Identification of amino acid residues involved in heme binding and hemoprotein utilization in the Porphyromonas gingivalis heme receptor HmuR.
We have previously identified and characterized a heme/hemoglobin receptor, HmuR, in Porphyromonas gingivalis. To analyze the conserved amino acid residues of HmuR that may be involved in hemin/hemoprotein binding and utilization, we constructed a series of P. gingivalis A7436 hmuR mutants with amino acid replacements and characterized the ability of these mutants to utilize hemin and hemoproteins. Site-directed mutagenesis was employed to introduce mutations H95A, H434A, H95A-H434A, YRAP420-423YAAA, and NPDL442-445NAAA into HmuR in both P. gingivalis and Escherichia coli. Point mutations at H95 and H434 and in the NPDL motif of HmuR resulted in decreased binding to hemin, hemoglobin, and human serum albumin-hemin complex. Notably, mutations of these conserved sites and motifs led to reduced growth of P. gingivalis when human serum was used as the heme source. Analysis using a three-dimensional homology model of HmuR indicated that H95, H434, and the NPDL motif are present on apical or extracellular loops of HmuR, while the YRAP motif is present on the barrel wall. Taken together, these results support a role for H95, H434, and the NPDL motif of the P. gingivalis HmuR protein in heme binding and utilization of serum hemoproteins and the HmuR YRAP motif in serum hemoprotein utilization.
Liu
Xinyan
X
Department of Medicine, Section of Infectious Diseases, Boston University School of Medicine, 650 Albany St., Boston, MA 02118, USA.
Olczak
Teresa
T
Guo
Hwai-Chen
HC
Dixon
Dabney W
DW
Genco
Caroline Attardo
CA
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006420:Hemeproteins / Q000378:metabolism*; D006427:Hemin / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D016966:Porphyromonas gingivalis / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D006420:Hemeproteins; D011956:Receptors, Cell Surface; C028027:heme receptor; D006427:Hemin