Payne John C, Rous Brian W, Tenderholt Adam L, Godwin Hilary Arnold
Department of Chemistry, Northwestern University, 2145 Sheridan Road, Evanston, Illinois 60208-3113, USA.
Biochemistry. 2003 Dec 9;42(48):14214-24. doi: 10.1021/bi035002l.
Zinc binding to the two Cys(4) sites present in the DNA-binding domain (DBD) of nuclear hormone receptor proteins is required for proper folding of the domain and for protein activity. By utilizing Co(2+) as a spectroscopic probe, we have characterized the metal-binding properties of the two Cys(4) structural zinc-binding sites found in the DBD of human estrogen receptor alpha (hERalpha-DBD) and rat glucocorticoid receptor (GR-DBD). The binding affinity of Co(2+) to the two proteins was determined relative to the binding affinity of Co(2+) to the zinc finger consensus peptide, CP-1. Using the known dissociation constant of Co(2+) from CP-1, the dissociation constants of cobalt from hERalpha-DBD were calculated: K(d1)(Co) = 2.2 (+/- 1.0) x 10(-7) M and K(d2)(Co) = 6.1 (+/- 1.5) x 10(-7) M. Similarly, the dissociation constants of Co(2+) from GR-DBD were calculated: K(d1)(Co) = 4.1 (+/- 0.6) x 10(-7) M and K(d2)(Co) = 1.7 (+/- 0.3) x 10(-7) M. Metal-binding studies conducted in which Zn(2+) displaces Co(2+) from the metal-binding sites of hERalpha-DBD and GR-DBD indicate that Zn(2+) binds to each of the Cys(4) metal-binding sites approximately 3 orders of magnitude more tightly than Co(2+) does: the stoichiometric dissociation constants are K(d1)(Zn) = 1 (+/- 1) x 10(-10) M and K(d2)(Zn) = 5 (+/- 1) x 10(-10) M for hERalpha-DBD and K(d1)(Zn) = 2 (+/- 1) x 10(-10) M and K(d2)(Zn) = 3 (+/- 1) x 10(-10) M for GR-DBD. These affinities are comparable to those observed for most other naturally occurring structural zinc-binding sites. In contrast to the recent prediction by Low et. al. that zinc binding in these systems should be cooperative [Low, L. Y., Hernández, H., Robinson, C. V., O'Brien, R., Grossmann, J. G., Ladbury, J. E., and Luisi, B. (2002) J. Mol. Biol. 319, 87-106], these data suggest that the zincs that bind to the two sites in the DBDs of hERalpha-DBD and GR-DBD do not interact.
锌与核激素受体蛋白DNA结合结构域（DBD）中存在的两个Cys(4)位点结合，是该结构域正确折叠和蛋白质活性所必需的。通过使用Co(2+)作为光谱探针，我们表征了在人雌激素受体α（hERα-DBD）和大鼠糖皮质激素受体（GR-DBD）的DBD中发现的两个Cys(4)结构锌结合位点的金属结合特性。相对于Co(2+)与锌指共有肽CP-1的结合亲和力，测定了Co(2+)与这两种蛋白质的结合亲和力。利用已知的Co(2+)从CP-1的解离常数，计算了钴从hERα-DBD的解离常数：K(d1)(Co) = 2.2（±1.0）×10(-7) M和K(d2)(Co) = 6.1（±1.5）×10(-7) M。同样，计算了Co(2+)从GR-DBD的解离常数：K(d1)(Co) = 4.1（±0.6）×10(-7) M和K(d2)(Co) = 1.7（±0.3）×10(-7) M。在hERα-DBD和GR-DBD的金属结合位点上进行的Zn(2+)取代Co(2+)的金属结合研究表明，Zn(2+)与每个Cys(4)金属结合位点的结合比Co(2+)紧密约3个数量级：hERα-DBD的化学计量解离常数为K(d1)(Zn) = 1（±1）×10(-10) M和K(d2)(Zn) = 5（±1）×10(-10) M，GR-DBD的为K(d1)(Zn) = 2（±1）×10(-10) M和K(d2)(Zn) = 3（±1）×10(-10) M。这些亲和力与大多数其他天然存在的结构锌结合位点所观察到的亲和力相当。与Low等人最近的预测相反，即这些系统中的锌结合应该是协同的[Low, L. Y., Hernández, H., Robinson, C. V., O'Brien, R., Grossmann, J. G., Ladbury, J. E., and Luisi, B. (2002) J. Mol. Biol. 319, 87 - 106]，这些数据表明，与hERα-DBD和GR-DBD的DBD中两个位点结合的锌不相互作用。
