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Axial Heme Coordination by the Tyr-His Motif in the Extracellular Hemophore HasAp Is Critical for the Release of Heme to the HasR Receptor of Pseudomonas aeruginosa.

ABSTRACT: Pseudomonas aeruginosa senses extracellular heme via an extra cytoplasmic function σ factor that is activated upon interaction of the hemophore holo-HasAp with the HasR receptor. Herein, we show Y75H holo-HasAp interacts with HasR but is unable to release heme for signaling and uptake. To understand this inhibition, we undertook a spectroscopic characterization of Y75H holo-HasAp by resonance Raman (RR), electron paramagnetic resonance (EPR), and X-ray crystallography. The RR spectra are consistent with a mixed six-coordinate high-spin (6cHS), six-coordinate low-spin (6cLS) heme configuration and an H₂¹⁸O exchangeable Fe^III-O stretching frequency with ¹⁶O/¹⁸O and H/D isotope shifts that support a two-body Fe-OH₂ oscillator with (iron-hydroxy)-like character as both hydrogen atoms are engaged in short hydrogen bond interactions with protein side chains. Further support comes from the EPR spectrum of Y75H holo-HasAp that shows a LS rhombic signal with ligand-field splitting values intermediate between those of His-hydroxy and bis-His ferric hemes. The crystal structure of Y75H holo-HasAp confirmed the coordinated solvent molecule hydrogen bonded through H75 and H83. The long-range conformational rearrangement of HasAp upon heme binding can still take place in Y75H holo-HasAp, because the intercalation of a hydroxy ligand between the heme iron and H75 allows the variant to reproduce the heme binding pocket observed in wild-type holo-HasAp. However, in the absence of a covalent linkage to the Y75 loop combined with the malleability provided by the bracketing H75 and H83 hydrogen bonds, either the hydroxy sixth ligand remains bound after complexation of Y75H holo-HasAp with HasR or rearrangement and coordination of H85 prevent heme transfer.

SUBMITTER: Dent AT

PROVIDER: S-EPMC8672344 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Hemophores from Pseudomonas aeruginosa (HasAp), Serratia marcescens (HasAsm), and Yersinia pestis (HasAyp) bind hemin between two loops. One of the loops harbors conserved axial ligand Tyr75 (Y75 loop) in all three structures, whereas the second loop (H32 loop) contains axial ligand His32 in HasAp and HasAsm, but a noncoordinating Gln32 in HasAyp. Binding of hemin to the Y75 loop of HasAp or HasAsm causes a large rearrangement of the H32 loop that allows His32 coordination. The Q32 loop in apo-HasAyp is already in the closed conformation, such that binding of hemin to the conserved Y75 loop occurs with minimal structural rearrangement and without coordinative interaction with the Q32 loop. In this study, structural and spectroscopic investigations of the hemophore HasAp were conducted to probe (i) the role of the conserved Tyr75 loop in hemin binding and (ii) the proposed requirement of the His83-Tyr75 hydrogen bond to allow the coordination of hemin by Tyr75. High-resolution crystal structures of H83A holo-HasAp obtained at pH 6.5 (0.89 Å) and pH 5.4 (1.25 Å) show that Tyr75 remains coordinated to the heme iron, and that a water molecule can substitute for Nδ of His83 to interact with the Oη atom of Tyr75, likely stabilizing the Tyr75-Fe interaction. Nuclear magnetic resonance spectroscopy revealed that in apo-Y75A and apo-H83A HasAp, the Y75 loop is disordered, and that disorder propagates to nearby elements of secondary structure, suggesting that His83 Nδ-Tyr75 Oη interaction is important to the organization of the Y75 loop in apo-HasA. Kinetic analysis of hemin loading conducted via stopped-flow UV-vis and rapid-freeze-quench resonance Raman shows that both mutants load hemin with biphasic kinetic parameters that are not significantly dissimilar from those previously observed for wild-type HasAp. When the structural and kinetic data are taken together, a tentative model emerges, which suggests that HasA hemophores utilize hydrophobic, π-π stacking, and van der Waals interactions to load hemin efficiently, while axial ligation likely functions to slow hemin release, thus allowing the hemophore to meet the challenge of capturing hemin under inhospitable conditions and delivering it selectively to its cognate receptor.

Project description:When challenged by low-iron conditions several Gram-negative pathogens secrete a hemophore (HasA) to scavenge hemin from its host and deliver it to a receptor (HasR) on their outer membrane for internalization. Here we report results from studies aimed at probing the structural and dynamic processes at play in the loading of the apo-hemophore secreted by P. aeruginosa (apo-HasAp) with hemin. The structure of apo-HasAp shows a large conformational change in the loop harboring axial ligand His32 relative to the structure of holo-HasAp, whereas the loop bearing the other axial ligand, Tyr75, remains intact. To investigate the role played by the axial ligand-bearing loops in the process of hemin capture we investigated the H32A mutant, which was found to exist as a monomer in its apo-form and as a mixture of monomers and dimers in its holo-form. We obtained an X-ray structure of dimeric H32A holo-HasAp, which revealed that the two subunits are linked by cofacial interactions of two hemin molecules and that the conformation of the Ala32 loop in the dimer is identical to that exhibited by the His32 loop in wild type apo-HasAp. Additional data suggest that the conformation of the Ala32 loop in the dimer is mainly a consequence of dimerization. Hence, to investigate the effect of hemin loading on the topology of the His32 loop we also obtained the crystal structure of monomeric H32A holo-HasAp coordinated by imidazole (H32A-imidazole) and investigated the monomeric H32A HasAp and H32A-imidazole species in solution by NMR spectroscopy. The structure of H32A-imidazole revealed that the Ala32 loop attains a "closed" conformation nearly identical to that observed in wild type holo-HasAp, and the NMR investigations indicated that this conformation is maintained in solution. The NMR studies also highlighted conformational heterogeneity at the H32 loop hinges and in other key sections of the structure. Targeted molecular dynamics simulations allowed us to propose a possible path for the closing of the His32 loop upon hemin binding and identified molecular motions that are likely important in transmitting the presence of hemin in the Tyr75 loop to the His32 loop to initiate its closing. Importantly, residues implicated as undergoing motions in the computations are also observed as being dynamic by NMR. Taken together, these observations provide direct experimental evidence indicating that hemin loads onto the Tyr75 loop of apo-HasAp, which triggers the closing of the His32 loop.

Project description:BackgroundPseudomonas aeruginosa (PA) is a Gram-negative pathogen responsible for fatal nosocomial infections worldwide. Iron is essential for Gram-negative bacteria to establish an infection. Therefore, iron acquisition proteins (IAPs) of bacteria are attractive vaccine targets.MethodologyA "Reverse Vaccinology" approach was employed in the current study. Expression levels of 37 IAPs in various types of PA infections were analyzed in seven previously published studies. The IAP vaccine candidate was selected based on multiple criteria, including a high level of expression, high antigenicity, solubility, and conservation among PA strains, utilizing suitable bioinformatics analysis tools. The selected IAP candidate was recombinantly expressed in Escherichia coli and purified using metal affinity chromatography. It was further evaluated in vivo for protection efficacy. The novel immune adjuvant, naloxone (NAL), was used.Results and discussionHasAp antigen met all the in silico selection criteria, being highly antigenic, soluble, and conserved. In addition, it was the most highly expressed IAP in terms of average fold change compared to control. Although HasAp did excel in the in silico evaluation, subcutaneous immunization with recombinant HasAp alone or recombinant HasAp plus NAL (HasAP-NAL) did not provide the expected protection compared to controls. Immunized mice showed a low IgG2a/IgG1 ratio, indicating a T-helper type 2 (Th2)-oriented immune response that is suboptimal for protection against PA infections. Surprisingly, the bacterial count in livers of both NAL- and HasAp-NAL-immunized mice was significantly lower than the count in the HasAp and saline groups. The same trend was observed in kidneys and lungs obtained from these groups, although the difference was not significant. Such protection could be attributed to the enhancement of innate immunity by NAL.ConclusionsWe provided a detailed in silico analysis of IAPs of PA followed by in vivo evaluation of the best IAP, HasAp. Despite the promising in silico results, HasAp did not provide the anticipated vaccine efficacy. HasAp should be further evaluated as a vaccine candidate through varying the immunization regimens, models of infection, and immunoadjuvants. Combination with other IAPs might also improve vaccination efficacy. We also shed light on several highly expressed promising IAPs whose efficacy as vaccine candidates is worthy of further investigation.

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Axial Heme Coordination by the Tyr-His Motif in the Extracellular Hemophore HasAp Is Critical for the Release of Heme to the HasR Receptor of Pseudomonas aeruginosa.

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