NMR experiments redefine the hemoglobin binding properties of bacterial NEAr-iron Transporter domains

Abstract

Iron is a versatile metal cofactor that is used in a wide range of essential cellular processes. During infections, many bacterial pathogens acquire iron from human hemoglobin (Hb), which contains the majority of the body's total iron content in the form of heme (iron protoporphyrin IX). Clinically important Gram-positive bacterial pathogens scavenge heme using an array of secreted and cell-wall-associated receptors that contain NEAr-iron Transporter (NEAT) domains. Experimentally defining the Hb binding properties of NEAT domains has been challenging, limiting our understanding of their function in heme uptake. Here we show that solution-state NMR spectroscopy is a powerful tool to define the Hb binding properties of NEAT domains. The utility of this method is demonstrated using the NEAT domains from Bacillus anthracis and Listeria monocytogenes. Our results are compatible with the existence of at least two types of NEAT domains that are capable of interacting with either Hb or heme. These binding properties can be predicted from their primary sequences, with Hb- and heme-binding NEAT domains being distinguished by the presence of (F/Y)YH(Y/F) and S/YXXXY motifs, respectively. The results of this work should enable the functions of a wide range of NEAT domain containing proteins in pathogenic bacteria to be reliably predicted.

Keywords: NEAT domain; NMR spectroscopy; bacteria; heme; hemoglobin; pathogen.

Figure 1

Sequence alignment identifies key residues that define NEAT domain function. (a) Representative structure of a Hb binding NEAT domain with residues from the (F/Y)YH(Y/F) aromatic motif shown in green (the IsdH^N2 NEAT domain, PDB: 4FC3). (b) Representative structure of a heme‐binding NEAT domain with residues from the S/YXXXY motif shown in red (the IsdA NEAT domain, PDB: 2ITF). (c) Sequence alignment showing the aromatic and X/YXXXY motifs for select NEAT domains. Conserved residues implicated in Hb and heme binding are highlighted in green and red, respectively. Columns on the right provide the PDB accession codes for structures of NEAT domains determined in complex with either heme or Hb. Domains that have been proposed to have “dual” Hb/heme‐binding functions are highlighted in blue and are the subject of this investigation.

Figure 2

Characterization of purified Hb and NMR control experiments. Hb was purified from human blood and its integrity verified by (a) SDS‐PAGE and (b) SEC‐MALS. In the SDS‐PAGE, the α and β globin chains of Hb are monomeric and appear as a single band because they have similar molecular weights. Lanes: (left) molecular weight markers, (middle) 10 μg of purified Hb, (right) 40 μg of purified Hb. As expected, in the SEC‐MALS the measured molecular weight indicates that Hb is a tetramer with a mass that is slightly less than its theoretical value.47 (c) Control Hb titration experiments using the established Hb binding [¹⁵N]IsdH^N2 domain.18 Panels show the HSQC spectrum before and after adding one molar equivalent of Hb. Extensive signal broadening indicates that the domain binds to Hb. (d) Control heme titration experiments using the established heme‐binding IsdC^N NEAT domain. A half molar equivalent of heme was added.15 Binding is indicated by the appearance of a separate set of cross peaks (inset) when sub‐stoichiometric amounts of heme are added.

Figure 3

NMR spectra of ^LmHbp1^N in the presence and absence of Hb and hemin. (a) 2‐D ¹⁵N‐¹H HSQC spectra overlay of ^LmHbp1^N in its apo‐, heme added, and Hb added forms. Three regions of interest are highlighted and expanded in the panels shown beneath. Expanded view of each region is shown for: (b) the apo‐form of ^LmHbp1^N, (c) ^LmHbp1^N in the presence of eightfold molar excess hemin, (d) ^LmHbp1^N in the presence of fourfold molar excess Hb, and (e) overlay of the spectrum of apo‐^LmHbp1^N and spectra obtained in the presence of excess heme and Hb.

Figure 4

Putative dual Hb/Heme‐binding NEAT domains do not interact with Hb with strong affinity. (a) ¹⁵N‐¹H HSQC spectrum of the B. anthracis [¹⁵N]IsdX2^N5 NEAT domain before (left) and after adding heme (top right) or Hb (bottom right). No significant resonance broadening is observed when Hb is added at a 20‐fold molar excess, indicating that this NEAT domain does not bind to Hb with strong affinity. However, IsdX2^N5 does bind to heme based on the appearance of a second set of NMR resonances when this ligand is added. (b) Similar experimental data for the B. anthracis IsdX1^N NEAT domain. These putative dual binding domains do not interact with Hb with strong affinity, but they do bind to heme.

Figure 5

IsdX1^N binds commercial Hb, but not to freshly purified Hb. The results of an ELISA experiment showing His₆‐SUMO‐IsdX1^N binding to wells coated with commercial‐sourced Hb (sigma) (red trace). Little or no binding occurs to wells coated with freshly purified Hb (yellow trace), heme alone (purple trace) or buffer (blue trace). Error bars represent SD from three separate experiments.