Novel Mechanism of Hemin Capture by Hbp2, the Hemoglobin-binding Hemophore from Listeria monocytogenes

G Reza Malmirchegini,Megan Sjodt,Sergey Shnitkind,Michael R Sawaya,Justin Rosinski,Salete M Newton,Phillip E Klebba,Robert T Clubb

doi:10.1074/jbc.m114.583013

G Reza Malmirchegini, Megan Sjodt + Show 6 more

Open Access

https://doi.org/10.1074/jbc.m114.583013

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Abstract

Iron is an essential nutrient that is required for the growth of the bacterial pathogen Listeria monocytogenes. In cell cultures, this microbe secretes hemin/hemoglobin-binding protein 2 (Hbp2; Lmo2185) protein, which has been proposed to function as a hemophore that scavenges heme from the environment. Based on its primary sequence, Hbp2 contains three NEAr transporter (NEAT) domains of unknown function. Here we show that each of these domains mediates high affinity binding to ferric heme (hemin) and that its N- and C-terminal domains interact with hemoglobin (Hb). The results of hemin transfer experiments are consistent with Hbp2 functioning as an Hb-binding hemophore that delivers hemin to other Hbp2 proteins that are attached to the cell wall. Surprisingly, our work reveals that the central NEAT domain in Hbp2 binds hemin even though its primary sequence lacks a highly conserved YXXXY motif that is used by all other previously characterized NEAT domains to coordinate iron in the hemin molecule. To elucidate the mechanism of hemin binding by Hbp2, we determined crystal structures of its central NEAT domain (Hbp2(N2); residues 183-303) in its free and hemin-bound states. The structures reveal an unprecedented mechanism of hemin binding in which Hbp2(N2) undergoes a major conformational rearrangement that facilitates metal coordination by a non-canonical tyrosine residue. These studies highlight previously unrecognized plasticity in the hemin binding mechanism of NEAT domains and provide insight into how L. monocytogenes captures heme iron.

Highlights

Listeria monocytogenes scavenges iron from heme and human hemoglobin using the Hbp1 and hemoglobin-binding protein 2 (Hbp2) proteins
Hbp1 and Hbp2 Bind Hemin with Nanomolar Affinity— Inspection of the primary sequences of Hbp1 and Hbp2 reveals that they contain NEAr transporter (NEAT) domains (Fig. 1B)
Some NEAT domains interact with only host hemoproteins or only hemin, or they bind to hemin and additional ligands including plasma and extracellular matrix proteins [39]

Summary

Background

Listeria monocytogenes scavenges iron from heme and human hemoglobin using the Hbp and Hbp proteins. Iron is an essential nutrient that is required for the growth of the bacterial pathogen Listeria monocytogenes In cell cultures, this microbe secretes hemin/hemoglobin-binding protein 2 (Hbp; Lmo2185) protein, which has been proposed to function as a hemophore that scavenges heme from the environment. Hbp is expressed under iron-deficient conditions from the svpA-srtB operon, which encodes a sortase (SrtB), components of an ABC transporter of unknown function (Lmo2182, Lmo2183, and Lmo2184), and three uncharacterized proteins (Lmo2178, Lmo2179, and Lmo2180) (see Fig. 1A) Based on their primary sequences, they are predicted to be covalently attached to the cell wall by the SrtB sortase because they contain appropriate C-terminal sorting signals (NPKSS and NAKTN, respectively) [7]. The results of these studies provide new insight into the mechanism through which L. monocytogenes and other Gram-positive pathogens capture heme iron

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION