5iqx Citations

Replacing Arginine 33 for Alanine in the Hemophore HasA from Pseudomonas aeruginosa Causes Closure of the H32 Loop in the Apo-Protein.

Abstract

Previous characterization of hemophores from Serratia marcescens (HasAs), Pseudomonas aeruginosa (HasAp), and Yersinia pestis (HasAyp) showed that hemin binds between two loops, where it is axially coordinated by H32 and Y75. The Y75 loop is structurally conserved in all three hemophores and harbors conserved ligand Y75. The other loop contains H32 in HasAs and HasAp, but a noncoordinating Q32 in HasAyp. The H32 loop in apo-HasAs and apo-HasAp is in an open conformation, which places H32 about 30 Å from the hemin-binding site. Hence, hemin binding onto the Y75 loop of HasAs or HasAp triggers a large relocation of the H32 loop from an open- to a closed-loop conformation and enables coordination of the hemin-iron by H32. In comparison, the Q32 loop in apo-HasAyp is in the closed conformation, and hemin binding occurs with minimal reorganization and without coordinative interactions with the Q32 loop. Studies in crystallo and in solution have established that the open H32 loop in apo-HasAp and apo-HasAs is well structured and minimally affected by conformational dynamics. In this study we address the intriguing issue of the stability of the H32 loop in apo-HasAp and how hemin binding triggers its relocation. We address this question with a combination of NMR spectroscopy, X-ray crystallography, and molecular dynamics simulations and find that R33 is critical to the stability of the open H32 loop. Replacing R33 with A causes the H32 loop in R33A apo-HasAp to adopt a conformation similar to that of holo-HasAp. Finally, stopped-flow absorption and resonance Raman analyses of hemin binding to apo-R33A HasAp indicate that the closed H32 loop slows down the insertion of the heme inside the binding pocket, presumably as it obstructs access to the hydrophobic platform on the Y75 loop, but accelerates the completion of the heme iron coordination.

Articles - 5iqx mentioned but not cited (1)

  1. Replacing Arginine 33 for Alanine in the Hemophore HasA from Pseudomonas aeruginosa Causes Closure of the H32 Loop in the Apo-Protein. Kumar R, Qi Y, Matsumura H, Lovell S, Yao H, Battaile KP, Im W, Moënne-Loccoz P, Rivera M. Biochemistry 55 2622-2631 (2016)


Reviews citing this publication (2)

  1. Extracellular Heme Uptake and the Challenge of Bacterial Cell Membranes. Huang W, Wilks A. Annu Rev Biochem 86 799-823 (2017)
  2. Heme Uptake and Utilization by Gram-Negative Bacterial Pathogens. Richard KL, Kelley BR, Johnson JG. Front Cell Infect Microbiol 9 81 (2019)

Articles citing this publication (5)

  1. Gallium(III)-Salophen as a Dual Inhibitor of Pseudomonas aeruginosa Heme Sensing and Iron Acquisition. Centola G, Deredge DJ, Hom K, Ai Y, Dent AT, Xue F, Wilks A. ACS Infect Dis 6 2073-2085 (2020)
  2. Extracellular haem utilization by the opportunistic pathogen Pseudomonas aeruginosa and its role in virulence and pathogenesis. Mouriño S, Wilks A. Adv Microb Physiol 79 89-132 (2021)
  3. Structural and mutational analyses of the Leptospira interrogans virulence-related heme oxygenase provide insights into its catalytic mechanism. Soldano A, Klinke S, Otero LH, Rivera M, Catalano-Dupuy DL, Ceccarelli EA. PLoS One 12 e0182535 (2017)
  4. 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. Dent AT, Brimberry M, Albert T, Lanzilotta WN, Moënne-Loccoz P, Wilks A. Biochemistry 60 2549-2559 (2021)
  5. Getting Deeper into the Molecular Events of Heme Binding Mechanisms: A Comparative Multi-level Computational Study of HasAsm and HasAyp Hemophores. Tiessler-Sala L, Sciortino G, Alonso-Cotchico L, Masgrau L, Lledós A, Maréchal JD. Inorg Chem 61 17068-17079 (2022)