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.Additional Supporting Information may be found in the online version of this article.Significance statement: During infections, many bacterial pathogens use NEAr-iron Transporter (NEAT) domains to acquire iron, an essential nutrient that is required for microbial growth. Here we show that NMR is a powerful tool to assess the hemoglobin binding properties of these domains. Our results resolve prior discrepancies in the literature and help to define the functions of a wide range of NEAT domains based on their amino acid primary sequences.

The Shr receptor from Streptococcus pyogenes uses a cap and release mechanism to acquire heme–iron from human hemoglobin

“…Adding unlabeled tetrameric Hb that is saturated with heme (holo-Hb) to [U- 15 N] Shr H2 causes significant line broadening of its 1 H- 15 N heteronuclear single-quantum coherence (HSQC) spectrum, consistent with Shr H2 forming a high molecular weight complex with Hb (compare Fig. 2 A and B ) ( 19 , 29 ). Interestingly, the heme-capping interactions observed in the crystal structure are essential for receptor binding, as only minimal spectral changes are observed when Hb lacking heme (apo-Hb) is added to [U- 15 N] Shr H2 (compare Fig.…”

NEAr Transporter (NEAT) Domains: Unique Surface Displayed Heme Chaperones That Enable Gram-Positive Bacteria to Capture Heme-Iron From Hemoglobin

“…Hb contains α- and β-globin chains that each bind to heme. Structures of isolated IsdB and IsdH NEAT domains free and bound to tetrameric Hb have revealed that residues within their aromatic motifs undergo a disordered-to-ordered transition upon binding, forming a 3 10 helix that interacts with the A- and E-helices in the globin ( Pilpa et al, 2006 ; Krishna Kumar et al, 2011 ; Kumar et al, 2014 ; Macdonald et al, 2019 ). Heme-binding NEAT domains feature a similarly located binding surface to engage heme.…”

“…The results of in vitro binding experiments have led to the conclusion that some NEAT domains from B. anthracis (IsdX1N and IsdX2N5) and L. monocytogenes (Hbp1N) bind to Hb, even though they lack a (F/Y)YH(Y/F) motif ( Ekworomadu et al, 2012 ; Honsa et al, 2013 ; Malmirchegini et al, 2014 ). However, recent NMR studies have raised doubts about these findings, because the Hb used in these prior studies was obtained from commercial sources that are now known to contain breakdown products that can lead to erroneous conclusions about Hb binding and Hb-dependent microbial growth ( Pishchany et al, 2013 ; Macdonald et al, 2019 ). Thus, at present, it appears that only NEAT domains containing a (F/Y)YH(Y/F) motif function as Hb receptors.…”

Pirates of the haemoglobin

“…For Hb binding, ELISA experiments have been the main demonstrative tool. C. diphtheriae proteins HtaA and ChtB [34] [36], B. anthracis proteins IsdX1/X2 [57] and L. monocytogenes protein Hbp2 [63] are examples ELISA demonstrated Hb binding but follow up NMR experiments aimed at detecting haem binding directly failed to detect an Hb interaction for B. anthracis IsdX1/X2 [123]. One plausible explanation for this discrepancy is that in most ELISA experiments, the Hb used, usually commercially bought, contains a variety of breakdown products including multiple oligomeric and oxidation states and that immobilisation of In addition to this, there must be clarity when describing the ability of aforementioned Hb binding proteins to actually engage in the actively stripping of haem from Hb.…”