Heme binding mechanism of structurally similar iron-regulated surface determinant near transporter domains of Staphylococcus aureus exhibiting different affinities for heme.
Near transporter (NEAT) domains of the iron-regulated surface determinant (Isd) proteins are essential for the import of nutritional heme from host animals to Gram-positive pathogens such as Staphylococcus aureus. The order of transfer of heme between NEAT domains occurs from IsdH to IsdA to IsdC, without any energy input despite the similarity of their three-dimensional structures. We measured the free energy of binding of heme and various metalloporphyrins to each NEAT domain and found that the affinity of heme and non-iron porphyrins for NEAT domains increased gradually in the same order as that for heme transfer. To gain insight into the atomistic mechanism for the differential affinities, we performed in silico molecular dynamics simulation and in vitro site-directed mutagenesis. The simulations revealed that the negatively charged residues that are abundant in the loop between strand β1b and the 310 helix of IsdH-NEAT3 destabilize the interaction with the propionate group of heme. The higher affinity of IsdC was in part attributed to the formation of a salt bridge between its unique residue, Glu88, and the conserved Arg100 upon binding to heme. In addition, we found that Phe130 of IsdC makes the β7-β8 hairpin less flexible in the ligand-free form, which serves to reduce the magnitude of the entropy loss on binding to heme. We confirmed that substitution of these key residues of IsdC decreased its affinity for heme. Furthermore, IsdC mutants, whose affinities for heme were lower than those of IsdA, transferred heme back to IsdA. Thus, NEAT domains have evolved the characteristic residues on the common structural scaffold such that they exhibit different affinities for heme, thus promoting the efficient transfer of heme.
Department of Biotechnology and ‡Agricultural Bioinformatics Research Unit, Graduate School of Agricultural and Life Sciences, The University of Tokyo , 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
Yoshitaka
Y
Moriwaki
Tohru
T
Terada
Jose M M
JM
Caaveiro
Yousuke
Y
Takaoka
Itaru
I
Hamachi
Kouhei
K
Tsumoto
Kentaro
K
Shimizu
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000942:Antigens, Bacterial / Q000737:chemistry* / Q000235:genetics; D001059:Apoproteins / Q000737:chemistry / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics; D017124:Conserved Sequence; D019277:Entropy; D006418:Heme / Q000737:chemistry*; D006860:Hydrogen Bonding; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D025341:Principal Component Analysis; D011485:Protein Binding; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D011956:Receptors, Cell Surface / Q000737:chemistry* / Q000235:genetics; D013211:Staphylococcus aureus
D000942:Antigens, Bacterial; D001059:Apoproteins; D001426:Bacterial Proteins; D002352:Carrier Proteins; C512791:IsdA protein, Staphylococcus aureus; C488727:IsdC protein, Staphylococcus aureus; C512792:IsdH protein, Staphylococcus aureus; D011956:Receptors, Cell Surface; D006418:Heme