Affinity, Avidity, and Kinetics of Target Sequence Binding to LC8 Dynein Light Chain Isoforms

Abstract

LC8 dynein light chain (DYNLL) is a highly conserved eukaryotic hub protein with dozens of binding partners and various functions beyond being a subunit of dynein and myosin Va motor proteins. Here, we compared the kinetic and thermodynamic parameters of binding of both mammalian isoforms, DYNLL1 and DYNLL2, to two putative consensus binding motifs (KXTQTX and XG(I/V)QVD) and report only subtle differences. Peptides containing either of the above motifs bind to DYNLL2 with micromolar affinity, whereas a myosin Va peptide (lacking the conserved Gln) and the noncanonical Pak1 peptide bind with K d values of 9 and 40 M, respectively. Binding of the KXTQTX motif is enthalpy-driven, although that of all other peptides is both enthalpy-and entropy-driven. Moreover, the KXTQTX motif shows strikingly slower off-rate constant than the other motifs. As most DYNLL partners are homodimeric, we also assessed the binding of bivalent ligands to DYNLL2. Compared with monovalent ligands, a significant avidity effect was found as follows: K d values of 37 and 3.5 nM for a dimeric myosin Va fragment and a Leu zipper dimerized KXTQTX motif, respectively. Ligand binding kinetics of DYNLL can best be described by a conformational selection model consisting of a slow isomerization and a rapid binding step. We also studied the binding of the phosphomimetic S88E mutant of DYNLL2 to the dimeric myosin Va fragment, and we found a significantly lower apparent K d value (3 M). We conclude that the thermodynamic and kinetic fine-tuning of binding of various ligands to DYNLL could have physiological relevance in its interaction network.

Structural Analysis of the Regulation of the DYNLL/LC8 Binding to Nek9 by Phosphorylation

“…2 and 3), establishing several backbone hydrogen bonds and side chain interactions with the ␤3 strand of one of the LC8 subunit. The only interaction of the peptide with the opposite subunit of the LC8 homodimer is mainly established by Gln 947 from the highly conserved KXTQTX motif, with its side chain buried in a hydrophobic pocket formed by Ile 34 , Glu 35 , and Lys 36 from the ␣2 helix.…”

“…Only a few LC8 partners contain non-canonical binding motifs lacking the most conserved Gln residue (25). The binding affinity of LC8 to monomeric peptides is moderately weak (K d between 0.1 and 40 M) (13); however, bivalent peptides linking two consensus motifs have been shown to increase significantly the binding affinity for LC8 (34), indicating the importance of the dimer-dimer interaction in the complex of LC8 with protein partners. Despite the strong conservation in the LC8 binding region, recent biophysical data indicates an inherent plasticity that allows the interaction with these different consensus motifs (24).…”

DYNLL2 Dynein Light Chain Binds to an Extended Linear Motif of Myosin 5a Tail That Has Structural Plasticity

“…The IC84-143 segment contains the highly conserved "TQT box" and binding to LC8 caused broadening below detection limit of the peaks belonging to the residues involved in binding; a similar broadening effect that was observed for M L , while the peaks of the residues outside the binding sequence remained unperturbed and maintained the disorder state outside the binding region. On the contrary here we show not only the partial folding of the unbound M L , but also the structural transition into a β-sheet upon interaction with DYNLL2, an example of structural plasticity in a short linear motif (Figure 7).Previously we have shown by kinetic studies that the binding of DYNLL isoforms to their various partners can be best described by a conformational selection model (18). The region of the free myo5a peptide contains an inherent α-helix that should be in the unfolded state in order to fit into the binding groove of DYNLL2; this finding is consistent with a conformational selection mechanism for the formation of DYNLL complexes.…”

“…Binding of DYNLL2 stabilizes the flanking coiled-coil sequences (16,17). Detailed kinetic and thermodynamic characterization of this interaction using monoand dimeric myo5a fragments revealed that the dissociation constant is somewhat weaker than that of the canonical motifs(~1-5 M for a monomeric peptide) andbivalency of the ligand leads to pronounced avidity (18).…”

“…nNOS (21,22), Pak1 (23)(24)(25), Bmf (26), Bim (27), Bassoon (28), GKAP (29), and viral proteins (30,31)), it was hypothesized that DYNLL may provide direct physical linkage between the motor and the cargos (26,28,29). However, as the majority of the binding partners (including the motors) are dimeric, and therefore they are bivalent ligands of LC8 and the formation of highly stable dimer-to-dimer complexes is preferred (18). Therefore, the validity of the original cargo adaptor hypothesis is questioned.…”