NMR studies of the C-terminal secretion signal of the haem-binding protein, HasA.
HasA is a haem-binding protein which is secreted under iron-deficiency conditions by the gram-negative bacterium Serratia marcescens. It is a monomer of 19 kDa (187 residues) able to bind free haem as well as to capture it from haemoglobin. HasA delivers haem to a specific outer-membrane receptor HasR and allows the bacteria to grow in the absence of any other source of iron. It is secreted by a signal peptide-independent pathway which involves a C-terminal secretion signal and an ABC (ATP-binding cassette) transporter. The C-terminal region of the secretion signal containing the essential secretion motif is cleaved during or after the secretion process by proteases secreted by the bacteria. In this work, we study by 1H NMR the conformation of the C-terminal extremity of HasA in the whole protein and that of the isolated secretion signal peptide in a zwitterionic micelle complex that mimicks the membrane environment. We identify a helical region followed by a random-coil C-terminus in the peptide-micelle complex and we show that in both the whole protein and the complex, the last 15 residues containing the motif essential for secretion are highly flexible and unstructured. This flexibility may be a prerequisite to the recognition of HasA by its ABC transporter. We determine the cleavage site of the C-terminal extremity of the protein and analyse the effect of the cleavage on the haem acquisition process.
Izadi-Pruneyre
N
N
Laboratoire de Résonance Magnétique Nucléaire, CNRS URA 1129, Institut Pasteur, Paris, France.nizadi@pasteur.fr
Wolff
N
N
Redeker
V
V
Wandersman
C
C
Delepierre
M
M
Lecroisey
A
A
D018528:ATP-Binding Cassette Transporters / Q000737:chemistry; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D002352:Carrier Proteins; D006418:Heme / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism; D008666:Metalloendopeptidases / Q000378:metabolism; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D010767:Phosphorylcholine / Q000031:analogs & derivatives / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D021382:Protein Sorting Signals / Q000737:chemistry; D017433:Protein Structure, Secondary; D016415:Sequence Alignment; D012706:Serratia marcescens / Q000737:chemistry*
D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D002352:Carrier Proteins; C093956:HasA protein, Serratia marcescens; D008565:Membrane Proteins; D010446:Peptide Fragments; D021382:Protein Sorting Signals; D010767:Phosphorylcholine; D006418:Heme; C028810:dodecylphosphocholine; D008666:Metalloendopeptidases