Abstract
BackgroundThe NIMA-related kinases (Neks) are widespread among eukaryotes. In mammalians they represent an evolutionarily conserved family of 11 serine/threonine kinases, with 40-45% amino acid sequence identity to the Aspergillus nidulans mitotic regulator NIMA within their catalytic domains. Neks have cell cycle-related functions and were recently described as related to pathologies, particularly cancer, consisting in potential chemotherapeutic targets. Human Nek6, -7 and -9 are involved in the control of mitotic spindle formation, acting together in a mitotic kinase cascade, but their mechanism of regulation remain elusive.ResultsIn this study we performed a biophysical and structural characterization of human Nek6 with the aim of obtaining its low resolution and homology models. SAXS experiments showed that hNek6 is a monomer of a mostly globular, though slightly elongated shape. Comparative molecular modeling together with disorder prediction analysis also revealed a flexible disordered N-terminal domain for hNek6, which we found to be important to mediate interactions with diverse partners. SEC-MALS experiments showed that hNek6 conformation is dependent on its activation/phosphorylation status, a higher phosphorylation degree corresponding to a bigger Stokes radius. Circular dichroism spectroscopy confirmed our in silico predictions of secondary structure content and thermal stability shift assays revealed a slightly higher stability of wild-type hNek6 compared to the activation loop mutant hNek6(S206A).ConclusionsOur data present the first low resolution 3D structure of hNek6 protein in solution. SAXS, comparative modeling and SEC-MALS analysis revealed that hNek6 is a monomeric kinase of slightly elongated shape and a short unfolded N-terminal domain.
Highlights
The NIMA-related kinases (Neks) are widespread among eukaryotes
Human Nek6 is predicted to be phosphorylated at various sites and has an unfolded short N-terminal domain Human Nek6 amino acid sequence was analyzed considering its secondary structure, disordered regions, conserved motifs and putative phosphorylation sites by upstream kinases, resulting in a linear representation of its main structure predictions (Figure 1A)
We found that hNek6 unfolded short N-terminal region is important to mediate interactions with diverse partners [41] and, since hNek6 and hNek7 are similar in their catalytic domain sequences (~86% identity), but different in their N-terminal extensions (~20% identity) (Figure 1C), it is possible that both proteins depend on their disordered N-terminal domain to regulate the interactions with specific/different partners
Summary
The NIMA-related kinases (Neks) are widespread among eukaryotes In mammalians they represent an evolutionarily conserved family of 11 serine/threonine kinases, with 40-45% amino acid sequence identity to the Aspergillus nidulans mitotic regulator NIMA within their catalytic domains. The founding member of Nek family, the NIMA kinase of Aspergillus nidulans, contributes to multiple aspects of mitotic progression including the timing of mitotic entry, chromatin condensation, spindle organization and cytokinesis. Nek and Nek are significant exceptions to this, in that they are the smallest of the kinases and consist only of a catalytic domain with a relatively short N-terminal extension [4] They share significant similarity with each other, being ~86% identical within their catalytic domains, the N-terminal extensions are not conserved, and it has been suggested that they may play a role in differential regulation of the kinases [25]
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