title: Mutational analysis of hemoglobin binding and heme utilization by a bacterial hemoglobin receptor.
abstract: Iron is an essential nutrient for most living organisms. To acquire iron from their environment, Gram-negative bacteria use TonB-dependent transporters that bind host proteins at the bacterial surface and transport iron or heme to the periplasm via the Ton machinery. TonB-dependent transporters are barrel-shaped outer membrane proteins with 22 transmembrane domains, 11 surface-exposed loops, and a plug domain that occludes the pore. To identify key residues of TonB-dependent transporters involved in hemoglobin binding and heme transport and thereby locate putative protective epitopes, the hemoglobin receptor of Haemophilus ducreyi HgbA was used as a model of iron/heme acquisition from hemoglobin. Although all extracellular loops of HgbA are required by H. ducreyi to use hemoglobin as a source of iron/heme, we previously demonstrated that hemoglobin binding by HgbA only involves loops 5 and 7. Using deletion, substitution, and site-directed mutagenesis, we were able to differentiate hemoglobin binding and heme acquisition by HgbA. Deletion or substitution of the GYEAYNRQWWA region of loop 5 and alanine replacement of selected histidines affected hemoglobin binding by HgbA. Conversely, mutation of the phenylalanine in the loop 7 FRAP domain or substitution of the NRQWWA motif of loop 5 significantly abrogated utilization of heme from hemoglobin. Our findings show that hemoglobin binding and heme utilization by a bacterial hemoglobin receptor involve specific motifs of HgbA.
authors.affiliation: Departments of Medicine.
authors.forename: W G
authors.initials: WG
authors.lastname: Fusco
authors.forename: N R
authors.initials: NR
authors.lastname: Choudhary
authors.forename: S E
authors.initials: SE
authors.lastname: Council
authors.forename: E J
authors.initials: EJ
authors.lastname: Collins
authors.forename: I
authors.initials: I
authors.lastname: Leduc
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006191:Haemophilus ducreyi / Q000235:genetics / Q000378:metabolism*; D006418:Heme / Q000378:metabolism*; D006454:Hemoglobins / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid
chemical_list: D001425:Bacterial Outer Membrane Proteins; D006454:Hemoglobins; D006418:Heme