P. aeruginosa PilT structures with and without nucleotide reveal a dynamic type IV pilus retraction motor - PubMed (original) (raw)

P. aeruginosa PilT structures with and without nucleotide reveal a dynamic type IV pilus retraction motor

Ana M Misic et al. J Mol Biol. 2010.

Abstract

Type IV pili are bacterial extracellular filaments that can be retracted to create force and motility. Retraction is accomplished by the motor protein PilT. Crystal structures of Pseudomonas aeruginosa PilT with and without bound beta,gamma-methyleneadenosine-5'-triphosphate have been solved at 2.6 A and 3.1 A resolution, respectively, revealing an interlocking hexamer formed by the action of a crystallographic 2-fold symmetry operator on three subunits in the asymmetric unit and held together by extensive ionic interactions. The roles of two invariant carboxylates, Asp Box motif Glu163 and Walker B motif Glu204, have been assigned to Mg(2+) binding and catalysis, respectively. The nucleotide ligands in each of the subunits in the asymmetric unit of the beta,gamma-methyleneadenosine-5'-triphosphate-bound PilT are not equally well ordered. Similarly, the three subunits in the asymmetric unit of both structures exhibit differing relative conformations of the two domains. The 12 degrees and 20 degrees domain rotations indicate motions that occur during the ATP-coupled mechanism of the disassembly of pili into membrane-localized pilin monomers. Integrating these observations, we propose a three-state "Ready, Active, Release" model for the action of PilT.

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Figures

FIGURE 1

FIGURE 1. The structure of AMP-PCP bound PilT

a) AMP-PCP (yellow carbons, blue nitrogens, red oxygens, orange phosphates) is bound at the interface of the two domains, surrounded by conserved motifs of the secretion ATPases (Walker A, α6, red; Walker B, β10, blue; Asp Box, β8, yellow; His Box, β11, orange; all shown on subunit B). AMP-PCP is distant from the AIRNLIRE helix required for retraction (cyan). b) The PilT hexamer, looking down the crystallographic 2-fold axis with the N-terminal domains oriented towards the reader (subunit A, green; B, blue; C, purple; the second subunit C is grey to match the subunit shown in panel a; ligands colored as in panel a).

FIGURE 2

FIGURE 2. Conservation of PilT residues among secretion ATPases

Among 27 secretion ATPases, as mapped onto PilT, the most conserved residues (reddish-brown) are in the active site cleft or in proximity to the active site cleft of the neighboring subunit (grey ribbons). The least conserved residues (deep teal) are located on the periphery of the subunits. The active site ligands are colored as in Figure 1.

FIGURE 3

FIGURE 3. The nucleotide binding site and the His Box of PilT

a) Wall-eyed stereo view of the active sites of liganded (blue subunit B, green subunit A, grey Mg2+, red water) and apo (light blue subunit B, light green subunit A) PilT, aligned using the conserved RecA fold of the CTD (residues 106–301). In addition to residues discussed in the text, Arg276 coordinates the ribose while Leu109 and Leu268 sandwich the adenine moiety of the AMP-PCP. The Fo−Fc omit map was calculated from the ligand bound structure without AMP-PCP or Mg2+ ion and is contoured at 3σ. b) Thr220 and His222 (green, subunit A) and Thr132 and His229 (blue, subunit B) form a 3D His Box in the crystal structure.

FIGURE 4

FIGURE 4. Ready, Active, Release conformations of the clamp arginines of the 3 PilT subunits

a) Arginines 82 and 97 of subunit A are in a Ready conformation to bind the nucleotide. (Colors as in Fig. 1b and 3). b) The clamp arginines of subunit B are in an Active conformation, coordinating the γ-phosphate of the nucleotide. c) The clamp arginines have Released their hold on the nucleotide. d) A cartoon schematic representing the orientation of the NTDs with respect to the superimposed RecA CTDs. (Green, subunit A; blue, subunit B; purple, subunit C.)

FIGURE 5

FIGURE 5. Force generation by large domain motions among hexameric ATPase proteins

ATP binding leads to large domain movements (red arrows) within the subunit (shown in light blue cartoon). Left panels show two separate hexamer views with each subunit individually colored. The zoom in is of a single subunit (light blue) illustrating the motions of the moving domain during ATP binding, with the RecA domains fixed. Secretion ATPases (a) PilT (Chain A apo and Chain C – AMP-PCP), (b) GspE (2OAP, 2OAQ) and (c) HP0525 (1NLY, 1NLZ) exhibit domain motions that are diagonal to the central ring axis, while (d) HslU (1G3I, 1DO2) and (e) F1-ATPase (1BMF) have domain motions which are parallel and perpendicular to the ring axis, respectively.

FIGURE 6

FIGURE 6. Schematic model for pilus retraction

(A) If PilT were to act on pilin directly, the NTD of PilT (blue) would contact the N-terminal tail of the bottom-most pilin subunit (red, modeled with a PilT-induced kink at proline 22) in a Type IV pilus filament (2HIL27) undergoing disassembly across the inner membrane and guided through the outer membrane by the PilQ secretin (brown). (B) More likely, inner membrane proteins are also involved in the pilus retraction pathway, and the force generated upon domain closure by PilT is transferred through an inner membrane protein to the pilin subunits, thereby providing the energy needed to disrupt hydrophobic and polar pilin:pilin interactions. The association of PilT with any particular inner membrane protein remains an unproven hypothesis. Shown are one pilus filament, one PilM, a heterodimer of PilN:PilO and one PilC (for simplicity; PilC is likely a dimer 45). Based on membrane topology predictions for the 406 residue P. aeruginosa strain PA01 inner membrane protein PilC, we have represented PilC with three full transmembrane helices and two stubby membrane-embedded reentrant helices. This prediction should be viewed with caution, as other publicly available topology analysis algorithms yield varying results.

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