Lee Sunbae, Arunkumar Alphonse I, Chen Xiaohua, Giedroc David P
Department of Biochemistry and Biophysics, Texas A&M University, College Station, TX 77843-2128, USA.
J Am Chem Soc. 2006 Feb 15;128(6):1937-47. doi: 10.1021/ja0546828.
The Zn(II)/Co(II)-sensing transcriptional repressor, Staphylococcus aureus CzrA, is a homodimer containing a symmetry-related pair of subunit-bridging tetrahedral N(3)O metal sensor coordination sites. A metal-induced quaternary structural change within the homodimer is thought to govern the biological activity of this and other metal sensor proteins. Here, we exploit covalent (Gly(4)Ser)(n)() linkers of variable length in "fused" CzrAs, where n = 1 (designated 5L-fCzrA), 2 (10L-fCzrA), or 3 (15L-fCzrA), as molecular rulers designed to restrict any quaternary structural changes that are associated with metal binding and metal-mediated allosteric regulation of DNA binding to varying degrees. While 15L-fCzrA exhibits properties most like homodimeric CzrA, shortening the linker in 10L-fCzrA abolishes negative homotropic cooperativity of Zn(II) binding and reduces DNA binding affinity of the apoprotein significantly. Decreasing the linker length further in 5L-fCzrA effectively destroys one metal site altogether and further reduces DNA binding affinity. However, Zn(II) negatively regulates DNA binding of all fCzrAs, with allosteric coupling free energies (DeltaG(1)(c)) of 4.6, 3.1, and 2.7 kcal mol(-1) for 15L-, 10L-, and 5L-fCzrAs, respectively. Introduction of a single nonliganding H97N substitution into either the N-terminal or C-terminal protomer domain in 10L-fCzrA results in DeltaG(1)(c) = 2.6 kcal mol(-1) or approximately 83% that of 10L-fCzrA; in contrast, homodimeric H97N CzrA gives DeltaG(1)(c) = 0. (1)H-(15)N HSQC spectra acquired for wt-, 10L-fCzrA and H97N 10L-fCzrA in various Zn(II) ligation states reveal that the allosteric change of the protomer domains within the fused dimer is independent and not concerted. Thus, occupancy of a single metal site by Zn(II) introduces asymmetry into the CzrA homodimer that leads to significant allosteric regulation of DNA binding.
锌（II）/钴（II）感应转录阻遏物金黄色葡萄球菌CzrA是一种同型二聚体，含有一对与对称相关的亚基桥连四面体N(3)O金属传感器配位位点。同型二聚体内金属诱导的四级结构变化被认为控制着这种及其他金属传感器蛋白的生物活性。在此，我们在“融合”的CzrA中利用可变长度的共价（Gly(4)Ser）(n)连接子，其中n = 1（命名为5L-fCzrA）、2（10L-fCzrA）或3（15L-fCzrA），作为分子标尺，旨在不同程度地限制与金属结合及金属介导的DNA结合变构调节相关的任何四级结构变化。虽然15L-fCzrA表现出最类似于同型二聚体CzrA的性质，但缩短10L-fCzrA中的连接子会消除锌（II）结合的负协同性，并显著降低脱辅基蛋白的DNA结合亲和力。在5L-fCzrA中进一步缩短连接子长度会完全破坏一个金属位点，并进一步降低DNA结合亲和力。然而，锌（II）对所有fCzrA的DNA结合均有负调节作用，15L-fCzrA、10L-fCzrA和5L-fCzrA的变构偶联自由能（ΔG(1)(c)）分别为4.6、3.1和2.7 kcal mol(-1)。在10L-fCzrA的N端或C端原体结构域中引入单个非配位H97N取代，导致ΔG(1)(c) = 2.6 kcal mol(-1)，约为10L-fCzrA的83%；相比之下，同型二聚体H97N CzrA的ΔG(1)(c) = 0。在各种锌（II）配位状态下为野生型、10L-fCzrA和H97N 10L-fCzrA采集的（1）H-（15）N HSQC谱表明，融合二聚体内原体结构域的变构变化是独立的，而非协同的。因此，锌（II）占据单个金属位点会给CzrA同型二聚体引入不对称性，从而导致对DNA结合的显著变构调节。
