Induced fit on heme binding to the Pseudomonas aeruginosa cytoplasmic protein (PhuS) drives interaction with heme oxygenase (HemO)

Abstract

Iron, an essential nutrient with limited bioavailability, requires specialized cellular mechanisms for uptake. Although iron uptake into the cytoplasm in the form of heme has been well characterized in many bacteria, the subsequent trafficking is poorly understood. The cytoplasmic heme-binding proteins belong to a structurally related family thought to have evolved as “induced fit” ligand-binding macromolecules. One member, Pseudomonas aeruginosa cytoplasmic protein (PhuS), has previously been shown to be important for delivering heme to the iron regulated heme oxygenase (HemO). Spectroscopic investigations of the holo-PhuS complex revealed a dynamic heme environment with overlapping but distinct heme-binding sites with alternative coordinating heme ligands, His-209 or His-212. In the present work we establish a mechanism for how heme is transferred from PhuS to its partner, HemO. Using surface plasmon resonance and isothermal titration calorimetry, we have discovered that holo-PhuS, but not apo-PhuS, forms a 1:1 complex with HemO. Sedimentation velocity and limited proteolysis experiments suggest that heme binding to PhuS induces a conformational rearrangement that drives the protein interaction with HemO. Hydrodynamic analysis reveals that the holo-PhuS displays a more expanded hydrodynamic envelope compared with apo-PhuS, and we propose that this conformational change drives the interaction with HemO. We further demonstrate that replacement of His-212 by Ala disrupts the interaction of holo-PhuS with HemO; in contrast, the His-209-Ala variant can still complex with HemO, albeit more weakly. Together, the present studies reveal a mechanism that couples a heme-dependent conformational switch in PhuS to protein–protein interaction, the subsequent free energy of which drives heme release to HemO.

Ligand-induced allostery in the interaction of the Pseudomonas aeruginosa heme binding protein with heme oxygenase

“…2 A, Upper and B, Upper). These data are consistent with previous solution studies showing that heme binding shifts the sedimentation coefficient distribution for holo-PhuS and further protects the C-terminal domain from proteolysis (10). Interestingly, the HDX-MS data are not consistent with the recent apo-PhuS crystal structures, where the overall structural fold is almost identical to that of holo-PhuS, suggesting that the apoPhuS crystal structures are more representative of the ligandbound state (12,13).…”

“…Previous studies from our laboratory have shown the interaction of PhuS with HemO is driven by a conformational change on ligand binding (6,10). Furthermore, the previously reported kinetics of heme transfer from PhuS to HemO are slow: on the second timescale at 34°C (11).…”

“…Titrations of apo-HemO with holo-PhuS WT and H212R were performed on a MicroCal MCS Titration Calorimeter (MicroCal Inc.) as previously described (10). A detailed description is provided in SI Experimental Procedures.…”

Differential Contributions of the Outer Membrane Receptors PhuR and HasR to Heme Acquisition in Pseudomonas aeruginosa

“…Once internalized heme is sequestered by the cytoplasmic heme-binding protein PhuS. In a series of in vitro studies we have shown that the cytoplasmic heme-binding protein PhuS forms a specific protein complex with the ironregulated heme oxygenase, HemO (7)(8)(9). Heme is transferred to HemO for further degradation with the release of iron, CO, and biliverdin IX (BVIX) ␦-and ␤-isomers (10,11).…”

In vitro heme biotransformation by the HupZ enzyme from Group A streptococcus

“…Several cytoplasmic hemebinding proteins share sequence homology with ChuS including HemS from Yersinia enterocolitica (Schneider and Paoli 2005;Stojiljkovic and Hantke 1994), Bartonella henselae (Liu et al 2012a) and PhuS from P. aeruginosa (Lansky et al 2006). It has been established however, that PhuS main role is not heme degradation rather than it binds heme and delivers it to its partner HO, HemO for degradation (Lansky et al 2006;O'Neill et al 2012). PhuS was also shown to convert heme to verdoheme in vitro, and the possibility that it delivers verdoheme to HemO in vivo was raised (Lee et al 2014).…”