Modeling of the structure of the Haemophilus influenzae heme‐binding protein suggests a mode of heme interaction

Abstract

The structure and function of the periplasmic heme-binding protein HbpA ofHaemophilus influenzae were investigated. This protein is involved in the import of heme into the bacteria through the inner membrane, and thus is a key element of the organism's ability to survive in blood. A high degree of sequence similarity between HbpA and the dipeptide-binding protein of Escherichia coli is suggested to be the result of a functional relationship. An HbpA model built using the dipeptide-binding protein suggests a mode of heme binding that is distinct from those known in proteins of the human host. These results provide a starting point for rational drug design.Keywords: heme; modeling; periplasmic binding protein; protein structure; transport Haemophilus influenzae is an important cause of meningitis, as well as a major agent of lower respiratory tract and other diseases. The organism has an absolute requirement for exogenously supplied heme, the import of which utilizes a periplasmic lipoprotein, HbpA (Hanson & Hansen, 1991). This heme(haemin)-binding protein is the primary receptor for a heme transport system in the cytoplasmic membrane and, as a key element of the organism's ability to survive in blood, is a desirable target for drugs that would inhibit heme transport. As part of a transport system, it could also be used as a means of selective delivery of toxic compounds into the bacterial cell. The sequence is strongly conserved among various Haemophilus strains (Hanson et al., 1992). A similar protein has also been found in Yersinia enterocolitica (Stojiljkovic & Hantke, 1994) and proposed to exist in Neisseria meningitidis (Stojiljkovic et al., 1995), other hemerequiring pathogens. These facts suggest that drugs aimed at HbpA might be generally useful for a variety of disease-causing bacteria. The success of such a strategy depends largely on the ability to design drugs that do not interfere with the normal roles of heme in the host, and thus also on a knowledge of the modes of heme binding in the protein structures involved. The X-ray Reprint requests to: Sherry L. Mowbray, Department of Molecular Biology, BioMedical Center, Box 590, Swedish University of Agricultural Sciences, 75124 Uppsala, Sweden; e-mail: mowbray@xray.bmc.uu.se.Abbreviations: HbpA, the heme-binding protein of Haemophilus influenzae; DBP, the dipeptide-binding protein of Escherichia coli.

Heme Utilization by Nontypeable Haemophilus influenzae Is Essential and Dependent on Sap Transporter Function

“…Comparison of the deduced SapA amino acid sequence with those of other periplasmic binding proteins revealed a cluster of proteins that were 100 residues larger than other binding proteins of this classification and predicted to contain three structural domains (7,8,19). SapA is predicted to be structurally similar to the heme-binding lipoprotein (HbpA) from Haemophilus and the E. coli dipeptide-binding protein (DppA) in both domain organization and size.…”

“…DppA shares 55% identity with HbpA, which suggests a conserved functional relationship. The known crystal structure of DppA reveals a binding site for dipeptide ligands that also shares amino acid residues predicted to mediate binding to heme (7,8,19). An HbpA model built upon the DppA structure suggests a mode of heme binding by the formation of a heme-binding pocket (7,8).…”

“…The known crystal structure of DppA reveals a binding site for dipeptide ligands that also shares amino acid residues predicted to mediate binding to heme (7,8,19). An HbpA model built upon the DppA structure suggests a mode of heme binding by the formation of a heme-binding pocket (7,8). Since SapA shares 31% identity (50% similarity) with HbpA and 29% identity (50% similarity) with DppA, we examined whether amino acid residues predicted to mediate the formation of a heme-binding pocket in both DppA and HbpA were also conserved in SapA.…”

The housekeeping dipeptide permease is the Escherichia coli heme transporter and functions with two optional peptide binding proteins

“…An H. influenzae hbpA insertion mutant is strongly affected in heme acquisition, indicating that the HbpA protein is important but not essential for heme acquistion (30). HbpA has been modeled with heme in the putative binding pocket (31). Heme binding to DppA was not detected in one previous study (32), although, under some purification conditions, DppA samples contain bound peptides competing with heme binding (20).…”

Glutathione import in Haemophilus influenzae Rd is primed by the periplasmic heme-binding protein HbpA

“…It has long been recognized that HbpA shares close homology with bacterial dpp-like proteins (typically >50% sequence identity), which led to proposals about how it could serve as a binding platform for heme (36). Although HbpA has been implicated in heme acquisition, its affinity for heme was never quantified in order to place the role of HbpA in a physiological context.…”