Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, Baltimore, Maryland.
Department of Chemistry, King's College London, London, United Kingdom.
Biophys J. 2021 Dec 7;120(23):5141-5157. doi: 10.1016/j.bpj.2021.11.010. Epub 2021 Nov 10.
The cytoplasmic heme binding protein from Pseudomonas aeruginosa, PhuS, plays two essential roles in regulating heme uptake and iron homeostasis. First, PhuS shuttles exogenous heme to heme oxygenase (HemO) for degradation and iron release. Second, PhuS binds DNA and modulates the transcription of the prrF/H small RNAs (sRNAs) involved in the iron-sparing response. Heme binding to PhuS regulates this dual function, as the unliganded form binds DNA, whereas the heme-bound form binds HemO. Crystallographic studies revealed nearly identical structures for apo- and holo-PhuS, and yet numerous solution-based measurements indicate that heme binding is accompanied by large conformational rearrangements. In particular, hydrogen-deuterium exchange mass spectrometry (HDX-MS) of apo- versus holo-PhuS revealed large differences in deuterium uptake, notably in α-helices 6, 7, and 8 (α6,7,8), which contribute to the heme binding pocket. These helices were mostly labile in apo-PhuS but largely protected in holo-PhuS. In contrast, in silico-predicted deuterium uptake levels of α6,7,8 from molecular dynamics (MD) simulations of the apo- and holo-PhuS structures are highly similar, consistent only with the holo-PhuS HDX-MS data. To rationalize this discrepancy between crystal structures, simulations, and observed HDX-MS, we exploit a recently developed computational approach (HDXer) that fits the relative weights of conformational populations within an ensemble of structures to conform to a target set of HDX-MS data. Here, a combination of enhanced sampling MD, HDXer, and dimensionality reduction analysis reveals an apo-PhuS conformational landscape in which α6, 7, and 8 are significantly rearranged compared to the crystal structure, including a loss of secondary structure in α6 and the displacement of α7 toward the HemO binding interface. Circular dichroism analysis confirms the loss of secondary structure, and the extracted ensembles of apo-PhuS and of heme-transfer-impaired H212R mutant, are consistent with known heme binding and transfer properties. The proposed conformational landscape provides structural insights into the modulation by heme of the dual function of PhuS.
铜绿假单胞菌的细胞质血红素结合蛋白 PhuS 在调节血红素摄取和铁稳态方面发挥着两个重要作用。首先，PhuS 将外源性血红素转运到血红素加氧酶（HemO）进行降解和铁释放。其次，PhuS 结合 DNA 并调节涉及铁节约反应的 prrF/H 小 RNA（sRNA）的转录。血红素与 PhuS 的结合调节这种双重功能，因为未配位的形式结合 DNA，而配位的形式结合 HemO。晶体结构研究揭示了 apo- 和 holo-PhuS 的结构几乎完全相同，但许多基于溶液的测量表明血红素结合伴随着大的构象重排。特别是，apo-PhuS 与 holo-PhuS 的氢氘交换质谱（HDX-MS）揭示了氘摄取的巨大差异，特别是在参与血红素结合口袋的α6、7 和 8（α6、7、8）螺旋中。这些螺旋在 apo-PhuS 中大多不稳定，但在 holo-PhuS 中基本受到保护。相比之下，从 apo-PhuS 和 holo-PhuS 结构的分子动力学（MD）模拟中预测的α6、7、8 的计算氘摄取水平非常相似，仅与 holo-PhuS HDX-MS 数据一致。为了使晶体结构、模拟和观察到的 HDX-MS 之间的差异合理化，我们利用了一种最近开发的计算方法（HDXer），该方法将构象种群的相对权重拟合到结构的集合中，以符合目标 HDX-MS 数据集。在这里，增强采样 MD、HDXer 和降维分析的组合揭示了 apo-PhuS 的构象景观，与晶体结构相比，α6、7 和 8 发生了明显的重排，包括α6 中二级结构的丧失和α7 向 HemO 结合界面的位移。圆二色性分析证实了二级结构的丧失，提取的 apo-PhuS 集合和血红素转移受损的 H212R 突变体的集合与已知的血红素结合和转移特性一致。所提出的构象景观为血红素对 PhuS 双重功能的调节提供了结构见解。
