Slupsky C M, Sykes B D
Department of Biochemistry, University of Alberta, Edmonton, Canada.
Biochemistry. 1995 Dec 12;34(49):15953-64. doi: 10.1021/bi00049a010.
Troponin C (TnC) is an 18 kDa (162-residue) thin-filament calcium-binding protein responsible for triggering muscle contraction upon the release of calcium from the sarcoplasmic reticulum. The structure of TnC with two calcium ions bound has previously been solved by X-ray methods. Shown here is the solution structure of TnC which has been solved using 3D and 4D heteronuclear nuclear magnetic resonance (NMR) spectroscopic techniques. The 1H, 13C, and 15N backbone chemical shifts have already been published [Slupsky, C. M., Reinach, F. C., Smillie, L. B., & Sykes, B. D. (1995) Protein Sci. 4, 1279-1290]. Presented herein are the 1H, 13C, and 15N side-chain chemical shifts which are 80% complete. The structure of calcium-saturated TnC was determined on the basis of 2106 NOE-derived distance restraints, 121 phi dihedral angle restraints, and 76 psi dihedral angle restraints. The appearance of calcium-saturated TnC reveals a dumbbell-shaped molecule with two globular domains connected by a linker. The structures of the N-terminal and C-terminal domains are highly converged [backbone atomic root mean square deviations (rmsd) about the mean atomic coordinate position for residues 10-80 and 98-155 are 0.66 +/- 0.17 and 0.69 +/- 0.18 A, respectively]; however, the orientation of one domain with respect to the other is not well-defined, and thus each domain appears to be structurally independent. Comparison of the calcium-saturated form of TnC determined herein with the half-saturated form determined by X-ray methods reveals two major differences. First, there is a major structural change which occurs in the N-terminal domain resulting in the opening of a hydrophobic pocket presumably to present itself to its target protein troponin I. This structural change appears to involve only helices B and C which move away from helices N/A/D by the alteration of the backbone phi, psi angles of glutamic acid 41 from irregular in the crystal structure (-97 degrees, -7 degrees) to helical in the NMR calcium-saturated structure (-60 degrees, -34 degrees). The other difference between the two structures is the presence of a flexible linker between the two domains in the NMR structure. This flexible linker allows the two domains of TnC to adopt any orientation with respect to one another such that they can interact with a variety of targets.
肌钙蛋白C（TnC）是一种18 kDa（162个残基）的细肌丝钙结合蛋白，负责在肌浆网释放钙时触发肌肉收缩。此前已通过X射线方法解析了结合两个钙离子的TnC的结构。此处展示的是利用三维和四维异核核磁共振（NMR）光谱技术解析得到的TnC的溶液结构。1H、13C和15N主链化学位移已发表 [斯卢普斯基，C.M.，雷纳赫，F.C.，斯米利，L.B.，& 赛克斯，B.D.（1995年）《蛋白质科学》4，1279 - 1290]。本文给出的是完成度达80%的1H、13C和15N侧链化学位移。基于2106个源自核Overhauser效应（NOE）的距离约束、121个φ二面角约束和76个ψ二面角约束，确定了钙饱和TnC的结构。钙饱和TnC呈现出哑铃状分子，由一个连接子连接两个球状结构域。N端和C端结构域的结构高度趋同 [残基10 - 80和98 - 155围绕平均原子坐标位置的主链原子均方根偏差（rmsd）分别为0.66±0.17 Å和0.69±0.18 Å]；然而，一个结构域相对于另一个结构域的取向并不明确，因此每个结构域在结构上似乎是独立的。将本文确定的钙饱和形式的TnC与通过X射线方法确定的半饱和形式进行比较，发现两个主要差异。首先，N端结构域发生了重大结构变化，导致一个疏水口袋打开，大概是为了与它的靶蛋白肌钙蛋白I相互作用。这种结构变化似乎仅涉及螺旋B和C，它们通过将谷氨酸41的主链φ、ψ角从晶体结构中的不规则值（-97°，-7°）改变为NMR钙饱和结构中的螺旋值（-60°，-34°），从而远离螺旋N/A/D。这两种结构的另一个差异是在NMR结构中两个结构域之间存在一个柔性连接子。这个柔性连接子使TnC的两个结构域能够相对于彼此采取任何取向，以便它们能够与多种靶标相互作用。
