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 Å 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 (kon ∼ 1 s-1 mM-1) in the monomer-dimer equilibrium of both wild-type LC8 and LC8S88E, possibly due to the strand-swapped architecture of the dimer. The Ser88Glu mutation raises the dimer dissociation constant (KD) through a combination of a higher koff and lower kon. Using a minimal model of titration linked to dimerization, we dissect the thermodynamics of dimerization of wild-type LC8 and LC8S88E in their various protonation states. When both Glu88 residues are protonated, the LC8S88E dimer is nearly as stable as the wild-type dimer, but deprotonation of one Glu88 residue raises KD by a factor of 400. We infer that phosphorylation of one subunit of wild-type LC8 raises KD 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.
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