess over nuclease); lane 5, 20-fold excess ArdB_YFJ; lane 6, 20-fold excess KlcA₁₃₆; lane 7, pBRsk1 in the presence of R subunit only; lane 8, pBRsk1 in the presence of Mtase only. M, 1 kbp size marker.

Figure 6.

2D ¹H-¹⁵N HSQC spectrum of KlcA₁₃₆ at 800 MHz.

Figure 7.

KlcA₁₃₆ secondary and tertiary structure. Colour code: alpha-helices, red; beta-strands, yellow and 3₁₀ helix, purple. (A) Secondary structure elements identified by STRIDE for the ensemble consensus shown as a topology fold schematic (55–57). (B) A cartoon representation of the closest-to-mean 3D structure in the ensemble. Note: loops are rendered smooth for clarity.

Figure 8.

Electrostatic surface representation of the KlcA₁₃₆ protein. Two views rotated by 180° about the y-axis of a GRASP electrostatic surface representation of the KlcA₁₃₆ protein. The molecule appears to expose many charged residues, labelled. Negative charge is coloured red and positive charge coloured blue, ranging from −10 k_BT to +10 k_BT (k_B: Boltzmann constant; T: temperature in Kelvin).

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.

Figure 10.

A PROMALS3D sequence alignment of KlcA and ArdB proteins. KlcA_RK2 and ArdB_pKM101 are the archetypal sequences for these two proteins (11,20). Yellow shading represents beta strands, red shading represents alpha helices and purple shading represents the 3₁₀ helix. The grey shading highlights the anti-restriction motif LLREYVNTL in ArdB from pKM101 (7,26). Consensus amino acid symbols are: conserved amino acids are in bold and uppercase letters; aliphatic (I, V, L): l; aromatic (Y, H, W, F): @; hydrophobic (W, F, Y, M, L, I, V, A, C, T, H): h; alcohol (S, T): o; polar residues (D, E, H, K, N, Q, R, S, T): p; tiny (A, G, C, S): t; small (A, G, C, S, V, N, D, T, P): s; bulky residues (E, F, I, K, L, M, Q, R, W, Y): b; positively charged (K, R, H): +; negatively charged (D, E): −; charged (D, E, K, R, H): c.

Figure 11.

Sequence conservation mapped onto KlcA₁₃₆ protein surface. Conservation surface representation based upon the PROMALS3D multiple sequence alignment of ArdB and KlcA sequences (Supplementary Figure S5) where largely and completely conserved residues are shown in lavender/plum/violet colour mapped onto the surface and variable residue positions coloured cyan.