Füzéry Anna K, Tonelli Marco, Ta Dennis T, Cornilescu Gabriel, Vickery Larry E, Markley John L
Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706, USA.
Biochemistry. 2008 Sep 9;47(36):9394-404. doi: 10.1021/bi800502r. Epub 2008 Aug 15.
The interaction between IscU and HscB is critical for successful assembly of iron-sulfur clusters. NMR experiments were performed on HscB to investigate which of its residues might be part of the IscU binding surface. Residual dipolar couplings ( (1) D HN and (1) D CalphaHalpha) indicated that the crystal structure of HscB [Cupp-Vickery, J. R., and Vickery, L. E. (2000) Crystal structure of Hsc20, a J-type cochaperone from Escherichia coli, J. Mol. Biol. 304, 835-845] faithfully represents its solution state. NMR relaxation rates ( (15)N R 1, R 2) and (1)H- (15)N heteronuclear NOE values indicated that HscB is rigid along its entire backbone except for three short regions which exhibit flexibility on a fast time scale. Changes in the NMR spectrum of HscB upon addition of IscU mapped to the J-domain/C-domain interface, the interdomain linker, and the C-domain. Sequence conservation is low in the interface and in the linker, and NMR changes observed for these residues likely result from indirect effects of IscU binding. NMR changes observed in the conserved patch of residues in the C-domain (L92, M93, L96, E97, E100, E104, and F153) were suggestive of a direct interaction with IscU. To test this, we replaced several of these residues with alanine and assayed for the ability of HscB to interact with IscU and to stimulate HscA ATPase activity. HscB(L92A,M93A,F153A) and HscB(E97A,E100A,E104A) both showed decreased binding affinity for IscU; the (L92A,M93A,F153A) substitution also strongly perturbed the allosteric interaction within the HscA.IscU.HscB ternary complex. We propose that the conserved patch in the C-domain of HscB is the principal binding site for IscU.
IscU与HscB之间的相互作用对于铁硫簇的成功组装至关重要。对HscB进行了核磁共振实验，以研究其哪些残基可能是IscU结合表面的一部分。剩余偶极耦合（（1）D HN和（1）D CalphaHalpha）表明，HscB的晶体结构[Cupp-Vickery, J. R., and Vickery, L. E. (2000) Crystal structure of Hsc20, a J-type cochaperone from Escherichia coli, J. Mol. Biol. 304, 835-845]如实地反映了其溶液状态。核磁共振弛豫率（（15）N R 1、R 2）和（1）H-（15）N异核NOE值表明，HscB除了三个在快速时间尺度上表现出灵活性的短区域外，其整个主链都是刚性的。加入IscU后HscB核磁共振谱的变化映射到J结构域/C结构域界面、结构域间连接子和C结构域。界面和连接子中的序列保守性较低，这些残基观察到的核磁共振变化可能是IscU结合的间接效应导致的。在C结构域中保守的残基区域（L92、M93、L96、E97、E100、E104和F153）观察到的核磁共振变化表明与IscU存在直接相互作用。为了验证这一点，我们将这些残基中的几个替换为丙氨酸，并检测HscB与IscU相互作用以及刺激HscA ATP酶活性的能力。HscB（L92A，M93A，F153A）和HscB（E97A，E100A，E104A）对IscU的结合亲和力均降低；（L92A，M93A，F153A）替换也强烈干扰了HscA.IscU.HscB三元复合物内的变构相互作用。我们提出，HscB的C结构域中的保守区域是IscU的主要结合位点。
