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 cond itions 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 fr om haemoglobin. HasA delivers haem to a specific outer-membrane receptor Ha sR and allows the bacteria to grow in the absence of any other source of ir on. 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 sec retion motif is cleaved during or after the secretion process by proteases secreted by the bacteria. In this work, we study by H-1 NMR the conformation of the C-terminal extrem ity of HasA in the whale protein and that of the isolated secretion signal peptide in a zwitterionic micelle complex that mimicks the membrane environ ment. 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 secret ion are highly flexible and unstructured. This flexibility may be a prerequ isite to the recognition of HasA by its ABC transporter. We determine the c leavage site of the C-terminal extremity of the protein and analyse the eff ect of the cleavage on the haem acquisition process.