Structural analysis of the regulation of the DYNLL/LC8 binding to Nek9 by phosphorylation

Abstract

The NIMA family protein kinases Nek9/Nercc1, Nek6, and Nek7 constitute a signaling module activated in early mitosis involved in the control of spindle organization. DYNLL/LC8 (dynein light chain 8) was originally described as a component of the dynein complex, but the recent discovery of multiple interaction partners for LC8 has suggested that it has a general role as a dimerization hub that organizes different protein partners. Recent experiments suggested that LC8 binding to Nek9 was regulated by Nek9 autophosphorylation on Ser(944), a residue immediately located N-terminal to the LC8 conserved (K/R)xTQT binding motif, and that this was crucial for the control of signal transduction through the Nek/Nek6/7 module. In the present work, we present two crystal structures of LC8 with a peptide corresponding to the Nek9 binding region with and without a phosphorylation on Ser(944). Structural analysis of LC8 with both Nek9 peptides, together with different biophysical experiments, explains the observed diminished binding affinity of Nek9 to LC8 upon phosphorylation on Ser(944) within the Nek9 sequence, thus shedding light into a novel phosphorylation regulatory mechanism that interferes with LC8 protein · protein complex formation.

FIGURE 1.

Circular dichroism analysis of LC8 binding to the Nek9 C-terminal region. a, schematic representation of the Nek9 protein. Amino acid sequence for the Nek9-CC construct is shown in one letter code. b, circular dichroism spectra of LC8 and Nek9-CC wild-type and single point mutants. c, circular dichroism spectra of the mixture of LC8 in complex with Nek9-CC and of the spectral sum of LC8 and Nek9-CC, for the Nek9-CC wild-type form and the Nek9-CC point mutants.

FIGURE 2.

Crystal structures of LC8 with Nek9 peptide and Nek9 P-peptide. a, schematic representation of the LC8·Nek9 peptide structure. α-Helices and β-strands of each LC8 subunit are labeled. The two Nek9 peptides binding the homodimer are depicted in red. b, surface representation of the LC8 homodimer (yellow and orange) in complex with the Nek9 peptide, which is labeled and shown in stick representation. c, surface representation of the LC8 homodimer (yellow and orange) in complex with the Nek9 P-peptide, which is labeled and shown in stick representation. d, detailed stick representation of the interaction between the N-terminal regions of the Nek9 peptide (gray) with LC8 (blue). e, detailed stick representation of the interaction between the N-terminal region of the Nek9 P-peptide (yellow) with LC8 (blue). The figure was prepared with PyMOL (43).

FIGURE 3.

Comparison of the interaction of LC8 with Nek9 peptide and Nek9 P-peptide. a, stereo representation of the superposition of the six Nek9 peptides from the asymmetric unit. Nek9 peptide and interacting LC8 residues are labeled and shown in stick representation. b, stereo representation of the superposition of the six Nek9 P-peptides from the asymmetric unit. Nek9 P-peptide and interacting LC8 residues are labeled and shown in stick representation. c, stereo representation of the ribbon superposition of the LC8·Nek9 peptide and LC8·Nek9 P-peptide structures. Both Nek9 peptides are labeled and shown in stick representation. The two LC8 subunits of the homodimer are labeled monomers 1 and 2.

FIGURE 4.

Circular dichroism analysis of LC8 binding to Nek9 peptide and Nek9 P-peptide. a, right: circular dichroism spectra of the titration of LC8 with an increasing concentration of the Nek9 peptide. Left: circular dichroism spectra of the titration of LC8 with an increasing concentration of the Nek9 P-peptide. b, plot of the variation of the ellipticity values from the CD spectra of a and b at 220 nm, which follows the change of β-secondary structure. Circles, Nek9 peptide; squares, Nek9 P-peptide.

FIGURE 5.

Thermal unfolding of LC8 followed by intrinsic fluorescence. Blue circles indicate the thermal unfolding kinetic of LC8 Red squares indicate the thermal unfolding kinetic of LC8 in the presence of 11 μm Nek9 peptide. Orange diamonds indicate the thermal unfolding kinetic of LC8 in the presence of 11 μm Nek9 P-peptide.

FIGURE 6.

Calorimetric titrations (isothermal titration calorimetry) for the LC8 binding to Nek9 peptides and Nek9 P-peptide. Shown are calorimetric thermograms (upper panels, thermal power versus time) and binding isotherms (lower panels, normalized heat versus molar ratio) for the titration of LC8 10 μm with 200 μm Nek9 peptide (a) and 300 μm Nek9 P-peptide (b), in 100 mm NaCl, 10 mm sodium phosphate, 7.5 and 1 mm β-mercaptoethanol, at 25 °C.