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首页> 外文期刊>Biochemistry >Structural Characterization of the Hemophore HasAp from Pseudomonas aeruginosa: NMR Spectroscopy Reveals Protein−Protein Interactions between Holo-HasAp and Hemoglobin,
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Structural Characterization of the Hemophore HasAp from Pseudomonas aeruginosa: NMR Spectroscopy Reveals Protein−Protein Interactions between Holo-HasAp and Hemoglobin,

机译：铜绿假单胞菌的荧光团HasAp的结构表征：NMR光谱揭示了Holo-HasAp和血红蛋白之间的蛋白质-蛋白质相互作用，

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Pseudomonas aeruginosa secretes a 205 residue long hemophore (full-length HasAp) that is subsequently cleaved at the C′-terminal domain to produce mainly a 184 residue long truncated HasAp that scavenges heme [Letoff, S., Redeker, V., and Wandersman, C. (1998) Mol. Microbiol. 28, 1223−1234]. HasAp has been characterized by X-ray crystallography and in solution by NMR spectroscopy. The X-ray crystal structure of truncated HasAp revealed a polypeptide αβ fold and a ferriheme coordinated axially by His32 and Tyr75, with the side chain of His83 poised to accept a hydrogen bond from the Tyr75 phenolic acid group. NMR investigations conducted with full-length HasAp showed that the carboxyl-terminal tail (21 residues) is disordered and conformationally flexible. NMR spectroscopic investigations aimed at studying a complex between apo-HasAp and human methemoglobin were stymied by the rapid heme capture by the hemophore. In an effort to circumvent this problem NMR spectroscopy was used to monitor the titration of 15N-labeled holo-HasAp with hemoglobin. These studies allowed identification of a specific area on the surface of truncated HasAp, encompassing the axial ligand His32 loop that serves as a transient site of interaction with hemoglobin. These findings are discussed in the context of a putative encounter complex between apo-HasAp and hemoglobin that leads to efficient hemoglobin−heme capture by the hemophore. Similar experiments conducted with full-length 15N-labeled HasAp and hemoglobin revealed a transient interaction site in full-length HasAp similar to that observed in the truncated hemophore. The spectral perturbations observed while investigating these interactions, however, are weaker than those observed for the interactions between hemoglobin and truncated HasAp, suggesting that the disordered tail in the full-length HasAp must be proteolyzed in the extracellular milieu to make HasAp a more efficient hemophore.
机译：铜绿假单胞菌分泌205个残基的长荧光团（全长HasAp），随后在C'末端域切割，主要产生184个残基的长截短的HasAp，它清除了血红素[Letoff，S.，Redeker，V.和Wandersman ，C.（1998）微生物。 28，1223−1234]。 HasAp已通过X射线晶体学表征，并在溶液中通过NMR光谱表征。截短的HasAp的X射线晶体结构显示了多肽αβ折叠和亚铁血红素，由His32和Tyr75轴向配位，His83的侧链准备好接受来自Tyr75酚酸基团的氢键。使用全长HasAp进行的NMR研究表明，羧基末端的尾巴（21个残基）无序且构象灵活。旨在研究载脂蛋白-HasAp与人高铁血红蛋白之间的复合物的NMR光谱学研究被血红素快速捕获血红素所阻碍。为了避免该问题，使用NMR光谱法监测了用血红蛋白滴定15N标记的HaloAp。这些研究允许鉴定截短的HasAp表面的特定区域，包括轴向配体His32环，该环用作与血红蛋白相互作用的瞬时位点。这些发现是在apo-HasAp与血红蛋白之间的假定相遇复合物的背景下进行讨论的，该复合物导致血红蛋白有效捕获血红蛋白-血红素。用全长15N标记的HasAp和血红蛋白进行的类似实验显示，全长HasAp中的瞬时相互作用位点与在截短的荧光团中观察到的相似。然而，研究这些相互作用时观察到的光谱扰动要弱于血红蛋白和截短的HasAp之间的相互作用，这表明全长HasAp中无序的尾巴必须在细胞外环境中进行蛋白水解，以使HasAp成为更有效的荧光团。

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