Crystal Structure of the Siderophore Binding Protein BauB Bound to an Unusual 2:1 Complex Between Acinetobactin and Ferric Iron

Abstract

The critical role that iron plays in many biochemical processes has led to an elaborate battle between bacterial pathogens and their hosts to acquire and withhold this critical nutrient. Exploitation of iron nutritional immunity is being increasingly appreciated as a potential antivirulence therapeutic strategy, especially against problematic multidrug resistant Gram-negative pathogens such as Acinetobacter baumannii. To facilitate iron uptake and promote growth, A. baumannii produces a nonribosomally synthesized peptide siderophore called acinetobactin. Acinetobactin is unusual in that it is first biosynthesized in an oxazoline form called preacinetobactin that spontaneously isomerizes to the final isoxazolidinone acinetobactin. Interestingly, both isomers can bind iron and both support growth of A. baumannii. To address how the two isomers chelate their ferric cargo and how the complexes are used by A. baumannii, structural studies were carried out with the ferric acinetobactin complex and its periplasmic siderophore binding protein BauB. Herein, we present the crystal structure of BauB bound to a bis-tridentate (Fe³⁺L₂) siderophore complex. Additionally, we present binding studies that show multiple variants of acinetobactin bind BauB with no apparent change in affinity. These results are consistent with the structural model that depicts few direct polar interactions between BauB and the acinetobactin backbone. This structural and functional characterization of acinetobactin and its requisite binding protein BauB provides insight that could be exploited to target this critical iron acquisition system and provide a novel approach to treat infections caused by this important multidrug resistant pathogen.

Figure 1.

Chemical structures of acinetobactin (1) and pre-acinetobactin (2) from A. baumannii.

Figure 2.

Ribbon representation of BauB bound to the Acb2•Fe complex. (A) Highlight of the two domains and the α helix that joins them (N-domain, yellow; C-domain pink; α helix green) (B) Orthogonal view, rotated approximately 90° around the horizontal axis. The single gap in the protein between residues 235 and 239 is indicated with the dashed line. The Acb molecules are shown in stick representation with magenta carbon, red oxygen, and blue nitrogen atoms.

Figure 3.

Siderophore binding pocket in BauB. (A) The Acb₂Fe ligand is shown with buried (green) and exposed (yellow) acinetobactin molecules with a surface representation of BauB. (B) Stereorepresentation of the residues that form the binding pocket.

Figure 4.

Structures of (Acb)₂Fe from (A) experimental fitting into the electron density of the BauB substrate binding pocket and (B) DFT calculated lowest energy structure for the monoanionic [(Acb)₂Fe]⁻ metal complex (PBE0/Def2-SVP; PBE0/Def2-TZVP; PBE1PBE = PBE0 in Gaussian). (C) Overlay of (Acb)₂Fe structures from experimental (grey) and DFT (salmon) modeling.