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

Abstract

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.

Structural basis for the recognition of human hemoglobin by the heme-acquisition protein Shr from Streptococcus pyogenes

“…We believe the stoichiometry Hb: HID2 (1:3) may reflect a particular obstacle of the complex to crystallize in the expected 1:4 complex because of unfavorable crystal contacts (Figure S2 ). The stoichiometry found in this crystal structure contrasts to that observed in another structure of the complex, in which only three molecules of HID2 were bound to two tetramers of Hb in the asymmetric unit (37.5% occupancy) 13 .

Figure 2 Crystal structure of HID2 bound to human Hb.

…”

“…In contrast, from the α- or β-chain of Hb, the presence of three Lys residues was predominant: three Lys residues from the α-chain and four Lys residues from the β-chain, although only two of them (Lys 60 of α-chain and Lys95 of the β-chain) engaged in salt bridges with residues of HID2 (distance < 4.5 Å, Table S1 , Table S2 ). These observations were similar to those made in the previous structure of the complex (PDB entry code: 8DOV) 13 . Additional polar interactions between the interacting proteins were scarce, and indeed only two H-bond (< 3.3 Å) were found, specifically between side chain of Asp64 of α-chain and Ser225 of HID2, and between the backbone oxygen of Ser44 and the side chain of Gln209 of HID2 (Fig.…”

“…No significant structural changes in HID2 bound to the β-chain of Hb are observed with respect to that of HID2 bound to the α-chain (RMSD < 0.5 Å). Similarly, the structure of HID2 was nearly indistinguishable from that of the unbound protein also determined herein (RMSD 0.6 Å, Figure S3 ) or to the previously reported structure of a slightly shorter construct (PDB entry code 6DKQ, RMSD = 0.56 Å) 11 or to the previously reported structure of HID2 in complex with Hb (PDB entry code 8DOV, RMSD < 0.3 Å) 13 . These results indicate that HID2 does not undergo significant conformational changes to recognize and stably bind to the target region in Hb, suggesting a mechanism of lock-and-key similar to that observed for some VHH antibodies (nanobodies) binding to their antigens 14 .…”

“…It is noteworthy that the later interaction between Ser225 of HID2 is specific of interface with the α-chain of Hb, whereas the interaction involving Gln209 of HID2 is specific in the interaction with β-chain of Hb. In addition, the previously reported crystal structure also indicated the presence of water molecules at the interface between the E-helix of Hb and several residues of HID2 that could support the primary interactions enumerated above 13 . Our structure was determined at lower resolution and no evidence of interfacial waters was found.…”

“…These results suggested a more prominent role of HID2 during heme acquisition by Shr. Indeed, a recent report of the crystal structures of HID2 bound to Hb suggested a different molecular mechanism for heme extraction by Shr from S. pyogenes with respect to that of other bacterial systems studied so far 13 .…”