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

Abstract

A heme-dependent conformational rearrangement of the C-terminal domain of heme binding protein (PhuS) is required for interaction with the iron-regulated heme oxygenase (HemO). Herein, we further investigate the underlying mechanism of this conformational rearrangement and its implications for heme transfer via site-directed mutagenesis, resonance Raman (RR), hydrogendeuterium exchange MS (HDX-MS) methods, and molecular dynamics (MD). HDX-MS revealed that the apo-PhuS C-terminal α6/α7/α8-helices are largely unstructured, whereas the apo-PhuS H212R variant showed an increase in structure within these regions. The increased rate of heme association with apo-PhuS H212R compared with the WT and lack of a detectable five-coordinate highspin (5cHS) heme intermediate are consistent with a more folded and less dynamic C-terminal domain. HDX-MS and MD of holo-PhuS indicate an overall reduction in molecular flexibility throughout the protein, with significant structural rearrangement and protection of the heme binding pocket. We observed slow cooperative unfolding/ folding events within the C-terminal helices of holo-PhuS and the N-terminal α1/α2-helices that are dampened or eliminated in the holo-PhuS H212R variant. Chemical cross-linking and MALDI-TOF MS mapped these same regions to the PhuS:HemO protein-protein interface. We previously proposed that the protein-protein interaction induces conformational rearrangement, promoting a ligand switch from His-209 to His-212 and triggering heme release to HemO. The reduced conformational freedom of holo-PhuS H212R combined with the increase in entropy and decrease in heme transfer on interaction with HemO further support this model. This study provides significant insight into the role of protein dynamics in heme binding and release in bacterial heme transport proteins.heme binding | allostery | protein-protein interaction | Pseudomonas aeruginosa | heme transfer

Interpretation of HDX Data by Maximum-Entropy Reweighting of Simulated Structural Ensembles

“…Because side-chain and terminal-amine deuterons exchange back relatively rapidly with protons during analysis, HDX-MS data reports exclusively on backbone-amide exchange. This ability to directly probe protein dynamics has led to diverse applications ( 4 ), including studies of allostery ( 5 , 6 , 7 ), epitope mapping for protein-protein or protein-lipid interactions ( 8 , 9 , 10 , 11 ), effects of ligand binding ( 12 , 13 , 14 , 15 ), mechanisms of membrane proteins ( 16 , 17 , 18 , 19 , 20 , 21 , 22 ), and dynamics of large macromolecular complexes ( 23 , 24 , 25 , 26 ). This progress notwithstanding, the interpretation of HDX-MS data in structural and mechanistic terms has been, generally speaking, largely qualitative and lacking objective metrics.…”

The Arabidopsis locus AT3G03890 encodes a dimeric β-barrel protein implicated in heme degradation

“…Instead, they are suggested to deliver heme to heme-degrading enzyme, acting as heme chaperones. An example of such a chaperone-HO interaction is the PhuS-HemO complex [49].…”

Heme dependent transcriptional regulation of thePseudomonas aeruginosatandem sRNAprrF1,F2locus by the cytoplasmic heme binding protein PhuS

“…The amplified fragment was cloned into pUC18, resulting in pUC18-5’-PhuS-3’. The mutant allele phuSH212R was obtained following digestion of plasmid pET21 phuS H212R (21) with Nru I and Stu I and subcloned into Nru I and Stu I digested pUC18-5’-PhuS-3’, replacing the wild type allele. The new construct pUC18-5’- phuS H212R-3’ was confirmed by sequencing ( Eurofins MWG Operon ).…”