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Conventional time-resolved methods such as photoactivation or temperature jumps cannot be used to initiate this binding reaction because the process is triggered exclusively by direct contact between the two proteins. Furthermore, tetrameric Hb can form multiple intermediates with IsdB, making it difficult to capture the sequence of structural changes during haem extraction. A clear, time-resolved description of this pathogen–host interaction is therefore essential for understanding how S. aureus acquires iron in vivo and for guiding the design of strategies to block this pathway.

The experiment

Time-resolved X-ray solution scattering (TR-XSS) at beamline ID09 was used to follow the interaction between IsdB and Hb with millisecond resolution and to reconstruct the structural pathway leading to haem capture. Stopped-flow mixing provided a label-free mechanical trigger, enabling the reaction between Hb and IsdB to be initiated by rapid physical mixing of the two proteins (Figure 8). The combination of stopped- flow delivery with a pulsed X-ray beam enabled genuine time-resolved solution scattering on a protein–protein reaction – an approach rarely possible elsewhere – linking structural snapshots directly to millisecond kinetics.

Time-resolved X-ray scattering tracks human haem extraction by Staphylococcus aureus

• Accessing iron from human haemoglobin is essential for the survival of the pathogen Staphylococcus aureus, but the molecular steps of haem extraction have remained unclear. • Time-resolved X-ray solution scattering at beamlines ID09 and BM29 enabled real-time tracking of structural changes during the interaction between the bacterial receptor IsdB and human haemoglobin. • The resulting mechanism identifies key steps in pathogen–host interaction and provides a foundation for developing inhibitors that block iron acquisition.

The challenge

To survive and proliferate, pathogens such as Staphylococcus aureus must obtain iron from their human host. In the bloodstream, iron is largely sequestered within the haem groups of haemoglobin (Hb). S. aureus expresses the surface receptor IsdB to bind Hb and extract its haem cofactor (Figure 7). Disrupting this process represents a potential antimicrobial strategy, particularly for drug-resistant strains of S. aureus. However, understanding the mechanism has been limited by the transient nature of the protein– protein interactions involved.

Fig. 7: Overview of iron acquisition by S. aureus from the human host. To access host iron, S. aureus lyses red blood cells via secreted

haemolysins, releasing Hb into the extracellular space. At the bacterial surface, the receptor IsdB

binds Hb and extracts its haem cofactor, which is then used as an iron source.