Department of Chemistry, Indiana University, Bloomington, Indiana 47405-7102, USA.
J Am Chem Soc. 2009 Dec 16;131(49):17860-70. doi: 10.1021/ja906131b.
The linked equilibria of an allosterically regulated protein are defined by the structures, residue-specific dynamics and global energetics of interconversion among all relevant allosteric states. Here, we use isothermal titration calorimetry (ITC) to probe the global thermodynamics of allosteric negative regulation of the binding of the paradigm ArsR-family zinc sensing repressor Staphylococcus aureus CzrA to the czr DNA operator (CzrO) by Zn(2+). Zn(2+) binds to the two identical binding sites on the free CzrA homodimer in two discernible steps. A larger entropic driving force Delta(-TDeltaS) of -4.7 kcal mol(-1) and a more negative DeltaC(p) characterize the binding of the first Zn(2+) relative to the second. These features suggest a modest structural transition in forming the Zn(1) state followed by a quenching of the internal dynamics on filling the second zinc site, which collectively drive homotropic negative cooperativity of Zn(2+) binding (Delta(DeltaG) = 1.8 kcal mol(-1)). Negative homotropic cooperativity also characterizes Zn(2+) binding to the CzrACzrO complex (Delta(DeltaG) = 1.3 kcal mol(-1)), although the underlying energetics are vastly different, with homotropic Delta(DeltaH) and Delta(-TDeltaS) values both small and slightly positive. In short, Zn(2+) binding to the complex fails to induce a large structural or dynamical change in the CzrA bound to the operator. The strong heterotropic negative linkage in this system (DeltaG(c)(t) = 6.3 kcal mol(-1)) therefore derives from the vastly different structures of the apo-CzrA and CzrACzrO reference states (DeltaH(c)(t) = 9.4 kcal mol(-1)) in a way that is reinforced by a global rigidification of the allosterically inhibited Zn(2) state off the DNA (TDeltaS(c)(t) = -3.1 kcal mol(-1), i.e., DeltaS(c)(t) > 0). The implications of these findings for other metalloregulatory proteins are discussed.
变构调节蛋白的连接平衡由所有相关变构状态之间转换的结构、残基特异性动力学和整体能量学定义。在这里,我们使用等温滴定量热法(ITC)来探测变构负调节金黄色葡萄球菌 CzrA 与 czr DNA 操纵子(CzrO)结合的整体热力学,Zn(2+)。Zn(2+)以两个可区分的步骤结合到游离 CzrA 同二聚体的两个相同结合位点上。更大的熵驱动力 -Delta(-TDeltaS)为-4.7 kcal mol(-1)和更负的 DeltaC(p) 特征是第一个 Zn(2+)相对于第二个的结合。这些特征表明在形成 Zn(1)状态时存在适度的结构转变,然后在填充第二个锌位点时内部动力学被猝灭,这共同驱动 Zn(2+)结合的同型负协同性(Delta(DeltaG) = 1.8 kcal mol(-1))。Zn(2+)与 CzrACzrO 复合物的结合也表现出负同型协同性(Delta(DeltaG) = 1.3 kcal mol(-1)),尽管基础能量学大不相同,同型 Delta(DeltaH)和 Delta(-TDeltaS)值均较小且略微为正。简而言之,Zn(2+)与结合到算子上的 CzrA 结合不会诱导结构或动力学的较大变化。因此,在这个系统中,强的异型负连接(DeltaG(c)(t) = 6.3 kcal mol(-1))源于 apo-CzrA 和 CzrACzrO 参考状态的结构差异很大(DeltaH(c)(t) = 9.4 kcal mol(-1)),以一种方式得到加强,即变构抑制的 Zn(2)状态远离 DNA 的整体僵化(TDeltaS(c)(t) = -3.1 kcal mol(-1),即 DeltaS(c)(t) > 0)。这些发现对其他金属调节蛋白的意义进行了讨论。