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

Streptococcus pyogenes (group A Streptococcus) is a clinically important microbial pathogen that requires iron in order to proliferate. During infections, S. pyogenes uses the surface displayed Shr receptor to capture human hemoglobin (Hb) and acquires its iron-laden heme molecules. Through a poorly understood mechanism, Shr engages Hb via two structurally unique N-terminal Hb-interacting domains (HID1 and HID2) which facilitate heme transfer to proximal NEAr Transporter (NEAT) domains. Based on the results of X-ray crystallography, small angle X-ray scattering, NMR spectroscopy, native mass spectrometry, and heme transfer experiments, we propose that Shr utilizes a “cap and release” mechanism to gather heme from Hb. In the mechanism, Shr uses the HID1 and HID2 modules to preferentially recognize only heme-loaded forms of Hb by contacting the edges of its protoporphyrin rings. Heme transfer is enabled by significant receptor dynamics within the Shr–Hb complex which function to transiently uncap HID1 from the heme bound to Hb’s β subunit, enabling the gated release of its relatively weakly bound heme molecule and subsequent capture by Shr’s NEAT domains. These dynamics may maximize the efficiency of heme scavenging by S. pyogenes, enabling it to preferentially recognize and remove heme from only heme-loaded forms of Hb that contain iron.

To successfully mount infections bacterial pathogens must overcome host nutritional immunity mechanisms that limit access to iron, an essential metal nutrient required for microbial survival because it functions as a cofactor in enzymes that mediate cellular metabolism. Human hemoglobin (Hb) contains ~75 to 80% of the body’s total iron in the form of heme (iron–protoporphyrin IX) and is thus a prime nutrient source for invading microbes (1–9). Bacteria gain access to Hb’s iron-laden heme molecules when erythrocytes are ruptured by bacterial cytotoxins or when they spontaneously lyse. In gram-positive monoderm bacteria, extracellular Hb is captured by surface-displayed microbial receptors. Hb’s heme molecules are then released and transferred via microbial heme-binding chaperones across the expanse of the peptidoglycan to the membrane, where they are imported into the cell and degraded to release iron. The acquisition mechanisms that many pathogens use to bind to Hb and remove its tightly bound heme molecules are not well understood. Streptococcus pyogenes (group A Streptococcus) colonizes the skin and mucosal surfaces in humans and is estimated to cause more than 500,000 deaths annually (10–12). It causes a range of illnesses, ranging from acute pharyngitis to life-threatening diseases such as scarlet fever, bacteremia, pneumonia, necrotizing fasciitis, myonecrosis, and streptococcal toxic shock syndrome (13, 14). S. pyogenes employs the streptococcal hemoprotein receptor (Shr) to capture Hb and acquire its heme molecules, and it is an important virulence factor that when genetically deleted reduces the ability of the pathogen to grow in human blood and to cause infections in murine and zebrafish models (15–17). Strategies that interfere with the ability of S. pyogenes and other pathogenic bacteria to harvest heme from Hb could be useful in treating infections, as they would effectively starve pathogens of iron.The S. pyogenes Shr protein is a structurally unique multidomain Hb receptor that is also found in other streptococci and clostridia species (e.g., Clostridium novyi, Streptococcus iniae, Streptococcus equi, and Streptococcus dysgalactiae) (Fig. 1A). Its N-terminal region (NTR, residues 26 to 364) binds to Hb using two Hb interacting domains (HIDs), called HID1 and HID2 (formally known as DUF1533 domains) (18, 19). The HIDs are structurally novel binding modules and are joined via a structured linker domain (L) to a C-terminal region (CTR, residues 365 to 1,275) which contains two heme-binding NEAr iron Transporter domains (NEAT domains N1 and N2) that are separated by a series of leucine-rich repeats (LRR). The NTR and N1 domain within Shr (called NTR-N1) preferentially bind to holo-Hb and remove its heme (18). In vitro, heme bound by the N1 domain is then readily transferred to either the C-terminal N2 domain, or to Shp, a cell wall-associated protein that relays heme to the membrane-associated HtsABC/SiaABC transporter that pumps heme into the cytoplasm (20–22). The N2 domain in Shr may act as a storage unit, since it binds to heme with much higher affinity than N1 and does not directly transfer heme to Shp (23). Shr also interacts via its N2 domain with the human extracellular matrix (ECM) proteins fibronectin and laminin (15, 16, 18), and its exposure on the cell surface may make it a useful epitope in S. pyogenes vaccines (24, 25). However, it remains poorly understood how Shr acquires heme from Hb. Here we show using a combination of biophysical and structural methods that Shr uses its HIDs to selectively bind to the heme-loaded form of Hb, slowing the rate of heme release by directly contacting the edges of its protoporphyrin rings. However, receptor dynamics within the Shr–Hb complex act to transiently uncap the HIDs from Hb’s β subunit, enabling heme’s gated release and subsequent capture by the receptor. This “cap and release” mechanism exploits the β subunit’s inherent weaker affinity for heme (26), allowing S. pyogenes to preferentially capture only heme-saturated forms of Hb that contain iron.Open in a separate windowFig. 1.Structure of the Hb–Shr^H2 complex. (A) Domain schematic of the Shr receptor. The polypeptide constructs used in this study are shown below. (B) Crystal structure of the Hb–Shr^H2 complex. The asymmetric unit of the crystal contains two tetramers of Hb that are bound by three molecules of Shr^H2. (C and D) HID2 binds over the heme pockets in both the α and β chains of Hb. These capping interactions directly contact both the heme and globin chain, burying an average of ~153 Ų and ~408 Ų of solvent-accessible surface area, respectively. Hb contacts originate from three surface loops in HID2: β2-α1, β4-β5, and β5-β6. (E) Expanded view of the Hb-receptor interface showing interactions with the heme molecule bound to the α subunit. The heme molecules are shown in stick format with oxygen and nitrogen atoms colored red and blue, respectively. Side chains in the receptor that interact with Hb are shown in stick format. (F) Identical to panel (E), except that receptor contacts to the β subunit in Hb are shown. Hb is in its ferric form. Color scheme: α subunit (salmon), β subunit (green), and HID2 (blue).