Dataset Information

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.

SUBMITTER: Gallego P

PROVIDER: S-EPMC3636912 | biostudies-literature | 2013 Apr

REPOSITORIES: biostudies-literature

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

Gallego Pablo P Velazquez-Campoy Adrian A Regué Laura L Roig Joan J Reverter David D

The Journal of biological chemistry 20130312 17

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 regulate ...[more]

PMID: 23482567

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Project description:Multistep protein-protein interactions underlie most biological processes, but their characterization through methods such as isothermal titration calorimetry (ITC) is largely confined to simple models that provide little information on the intermediate, individual steps. In this study, we primarily examine the essential hub protein LC8, a small dimer that binds disordered regions of 100+ client proteins in two symmetrical grooves at the dimer interface. Mechanistic details of LC8 binding have remained elusive, hampered in part by ITC data analyses employing simple models that treat bivalent binding as a single event with a single binding affinity. We build on existing Bayesian ITC approaches to quantify thermodynamic parameters for multi-site binding interactions impacted by significant uncertainty in protein concentration. Using a two-site binding model, we identify positive cooperativity with high confidence for LC8 binding to multiple client peptides. In contrast, application of an identical model to the two-site binding between the coiled-coil NudE dimer and the intermediate chain of dynein reveals little evidence of cooperativity. We propose that cooperativity in the LC8 system drives the formation of saturated induced-dimer structures, the functional units of most LC8 complexes. In addition to these system-specific findings, our work advances general ITC analysis in two ways. First, we describe a previously unrecognized mathematical ambiguity in concentrations in standard binding models and clarify how it impacts the precision with which binding parameters are determinable in cases of high uncertainty in analyte concentrations. Second, building on observations in the LC8 system, we develop a system-agnostic heat map of practical parameter identifiability calculated from synthetic data which demonstrates that the ability to determine microscopic binding parameters is strongly dependent on both the parameters themselves and experimental conditions. The work serves as a foundation for determination of multi-step binding interactions, and we outline best practices for Bayesian analysis of ITC experiments.

Dataset Information

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

Publications

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

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