Mapping the Interaction between the Hemophore HasA and Its Outer Membrane Receptor HasR Using CRINEPT−TROSY NMR Spectroscopy

• Published in: Journal of the American Chemical Society Vol. 131; no. 5; pp. 1736 - 1744
• Main Authors: Caillet-Saguy, Célia, Piccioli, Mario, Turano, Paola, Izadi-Pruneyre, Nadia, Delepierre, Muriel, Bertini, Ivano, Lecroisey, Anne
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 11.02.2009
• Subjects: Amino Acid Sequence; Apoproteins - chemistry; Apoproteins - metabolism; Bacterial Proteins - chemistry; Bacterial Proteins - metabolism; Biochemistry, Molecular Biology; Carrier Proteins - chemistry; Carrier Proteins - metabolism; Heme - chemistry; Heme - metabolism; Life Sciences; Membrane Proteins - chemistry; Membrane Proteins - metabolism; Micelles; Models, Molecular; Molecular Sequence Data; Nuclear Magnetic Resonance, Biomolecular - methods; Phosphorylcholine - analogs & derivatives; Phosphorylcholine - chemistry; Protein Conformation; Receptors, Cell Surface - chemistry; Receptors, Cell Surface - metabolism; Structural Biology
• DOI: 10.1021/ja804783x
• ISSN: 0002-7863

The first step of heme acquisition by Gram-negative pathogenic bacteria through the so-called heme acquisition system, Has, requires delivery of the heme from the extracellular hemophore protein HasA to a specific outer membrane receptor, HasR. CRINEPT−TROSY NMR experiments in DPC micelles were here used to obtain information on the intermediate HasA−HasR complex in solution. A stable protein−protein adduct is detected both in the presence and in the absence of heme. Structural information on the complexed form of HasA is obtained from chemical shift mapping and statistical analysis of the spectral fingerprint of the protein NMR spectra obtained under different conditions. This approach shows the following: (i) only three different conformations are possible for HasA in solution: one for the isolated apoprotein, one for the isolated holoprotein, and one for the complexed protein, that is independent of the presence of the heme; (ii) the structure of the hemophore in the complex resembles the open conformation of the apoprotein; (iii) the surface contact area between HasA and HasR is independent of the presence of the heme, involving loop L1, loop L2, and the β2−β6 strands; (iv) upon complex formation the heme group is transferred from holoHasA to HasR.