Figure 5. Hemin binding analysis of purified IlsA.

(A) Chemoluminescent detection showing binding of GST-IlsA to hemin (He). Purified GST-IlsA or GST incubated either with hemin (+) or without (−) as indicated on the top of the lane, were migrated on non-denaturing PAGE and transferred to a nitrocellulose membrane. Phosphate buffer alone was also incubated with hemin (+) as a control. After reaction with an ECL reagent system, proteins with hemin-binding properties are detected on chemoluminescent-sensitive film. Upper arrow corresponds to GST-IlsA binding to hemin and lower arrows to hemin alone. (B) Multiple sequence alignments of NEAT domains of IlsA from B. cereus and Isd proteins from S. aureus. Positions shaded in yellow are identical in 100% of the aligned sequences, blue and green are identical or similar, respectively, in at least 50% of the aligned sequences. The first four letters of the sequence represent the protein identity. Asterisks represent the amino acids required for heme binding. Numbers 1 to 3 represent the NEAT domain numbered from the N-terminus out of the total number of NEAT domains in that protein. The accession numbers of Isd proteins from S. aureus are as follows: IsdH (Q99TD3), IsdB (Q7A656), IsdA (Q7A655), IsdC (Q7A654). The accession number of IlsA from B. cereus ATCC 14579 is NP_831113. NEAT domains were identified in the Pfam database. Alignments were generated in vector NTI advance 10 (Invitrogen), using a BLOSUM matrix with gap opening and extension penalties of 15 and 2, respectively.