Aquifex aeolicus PilT, Homologue of a Surface Motility Protein, Is a Thermostable Oligomeric NTPase

Bacterial surface motility works by retraction of surface-attached type IV pili. This retraction requires the PilT protein, a member of a large family of putative NTPases from type II and IV secretion systems. In this study, the PilT homologue from the thermophilic eubacterium Aquifex aeolicus was cloned, overexpressed, and purified. A. aeolicus PilT was shown to be a thermostable ATPase with a specific activity of 15.7 nmol of ATP hydrolyzed/min/mg of protein. This activity was abolished when a conserved lysine in the nucleotide-binding motif was altered. The substrate specificity was low; UTP, CTP, ATP, GTP, dATP, and dGTP served as substrates, UTP having the highest activity of these in vitro. Based on sedimentation equilibrium and size exclusion chromatography, PilT was identified as a ≈5- to 6-subunit oligomer. Potential implications of the NTPase activity of PilT in pilus retraction are discussed

Structure and function of a fungal adhesin that binds heparin and mimics thrombospondin-1 by blocking T cell activation and effector function.

Blastomyces adhesin-1 (BAD-1) is a 120-kD surface protein on B. dermatitidis yeast. We show here that BAD-1 contains 41 tandem repeats and that deleting even half of them impairs fungal pathogenicity. According to NMR, the repeats form tightly folded 17-amino acid loops constrained by a disulfide bond linking conserved cysteines. Each loop contains a highly conserved WxxWxxW motif found in thrombospondin-1 (TSP-1) type 1 heparin-binding repeats. BAD-1 binds heparin specifically and saturably, and is competitively inhibited by soluble heparin, but not related glycosaminoglycans. According to SPR analysis, the affinity of BAD-1 for heparin is 33 nM±14 nM. Putative heparin-binding motifs are found both at the N-terminus and within each tandem repeat loop. Like TSP-1, BAD-1 blocks activation of T cells in a manner requiring the heparan sulfate-modified surface molecule CD47, and impairs effector functions. The tandem repeats of BAD-1 thus confer pathogenicity, harbor motifs that bind heparin, and suppress T-cell activation via a CD47-dependent mechanism, mimicking mammalian TSP-1

Solution Structure of CCL21 and Identification of a Putative CCR7 Binding Site

CCL21 is a human chemokine that recruits normal immune cells and metastasizing tumor cells to lymph nodes through activation of the G protein-coupled receptor CCR7. The CCL21 structure solved by NMR contains a conserved chemokine domain followed by an extended, unstructured C-terminus that is not typical of most other chemokines. A sedimentation equilibrium study showed CCL21 to be monomeric. Chemical shift mapping indicates that the CCR7 N-terminus binds to the N-loop and third β-strand of CCL21’s chemokine domain. Details of CCL21-receptor recognition may enable structure-based drug discovery of novel antimetastatic agents

NMR determination of the structure of the tandem repeats.

<p><b>(A)</b>¹H-¹⁵N HSQC spectrum of TR4. Peaks arising from backbone amides of the representative repeat are labeled, while peaks from side chain N-H groups are indicated with smaller labels. Note that the four tryptophan side chains yield four distinctive peaks indicating distinct chemical environments. <b>(B)</b>¹H-¹⁵N HSQC spectrum of full length BAD-1 protein. <b>(C)</b>¹H-¹⁵N HSQC spectra of TR4 and BAD-1 overlaid to show that the chemical shifts for the amino acids of the TR4 repeats are similar to those of the full length BAD-1 repeats. <b>(D)</b> Structure of one tandem repeat loop and its tryptophan residues. We determined the structure of one repeat, focusing upon the loop created by the disulfide bond between the two universally conserved cysteines. The repeat forms two tightly folded turns followed by a short α-helix. All but one of the tryptophan residues are buried in the center of the tandem repeat fold. <b>(E)</b> Structure of one tandem repeat loop and its acidic residues. Negatively charged residues are uniformly oriented on the external surface of the loop. <b>(F)</b> Overlapping image of the top 20 structural predictions by CYANA. Variability is seen primarily in externalized side-chains (depicted using thin green lines). <b>(G)</b> Theoretical structure of the BAD-1 molecule. Model is based on energy minimization of the hinge regions and the fact that extensive steric hindrance between the tandem-repeat loops further limits flexibility. In this model, tandem repeats lie along a helical twist, with roughly 3.2 repeats describing one full turn. Individual tandem repeat structures are sequentially colored: pink, yellow, green, blue (and repeated).</p

Influence of competitors on BAD-1 binding to heparin.

<p><b>(A)</b> Effect of a WxxW motif heparin-binding peptide on binding of BAD-1 (eFluor605) to immobilized heparin. “None” denotes BAD-1 binding to heparin agarose with no competitor. The WxxW peptide, or a mutant control peptide, was incubated with heparin resin at 1 mg/ml before addition of fluorescent BAD-1. Binding was quantitated by fluorescence units detected in a Filtermax F5 plate reader. Results are the mean ± SEM two experiments. <b>(B)</b> Effect of TR4 reduction on binding to heparin. Samples were incubated with resin directly or first boiled for 3 min in buffer alone or buffer with 5 mM DTT. TR4 has four copies of the BAD-1 tandem repeat. ΔCterm has all 41 repeats, but no C-terminal EGF-like domain. Binding was quantified by A280 measurement. Reduced TR4 bound significantly better than untreated TR4 or TR4 boiled without DTT (<b>*</b>, p<0.05). Results are the mean ± SEM of two experiments. <b>(C)</b> Effect of reduced TR4 on binding of BAD-1 (eFluor605) to immobilized heparin. BAD-1 binding to heparin agarose was quantified with or without pretreatment of resin with reduced TR4 as in panel C. BAD-1 binding was quantified by fluorescence units as above. Mannan resin is a background control. Heparin resin pre-treated with reduced TR4 at 0.2 and 0.1 mg/ml bound BAD-1 significantly better than untreated heparin resin (*, p<0.05). Soluble heparin significantly blocked binding of BAD-1 to both of these pre-treated resins (**, p<0.05).</p