Bergqvist Simon, Croy Carrie H, Kjaergaard Magnus, Huxford Tom, Ghosh Gourisankar, Komives Elizabeth A
Department of Chemistry and Biochemistry, University of California-San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0378, USA.
J Mol Biol. 2006 Jul 7;360(2):421-34. doi: 10.1016/j.jmb.2006.05.014. Epub 2006 May 19.
IkappaBalpha is an ankyrin repeat protein that inhibits NF-kappaB transcriptional activity by sequestering NF-kappaB outside of the nucleus in resting cells. We have characterized the binding thermodynamics and kinetics of the IkappaBalpha ankyrin repeat domain to NF-kappaB(p50/p65) using surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC). SPR data showed that the IkappaBalpha and NF-kappaB associate rapidly but dissociate very slowly, leading to an extremely stable complex with a K(D,obs) of approximately 40 pM at 37 degrees C. As reported previously, the amino-terminal DNA-binding domain of p65 contributes little to the overall binding affinity. Conversely, helix four of p65, which forms part of the nuclear localization sequence, was essential for high-affinity binding. This was surprising, given the small size of the binding interface formed by this part of p65. The NF-kappaB(p50/p65) heterodimer and p65 homodimer bound IkappaBalpha with almost indistinguishable thermodynamics, except that the NF-kappaB p65 homodimer was characterized by a more favorable DeltaH(obs) relative to the NF-kappaB(p50/p65) heterodimer. Both interactions were characterized by a large negative heat capacity change (DeltaC(P,obs)), approximately half of which was contributed by the p65 helix four that was necessary for tight binding. This could not be accounted for readily by the small loss of buried non-polar surface area and we hypothesize that the observed effect is due to additional folding of some regions of the complex.
IkappaBalpha是一种锚蛋白重复序列蛋白，在静息细胞中通过将NF-kappaB隔离在细胞核外而抑制其转录活性。我们使用表面等离子体共振（SPR）和等温滴定量热法（ITC）对IkappaBalpha锚蛋白重复结构域与NF-kappaB（p50/p65）的结合热力学和动力学进行了表征。SPR数据显示，IkappaBalpha与NF-kappaB迅速结合但解离非常缓慢，在37℃时形成了一个极其稳定的复合物，其表观解离常数（K(D,obs)）约为40 pM。如先前报道，p65的氨基末端DNA结合结构域对整体结合亲和力贡献不大。相反，构成核定位序列一部分的p65的螺旋4对于高亲和力结合至关重要。考虑到p65这部分形成的结合界面较小，这一点令人惊讶。NF-kappaB（p50/p65）异二聚体和p65同二聚体与IkappaBalpha结合时的热力学几乎无法区分，只是相对于NF-kappaB（p50/p65）异二聚体，NF-kappaB p65同二聚体的观察到的焓变（DeltaH(obs)）更有利。两种相互作用的特征都是较大的负热容变化（DeltaC(P,obs)），其中约一半是由紧密结合所必需的p65螺旋4贡献的。这不能通过掩埋的非极性表面积的少量损失轻易解释，我们推测观察到的效应是由于复合物某些区域的额外折叠所致。
