PilT is a hexameric ATPase required for bacterial type IV pilus retraction and surface motility. Crystal structures of ADP- and ATP-bound Aquifex aeolicus PilT at 2.8 and 3.2 A resolution show N-terminal PAS-like and C-terminal RecA-like ATPase domains followed by a set of short C-terminal helices. The hexamer is formed by extensive polar subunit interactions between the ATPase core of one monomer and the N-terminal domain of the next. An additional structure captures a nonsymmetric PilT hexamer in which approach of invariant arginines from two subunits to the bound nucleotide forms an enzymatically competent active site. A panel of pilT mutations highlights the importance of the arginines, the PAS-like domain, the polar subunit interface, and the C-terminal helices for retraction. We present a model for ATP binding leading to dramatic PilT domain motions, engagement of the arginine wire, and subunit communication in this hexameric motor. Our conclusions apply to the entire type II/IV secretion ATPase family.

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PilT is a biological motor required for the retraction of bacterial type IV pili. Nesseria gonorrhoeae PilT has been purified and its ultrastructure has been examined by freeze-etch electron microscopy, revealing a 115 A outer diameter, 15-35 A inner diameter ring. Aquifex aeolicus PilT crystals were obtained in a primitive hexagonal space group (unit-cell parameters a = b = 107.3, c = 68.5 A) and diffract to a minimum Bragg spacing of 2.8 A when PilT is co-crystallized with adenine nucleotides. Initial phases to 3.5 A resolution have been determined by multiwavelength anomalous dispersion and density modification. Resulting electron-density maps show a hexameric A. aeolicus PilT ring 105 A wide by 55 A high, with an inner cavity that varies in shape and width from 20 to 40 A over the height of the complex. Both PilT ultrastructures are very similar to type II and type IV secretion ATPases in overall shape, size and assembly.

Virulence factor regulator (Vfr) enhances Pseudomonas aeruginosa pathogenicity through its role as a global transcriptional regulator. The crystal structure of Vfr shows that it is a winged-helix DNA-binding protein like its homologue cyclic AMP receptor protein (CRP). In addition to an expected primary cyclic AMP-binding site, a second ligand-binding site is nestled between the N-terminal domain and the C-terminal helix-turn-helix domain. Unlike CRP, Vfr is a symmetric dimer in the absence of DNA. Removal of seven disordered N-terminal residues of Vfr prevents the growth of P. aeruginosa.Pseudomonas aeruginosa is a ubiquitous, Gram-negative opportunistic pathogen that is a leading source of morbidity and mortality for individuals with compromised immune systems or cystic fibrosis. P. aeruginosa switches from an environmental organism to a pathogen through the regulation and elaboration of multiple virulence factors. Virulence factor regulator (Vfr) is a central player in a transcriptional cascade that controls this transition.Vfr is a 24-kDa protein belonging to the winged-helix family of transcription regulators (40). Vfr positively regulates quorum sensing by promoting the transcription of lasR, upregulates transcription of the type III secretion system, represses flagellar gene transcription, and positively regulates twitching motility (1, 2, 9, 41). Vfr binds cyclic adenosine 3Ј5Ј monophosphate (cyclic AMP [cAMP]) in vitro with a dissociation constant of 1.6 M (39). Deletion of both cytoplasmic P. aeruginosa adenyl cyclases results in a transcription profile similar to a vfr deletion (41). Furthermore, like the strain carrying the vfr deletion, the double adenyl cyclase mutant has reduced virulence compared to the wild-type strain in a mouse pneumonia model (38). Thus, cAMP is a biologically relevant ligand for Vfr in vivo.The amino acid sequence of Vfr is 67% identical to that of cAMP receptor protein (CRP) from Escherichia coli (40), a long-standing model for the study of transcription regulation and a structurally well-characterized protein (22). When overexpressed, Vfr complements the ␤-galactosidase and tryptophanase-deficient phenotypes of a crp deletion in E. coli (40). Conversely, CRP expressed in a vfr deletion strain of P. aeruginosa promotes sub-wild-type levels of lasR transcription. However, CRP is unable to rescue the protease-or exotoxin A-deficient phenotypes of the vfr mutant (40). Recent work suggests that this discrepancy might be due at least in part to cAMPindependent regulation at the lasR promoter (15).Mounting evidence supports a model for CRP function in which a dimer of CRP binds a cAMP ligand in the N-terminal domain of each monomer to achieve the protein conformation needed for high-affinity specific DNA binding and DNA bending that precedes transcriptional activation. CRP demonstrates low affinity for specific DNA sequences, with a K d of ϳ10 Ϫ4 M, in the absence of cAMP (16). Its specificity and affinity for its promoter sites increase to 10 Ϫ7 to 10 Ϫ8 M in cAMP concentrations...

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