Sjodt Megan, Macdonald Ramsay, Spirig Thomas, Chan Albert H, Dickson Claire F, Fabian Marian, Olson John S, Gell David A, Clubb Robert T
Department of Chemistry and Biochemistry, University of California, Los Angeles, 611 Charles Young Drive East, Los Angeles, CA 90095, USA; University of California, Los Angeles-Department of Energy Institute of Genomics and Proteomics, University of California, Los Angeles, 611 Charles Young Drive East, Los Angeles, CA 90095, USA; Molecular Biology Institute, University of California, Los Angeles, 611 Charles Young Drive East, Los Angeles, CA 90095, USA.
Department of Chemistry and Biochemistry, University of California, Los Angeles, 611 Charles Young Drive East, Los Angeles, CA 90095, USA.
J Mol Biol. 2016 Mar 27;428(6):1107-1129. doi: 10.1016/j.jmb.2015.02.008. Epub 2015 Feb 14.
Staphylococcus aureus is a medically important bacterial pathogen that, during infections, acquires iron from human hemoglobin (Hb). It uses two closely related iron-regulated surface determinant (Isd) proteins to capture and extract the oxidized form of heme (hemin) from Hb, IsdH and IsdB. Both receptors rapidly extract hemin using a conserved tri-domain unit consisting of two NEAT (near iron transporter) domains connected by a helical linker domain. To gain insight into the mechanism of extraction, we used NMR to investigate the structure and dynamics of the 38.8-kDa tri-domain IsdH protein (IsdH(N2N3), A326-D660 with a Y642A mutation that prevents hemin binding). The structure was modeled using long-range paramagnetic relaxation enhancement (PRE) distance restraints, dihedral angle, small-angle X-ray scattering, residual dipolar coupling and inter-domain NOE nuclear Overhauser effect data. The receptor adopts an extended conformation wherein the linker and N3 domains pack against each other via a hydrophobic interface. In contrast, the N2 domain contacts the linker domain via a hydrophilic interface and, based on NMR relaxation data, undergoes inter-domain motions enabling it to reorient with respect to the body of the protein. Ensemble calculations were used to estimate the range of N2 domain positions compatible with the PRE data. A comparison of the Hb-free and Hb-bound forms reveals that Hb binding alters the positioning of the N2 domain. We propose that binding occurs through a combination of conformational selection and induced-fit mechanisms that may promote hemin release from Hb by altering the position of its F helix.
金黄色葡萄球菌是一种具有重要医学意义的细菌病原体，在感染过程中会从人类血红蛋白（Hb）中获取铁。它利用两种密切相关的铁调节表面决定簇（Isd）蛋白，即IsdH和IsdB，从Hb中捕获并提取氧化形式的血红素（高铁血红素）。这两种受体都使用一个由两个NEAT（近铁转运体）结构域通过螺旋连接结构域相连组成的保守三结构域单元快速提取高铁血红素。为了深入了解提取机制，我们利用核磁共振（NMR）研究了38.8 kDa的三结构域IsdH蛋白（IsdH(N2N3)，A326 - D660，带有阻止高铁血红素结合的Y642A突变）的结构和动力学。该结构通过长程顺磁弛豫增强（PRE）距离限制、二面角、小角X射线散射、残余偶极耦合和结构域间核Overhauser效应（NOE）数据进行建模。该受体呈现出一种伸展构象，其中连接结构域和N3结构域通过疏水界面相互堆积。相比之下，N2结构域通过亲水界面与连接结构域接触，并且根据NMR弛豫数据，会发生结构域间运动，使其能够相对于蛋白质主体重新定向。通过系综计算来估计与PRE数据兼容的N2结构域位置范围。对无Hb和结合Hb形式的比较表明，Hb结合会改变N2结构域的定位。我们提出，结合是通过构象选择和诱导契合机制的组合发生的，这可能通过改变Hb的F螺旋位置来促进高铁血红素从Hb中释放。
