4o6t
1.25A resolution structure of the hemophore HasA from Pseudomonas aeruginosa (H83A mutant, pH 5.4)
Structural highlights
FunctionPublication Abstract from PubMedHemophores from Pseudomonas aeruginosa (HasAp), Serratia marcescens (HasAsm), and Yersinia pestis (HasAyp) bind hemin between two loops. One of the loops harbors conserved axial ligand Ty75 (Y75 loop) in all three structures, whereas the second loop (H32 loop) contains axial ligand His32 in HasAp and HasAsm, but a non-coordinating Gln32 in HasAyp. Hemin binding to the Y75 loop of HasAp or HasAsm causes a large rearrangement of the H32 loop that enables His32 coordination. The Q32 loop in apo-HasAyp is already in the closed conformation, such that hemin binding 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 enable hemin coordination by Tyr75. High-resolution crystal structures of H83A holo-HasAp obtained at pH 6.5 (0.89 A) and 5.4 (1.25 A) show that Tyr75 remains coordinated to the heme iron, and that a water molecule can substitute for His83 Ndelta to interact with the Oeta atom of Tyr75, likely stabilizing the Tyr75-Fe interaction. NMR spectroscopy revealed that in apo-Y75A and H83A HasAp, the Y75 loop is disordered, and that disorder propagates to nearby elements of secondary structure, suggesting that the His83 Ndelta to Tyr75 Oeta interaction is important to the organization of the Y75 loop in apo-HasA. Kinetic analysis of hemin loading carried out with stopped flow UV-vis and rapid-freeze-quench resonance Raman show that both mutants load hemin with biphasic kinetic parameters that are not significantly dissimilar from those previously observed with wild type HasAp. When the structural and kinetic data are taken together a tentative model emerges, which suggests that HasA hemophores utilize hydrophobic, pi-pi stacking, and van der Waals interactions to load hemin efficiently, while axial ligation likely functions to slow hemin release, thus enabling the hemophore to meet the challenge of capturing hemin under inhospitable conditions and deliver it selectively to its cognate receptor.<i><i><i><i> Replacing the Axial Ligand Tyrosine 75 or its Hydrogen Bond Partner Histidine 83 Minimally Affects Hemin Acquisition by the Hemophore HasAp from <i>Pseudomonas aeruginosa</i>,Kumar R, Matsumura H, Lovell SW, Yao H, Rodriguez JC, Battaile KP, Moenne-Loccoz P, Rivera M Biochemistry. 2014 Mar 13. PMID:24625274[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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