Structure and function of Pseudomonas aeruginosa protein PA1324 (21-170)

Abstract

Pseudomonas aeruginosa is the prototypical biofilm-forming gram-negative opportunistic human pathogen. P. aeruginosa is causatively associated with nosocomial infections and with cystic fibrosis. Antibiotic resistance in some strains adds to the inherent difficulties that result from biofilm formation when treating P. aeruginosa infections. Transcriptional profiling studies suggest widespread changes in the proteome during quorum sensing and biofilm development. Many of the proteins found to be upregulated during these processes are poorly characterized from a functional standpoint. Here, we report the solution NMR structure of PA1324, a protein of unknown function identified in these studies, and provide a putative biological functional assignment based on the observed prealbumin-like fold and FAST-NMR ligand screening studies. PA1324 is postulated to be involved in the binding and transport of sugars or polysaccharides associated with the peptidoglycan matrix during biofilm formation.

Figure 1

Multiple sequence alignment of PA1324 and six homologues. Gene names and NCBI RefSeq numbers are indicated by three letter abbreviations as follows: Pae, Pseudomonas aeruginosa PA1324, NP_250015; Csa, Chromohalobacter salexigens Csal_0658, YP_572718; Sde, Saccharophagus degradans Sde_0431, YP_525907; Dvu, Desulfovibrio vulgaris Dvul_1060, YP_966507; She, Shewanella sp. W3-18-1 Sputw3181_2461, YP_963839; Sul, Sulfurovum sp. NBC37-1 SUN_1567, YP_001358874.

Figure 2

Structure of PA1324 21-170. Left: ribbon cartoon structure with rainbow coloring. The N- (blue) and C- (red) termini, the prominent loop (residues 71–107), and β-strands of the barrel (numbered 1–7) are indicated. Right: stereo view of backbone atoms for all 20 superimposed structures comprising the ensemble.

Figure 3

A 1D ¹H NMR line-broadening experiment showing an expanded reference mixture spectrum (A) and the mixture with PA1324 (B). The highlighted region indicates the observed line broadening and binding of suramin to PA1324.

Figure 4

A subset of the 20 compounds found to bind to PA1324 by 1D ¹H NMR line-broadening experiments was Aryl or benzyl sulfonyl, and sulfonate moieties are present in 7 of the 20 compounds.

Figure 5

Suramin binding to PA1324. (A) An expanded region of an overlay of the 2D ¹H-¹⁵N HSQC spectrum of free PA1324 (black) and PA1324 in the presence of suramin (red). (B) A GRASP electrostatic surface of PA1324 demonstrating the positive electrostatic potential of the suramin binding site. Blue and red indicate positively charged and negatively charged surfaces, respectively. (C) A docked model of suramin in the PA1324 binding site identified by FAST-NMR. Residues that incurred a CSP upon binding suramin are colored blue. The distal CSPs could be long-range effects of the binding interaction or potential suramin conformational exchange.

Figure 6

The CPASS comparison of PA1324 with suramin (left) to 1H7L, a spore coat polysaccharide biosynthesis protein bound to thymidine-5′-phosphate (right). The sequence alignment below the figures illustrates the similarities in the ligand binding sites.