Ionization of His 55 at the Dimer Interface of Dynein Light-Chain LC8 Is Coupled to Dimer Dissociation

Abstract

LC8 is a highly conserved light-chain subunit of cytoplasmic dynein that interacts with a wide variety of cellular proteins and is presumed to play a fundamental role in dynein assembly and cargo recruitment and in the assembly of protein complexes unrelated to dynein. LC8 is a dimer at physiological pH but dissociates to a folded monomer at pH < 4.8. We have suggested that acid-induced dimer dissociation is due to protonation of His 55, which is stacked against His 55' and completely buried in the dimer interface. In this work, we show that the pH-induced dissociation is reversible and indeed governed by the ionization state of His 55. Mutagenesis of His 55 to Lys results in a monomer in the pH range of 3-8, while the mutation to Ala results in a dimer in the same pH range. Mutations that disrupt intermolecular hydrogen bonds between Tyr 65 and Lys 44' and His 55 and Thr 67' do not change the association state of the dimer. Titration curves for His 55 and the two other histidines, His 72 and 68, were determined by (13)C-(1)H NMR for H55K and for WT-LC8 in the monomeric and dimeric states. The pK(a) values of His 72 and His 68 are 6 in the WT dimer and 6.2-6.5 in monomeric H55K, while the pK(a) of His 55 is about 4.5 in the WT dimer. These results indicate that deprotonation of His 55 is linked to dimer formation and that mutation of His 55 to a small neutral residue or to a positively charged residue uncouples the protonation and dissociation processes.