title: Human Nek6 is a monomeric mostly globular kinase with an unfolded short N-terminal domain.
abstract: BACKGROUND
  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. 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.
  
  
  RESULTS
  In 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).
  
  
  CONCLUSIONS
  Our 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.
authors.affiliation: Laboratório Nacional de Biociências, Centro Nacional de Pesquisa em Energia e Materiais, Campinas, SP, Brazil.
authors.forename: Gabriela V
authors.initials: GV
authors.lastname: Meirelles
authors.forename: Júlio C
authors.initials: JC
authors.lastname: Silva
authors.forename: Yuri de A
authors.initials: Yde A
authors.lastname: Mendonça
authors.forename: Carlos Hi
authors.initials: CH
authors.lastname: Ramos
authors.forename: Iris L
authors.initials: IL
authors.lastname: Torriani
authors.forename: Jörg
authors.initials: J
authors.lastname: Kobarg
mesh_terms: D000595:Amino Acid Sequence; D002850:Chromatography, Gel; D006801:Humans; D008027:Light; D008958:Models, Molecular; D008969:Molecular Sequence Data; D000072379:NIMA-Related Kinases; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry* / Q000378:metabolism; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D058767:Protein Unfolding; D053838:Scattering, Small Angle; D012996:Solutions; D014961:X-Ray Diffraction
chemical_list: D012996:Solutions; C459456:NEK6 protein, human; D000072379:NIMA-Related Kinases; D017346:Protein Serine-Threonine Kinases