Redefining the PF06864 Pfam family based on Burkholderia pseudomallei PilO2(Bp) S-SAD crystal structure

Abstract

Type IV pili are surface-exposed filaments and bacterial virulence factors, represented by the Tfpa and Tfpb types, which assemble via specific machineries. The Tfpb group is further divided into seven variants, linked to heterogeneity in the assembly machineries. Here we focus on PilO2(Bp), a protein component of the Tfpb R64 thin pilus variant assembly machinery from the pathogen Burkholderia pseudomallei. PilO2(Bp) belongs to the PF06864 Pfam family, for which an improved definition is presented based on newly derived Hidden Markov Model (HMM) profiles. The 3D structure of the N-terminal domain of PilO2(Bp) (N-PilO2(Bp)), here reported, is the first structural representative of the PF06864 family. N-PilO2(Bp) presents an actin-like ATPase fold that is shown to be present in BfpC, a different variant assembly protein; the new HMM profiles classify BfpC as a PF06864 member. Our results provide structural insight into the PF06864 family and on the Type IV pili assembly machinery.

Figure 1. Comparison of Tfpb machinery R64 thin pilus variant encoding operons for different microorganisms.

The alignment was performed using tblastx from the Blast suite, and visualized in Artemis Comparison Tool. Conserved protein regions are paired by color-shaded regions; the blue and red colors represent the reverse and forward matches, respectively, and color intensity is proportional to the sequence homology. Genes are represented by arrows; the same arrow color indicates putative orthologs. The grey arrows represent genes lacking homologs among represented pil clusters. The pil cluster sequences were retrieved from GenBank: Tfp7 locus from B. pseudomallei (Bp) chromosome 2 complete sequence, BX571966.1; PAPI-1 pil gene cluster from P. aeruginosa (Pa) PA14, AY273869.1; R64 transfer region, AB027308.1; and pil operon from Salmonella enterica (Se) subsp. enterica serovar Paratyphi C strain CN13/87, AY249242.1.

Figure 2. Schematic view of the sequential strategy applied to generate PF06864 Pfam family improved HMM profiles.

See the main text and Supplementary Fig. S1. for alignment coding.

Figure 3. N-PilO2_Bp protein.

A. Overall fold of N-PilO2_Bp composed of two α/β topology subdomains, each displaying a mixed β-sheet, separated by a (central) cleft. The bound phosphate ion is shown as spheres. B. Topology diagram of N-PilO2_Bp. This diagram was generated using PDBSum server (www.ebi.ac.uk/pdbsum/) . C. Crystal packing of the phosphate-containing N-PilO2_Bp structure, showing the three crystal packing dimers formed by alternative interactions between four symmetry-related monomers (green, blue, magenta and black). The three interfaces are highlighted by black, blue and red shading. The first ‘dimer’, is formed by the interaction between the green (or blue) and the magenta (or black) monomers and the light green (or light blue) phosphate. The second crystallographic dimer occurs between the magenta and black monomers. The third dimer is formed by the green and blue monomers. D. Stereo view of the electron density map for the residues building the phosphate ion binding pocket. The phosphate ion is shown as sphere; the electron density is contoured at 1.5 sigma level. E. Front and back view of N-PilO2_Bp electrostatic surface potential. The electrostatic potential was calculated using the CCP4MG viewer. Negative (red) and positive (blue) charges, and uncharged (white) surfaces are shown. F. Superposition of the 3D structures of N-PilO2_Bp (cyan; PDB codes 4BYZ and 4BZ0) and N-BfpC (chocolate; PDB code 3VHJ).