Benison Gregory, Chiodo Marcus, Karplus P Andrew, Barbar Elisar
Department of Biochemistry and Biophysics, Oregon State University, Corvallis, Oregon 97331, USA.
Biochemistry. 2009 Dec 8;48(48):11381-9. doi: 10.1021/bi901589w.
Dynein light chain LC8 is a small, dimeric, very highly conserved globular protein first identified as an integral part of the dynein and myosin molecular motors but now recognized as a dimerization hub with wider significance. Phosphorylation at Ser88 is thought to be involved in regulating LC8 in the apoptotic pathway. The phosphomimetic Ser88Glu mutation weakens dimerization of LC8 and thus its overall ligand-binding affinity, because only the dimer binds ligands. The 1.9 A resolution crystal structure of dimeric LC8(S88E) bound to a fragment of the ligand Swallow (Swa) presented here shows that the tertiary structure is identical to that of wild-type LC8/Swa, with Glu88 well accommodated sterically at the dimer interface. NMR longitudinal magnetization exchange spectroscopy reveals remarkably slow association kinetics (k(on) approximately 1 s(-1) mM(-1)) in the monomer-dimer equilibrium of both wild-type LC8 and LC8(S88E), possibly due to the strand-swapped architecture of the dimer. The Ser88Glu mutation raises the dimer dissociation constant (K(D)) through a combination of a higher k(off) and lower k(on). Using a minimal model of titration linked to dimerization, we dissect the thermodynamics of dimerization of wild-type LC8 and LC8(S88E) in their various protonation states. When both Glu88 residues are protonated, the LC8(S88E) dimer is nearly as stable as the wild-type dimer, but deprotonation of one Glu88 residue raises K(D) by a factor of 400. We infer that phosphorylation of one subunit of wild-type LC8 raises K(D) by at least as much to prevent dimerization of LC8 at physiological concentrations. Some LC8 binding partners may bind tightly enough to promote dimerization even when one subunit is phosphorylated; thus linkage between phosphorylation and dimerization provides a mechanism for differential regulation of binding of LC8 to its diverse partners.
动力蛋白轻链LC8是一种小的、二聚体的、高度保守的球状蛋白，最初被鉴定为动力蛋白和肌球蛋白分子马达的一个组成部分，但现在被认为是一个具有更广泛意义的二聚化中心。Ser88位点的磷酸化被认为参与了凋亡途径中对LC8的调节。模拟磷酸化的Ser88Glu突变削弱了LC8的二聚化，从而降低了其整体配体结合亲和力，因为只有二聚体才能结合配体。本文展示的与配体Swallow（Swa）片段结合的二聚体LC8（S88E）的1.9 Å分辨率晶体结构表明，其三级结构与野生型LC8/Swa相同，Glu88在二聚体界面处空间容纳良好。核磁共振纵向磁化交换光谱显示，野生型LC8和LC8（S88E）在单体 - 二聚体平衡中的缔合动力学非常缓慢（k(on)约为1 s(-1) mM(-1)），这可能是由于二聚体的链交换结构所致。Ser88Glu突变通过更高的k(off)和更低的k(on)组合提高了二聚体解离常数（K(D)）。使用与二聚化相关的滴定最小模型，我们剖析了野生型LC8和LC8（S88E）在其各种质子化状态下二聚化的热力学。当两个Glu88残基都被质子化时，LC8（S88E）二聚体几乎与野生型二聚体一样稳定，但一个Glu88残基的去质子化使K(D)增加了400倍。我们推断野生型LC8一个亚基的磷酸化使K(D)至少增加这么多，以防止LC8在生理浓度下二聚化。一些LC8结合伙伴可能结合得足够紧密，即使一个亚基被磷酸化也能促进二聚化；因此，磷酸化和二聚化之间的联系为差异调节LC8与其不同伙伴的结合提供了一种机制。
