Mahoney, B. J. | Search bioRxiv

bioRxiv2026

Cryo-EM provides insight into how the Staphylococcus aureus IsdH receptor removes hemin from the hemoglobin:haptoglobin complex

Staphylococcus aureus extracts hemin from human hemoglobin (Hb) to overcome host-imposed iron limitation. How it recovers Hb-bound hemin from the hemoglobin:haptoglobin (Hb:Hp) complex, the major circulating form of Hb outside red blood cells, remains unclear. Here we use cryo-electron microscopy, biophysical measurements, and solution kinetics to define how the S. aureus IsdH surface receptor extracts hemin from Hb:Hp. A 3.1 [A] cryo-EM structure of Hb:Hp bound by full-length IsdH reveals that its N-terminal NEAT domain (N1) anchors it to Hb, whereas its downstream N2N3 extraction unit engages {beta}Hb to remove its hemin. The receptor engages Hb:Hp differently than isolated Hb, because N-linked glycans on haptoglobin bias the extraction unit toward {beta}Hb, sterically occluding its access to Hb while still permitting engagement by N1. Kinetic assays show that IsdH actively accelerates hemin release from Hb:Hp. Three-dimensional variability analysis indicates that this likely occurs via a dynamic interface in which receptor motions reposition the extraction unit relative to {beta}Hb, collectively supporting a model in which IsdH transiently perturbs the F-helix to promote hemin extraction. Alignment of that model with a previously determined CD163:Hb:Hp structure shows how IsdH may disrupt Hb:Hp recognition by macrophage and monocyte CD163 receptors, helping to explain how it may hinder clearance of Hb:Hp from circulation. In aggregate, these results help define the structural basis for hemin extraction from Hb:Hp and how IsdH may subvert receptor-mediated clearance of the Hb:Hp complex. SignificanceStaphylococcus aureus scavenges hemin from host hemoglobin to proliferate, yet most extracellular hemoglobin is sequestered in hemoglobin:haptoglobin (Hb:Hp) complexes, which are rapidly cleared from circulation. To clarify how bacteria access hemin in this context, we now show that the IsdH surface receptor is structurally adapted to extract hemin from Hb:Hp. Our results indicate that IsdH uses distinct NEAT domains to anchor to Hb and selectively extract hemin from {beta}Hb. This {beta}Hb selectivity is shaped by haptoglobin N-glycans and enhances microbial access to iron. These findings provide a mechanistic framework for targeting heme acquisition as an anti-virulence strategy.

microbiology↗

bioRxiv2023

The Basal and Major Pilins in the Corynebacterium diphtheriae SpaA Pilus Adopt Similar Structures that Competitively React with the Pilin Polymerase

Many species of pathogenic gram-positive bacteria display covalently crosslinked protein polymers (called pili or fimbriae) that mediate microbial adhesion to host tissues. These structures are assembled by pilus-specific sortase enzymes that join the pilin components together via lysine-isopeptide bonds. The archetypal SpaA pilus from Corynebacterium diphtheriae is built by the CdSrtA pilus-specific sortase, which crosslinks lysine residues within the SpaA and SpaB pilins to build the shaft and base of the pilus, respectively. Here, we show that CdSrtA crosslinks SpaB to SpaA via a K139(SpaB)-T494(SpaA) lysine-isopeptide bond. Despite sharing only limited sequence homology, an NMR structure of SpaB reveals striking similarities with the N-terminal domain of SpaA (NSpaA) that is also crosslinked by CdSrtA. In particular, both pilins contain similarly positioned reactive lysine residues and adjacent disordered AB loops that are predicted to be involved in the recently proposed "latch" mechanism of isopeptide bond formation. Competition experiments using an inactive SpaB variant and additional NMR studies suggest that SpaB terminates SpaA polymerization by outcompeting NSpaA for access to a shared thioester enzyme-substrate reaction intermediate.

biochemistry↗