Solution Structure of Pyoverdin GM - IIf

The three-dimensional structure in solution of ferri-pyoverdin GM-I1 isolated from the culture medium of Pseudomonasfluorescens was determined by application of N M R methods to the Ga3+ analogue. Distance geometry calculations were performed with FILMAN using interproton distances and coupling constants as constraints. Further conformational analysis was carried out by energy minimization with MM2 and AMBER. Back-calculation of the NOESY spectra shows that the resulting structures are in agreement with the experimental data. Iron is a trace element essential for the redox systems in nearly all organisms. Under aerobic conditions its most stable oxidation state is Fe(III), whose oxide hydrates are poorly soluble in water at physiological pH values (KL = at pH 7). In order to overcome iron deficiency, many bacteria and fungi have developed highly specific iron acquisition systems. Most of them are based on complexation of the available Fe(111) by strong chelators called siderophores. Usually microorganisms recognize only their own siderophore by outer membrane proteins. Pseudomonas spp. of the so-called fluorescent group, to which belongs inter alia the human pathogenic Pseudomonas aeruginosa, produce peptidic siderophores named pyoverdins when grown in an iron-deficient medium. Their common feature is the chromophore (lS)-5-amin0-2,3-dihydroxy-lHpyrimido[ 1,2-a]quinolinel-carboxylic acid. Its 5-amino group is linked to a dicarboxylic acid belonging to the citric acid cycle while the l-carboxyl group is bound in most cases to the N-terminus of a linear or cyclic peptide containing 6-12 amino acids (Budzikiewicz, 1993). Cross-feeding experiments have shown that usually a given Pseudomonas strain accepts only its own pyoverdin. Strain specificity for pyoverdin-mediated iron uptake was systematically investigated for P. aeruginosa, P . fluorescens, P . put ida, P. chlororaphis, and P. tolasii by cross-feeding, iron-uptake studies, and binding experiments (Hohnadel & Meyer, 1988; Cornelis et al., 1989). However, nothing is known about the nature of the amino acids and the steric requirements for the interaction of a pyoverdin with the cell surface proteins. We intend, therefore, to investigate the strain specificity on the basis of the three-dimensional molecular structure of the pyoverdin complexes in solution as derived from NMR' spectroscopic data. So far it is only known that Fe(II1) forms octahedral complexes and thus provides six coordination sites which can accommodate three bidentate ligands. The catecholic moiety of the chromophore is one of the complexing groups. The other two (a-hydroxycarboxylic or hydroxamic acids) are located in the peptide residue. + This work was supported by a Merrell/Dow-Universitkhuis Pasteur Fellowship of the Acadkmie des Sciences. * To whom correspondence should be addressed. Present address: Bayer AG Wuppertal, Geb. 302, Friedrich-Ebert-Strasse, D-42117 Wuppertal, Germany. t CNRS. i Institut fur Organische Chemie der Universitit K61n. Abstract published in Advance ACS Abstracts, February 15, 1994. 0006-2960/94/0433-2843$04.5Q~0 Recently, the primary structure of pyoverdins GM-I and GM-I1 (Figure 1) was elucidated (Mohn et a1.,1990). In this paper we describe the determination of the three-dimensional structure of theGa(II1) complex of pyoverdin GM-I1 by NMR spectroscopic studies, distance geometry calculation, and restrained molecular mechanics. EXPERIMENTAL, PROCEDURES CD Spectroscopy of Ferri-GM-ZZ. CD spectra were recorded on a Dichrograph Mark I11 (ISA) in buffered aqueous solution (0.1 N Tris-HC1, pH 7.2). Preparation of the Gq'ZZZ) Complex. Ferri-GM-II(40 mg) was dissolved in 3 mL of 0.1 N acetic acid, and, after adjusting the pH to 3.0 with diluted aqueous NH3,lOO mg of gallium(111) nitrate and 300 mg of ascorbic acid in 3 mL of 0.1 N acetic acid were added. After the pH of the solution was adjusted to 5.0, the Ga(II1) complex was adsorbed on XAD-4 resin, washed with water, and desorbed with acetonitrile/ water (1: 1, v/v). The extract was purified by chromatography on Bio-Gel P-2 with a 0.1 M pyridinium acetate buffer (pH 6.5); the yield was 32 mg. The purity of the Ga complex was demonstrated by RP-HPLC on Polygosil60-C18, eluted with 5% acetonitrile in 0.1 M ammonium acetate buffer (pH 6.2). In the PI-FAB mass spectrum the masses of the [M+H]+ ions of the free ligand and the Ga(II1) complex differ by 66 amu (+69Ga 3H) and 68 amu (+'lGa 3H). NMR Spectroscopy. NMR experiments were performed on a Bruker AMX 500 spectrometer. Data processing was achieved with an Aspect X32 computer using UXNMR software or with FELIX (Biosym) running on Indigo (Silicon Graphics). The measurements were performed with 10 mM solutions of Ga pyoverdin GM-I1 or of the free ligand in CD3COONa/CDsCOOH buffered HzO/D20 95/5 (v/v) in a pH range between 2.0 and 6.5 and in a temperature range between 278 and 313 K. The water resonance was suppressed using the jump-and-return method (Plateau & Gubron, 1982) or by presaturation during the relaxation delay. COSY, RE' Abbreviations: CD, circular dichroism; Chr, chromophore; d, distance (A); COSY, correlated spectroscopy; DSS, 3-(trimethylsiiyl)-l-propane sulfonic acid; FAB-MS, fast atom bombardment mass spectrometry (PI = positive ion); HOHAHA, homonuclear Hartmann-Hahn spect"py; (OH)Asp, threo-3-hydroxyaspartic acid; NMR, nuclear magnetic reaonance; NOE, nuclear Overhauser enhancement; NOESY, nuclear Overhauser enhancement spectroscopy; (OH)Om, N-hydroxyornithine; rmsd, root mean square deviation; RP-HPLC, r e v d p h a s e highperformance liquid chromatography; SD, standard deviation of atomic positions; SUC, succinic acid. 1994 American Chemical Society 2844 Biochemistry, Vol. 33, No. 10, 1994