Human Nek6 is a monomeric mostly globular kinase with an unfolded short N-terminal domain
© Meirelles et al; licensee BioMed Central Ltd. 2011
Received: 10 September 2010
Accepted: 14 February 2011
Published: 14 February 2011
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.
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).
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.
Mitotic progression and assembly of the bipolar mitotic spindle are regulated by several serine/threonine protein kinases, including members of the cyclin-dependent kinase (Cdk), Polo-like kinase (Plk), Aurora, and NIMA-related kinase (Nek) families [1–4]. 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. Mammals contain a large family of eleven Neks, the catalytic domain of which is evolutionarily related to that of NIMA . Nek2 has a central role in centrosome maturation and disjunction , whereas Nek1 and Nek8 have been proposed to contribute to ciliary function [6, 7]. Besides Nek2, Nek1, -6, -7 and -9 were also described to participate in centrosomal regulation [7–11]. Nek6, Nek7  and Nek9  are involved in the control of mitotic spindle formation, acting together in a mitotic kinase cascade, with Nek9 being upstream of Nek6 and Nek7 . Nek kinases are also described as related to pathologies, particularly cancer, presenting thereby interesting potential chemotherapeutic targets [14–20]. Recently, hNek6 was described to have its transcript, protein, and/or kinase activity levels highly elevated in a number of tumors and human cancer cell lines, indicating an important role for hNek6 in tumorigenesis [21–24].
Structurally, Neks in general are characterized by having a conserved N-terminal catalytic domain, followed by a nonconserved C-terminal regulatory domain that varies in size and structure. However, Nek6 and Nek7 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 . Although 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 .
The mechanisms of regulation of hNek6, -7, and -9 kinases are currently unknown, and elucidating this pathway would provide relevant knowledge on early mitotic events as well as new hints for drug design and cancer therapy. However, hNek2 and hNek7 are the only NIMA-related kinases for which structures have been reported [26–28]. In this context, we present here the first low resolution three-dimensional structure of hNek6 protein in solution. SAXS experiments, together with SEC-MALS and comparative molecular modeling revealed a monomeric mostly globular, though slightly elongated conformation for hNek6, with a flexible disordered N-terminal domain.
Results and Discussion
Human Nek6 is predicted to be phosphorylated at various sites and has an unfolded short N-terminal domain
Our consensus of secondary structure was scored by the number of times (one to five times) the predicted secondary structure element (α-helices, β-strands or coils) scored positive from five predictions using different databases: PredictProtein/Prof, PSIPRED, SSpro, SOPMA and GOR4. In summary, the secondary structure analysis suggested that hNek6 was composed of approximately 34% α-helices, 12% β-strands and 54% coils. Our hNek7 consensus of predicted secondary structure is 80% identical to the author-approved secondary structure in PDB (2WQM) (Figure 1B).
In the case of the disordered regions predictions, our consensus was obtained following the same criteria as for the secondary structure predictions, except that we used here nine different databases: FoldIndex, GlobPlot Russell/Linding, PONDR VL-XT, DISpro, IUPred, DisEMBL Hot-loops, DisEMBL Remark-465, DisEMBL Loops/coils, and VSL2B. From this analysis, we were able to identify a short high scored segment of disorder covering the majority of hNek6 N-terminal extension before its catalytic domain, which we are calling here the regulatory domain. This characteristic is also present in our hNek7 consensus of disorder predictions and in its crystal structure , where amino acids 1-19 are missing residues (due to a flexible region) and amino acids 20-23 are coils. Notably, we found that hNek6 unfolded short N-terminal region is important to mediate interactions with diverse partners  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.
Prediction of putative phosphorylation sites in human Nek6.
Putative Upstream Kinaseb
NetPhosK (0.56)/NetPhos (0.72)
NetPhosK (0.65)/NetPhos (0.72)
NetPhosK (0.56)/NetPhos (0.79)
NetPhosK (0.50)/NetPhos (0.79)
NetPhosK (0.53)/NetPhos (0.99)
NetPhosK (0.50)/NetPhos (0.99)
NetPhosK (0.59)/NetPhos (0.99)
NetPhosK (0.55)/NetPhos (0.72)
Secondary structure analysis
Comparative molecular modeling of human Nek6
The absence of a three-dimensional structure of hNek6 and the increasing interest in studying Nek proteins within the context of drug design strategies prompted us to construct a homology model for the aforementioned protein. Here, we are interested in the activation loop mutant hNek6(S206A). The activation loop is a centrally located loop, typically 20-30 residues in length, that provides a platform for the peptide substrate. Activation of most protein kinases usually requires phosphorylation of a residue in this loop, which leads to a rearrangement of the loop and increase in enzymatic activity . In hNek6, S206 is an important residue, which phosphorylation leads to an increase in the activation status of the kinase [13, 41]. The activation loop has the capacity to undergo large conformational changes when the kinase switches between inactive and active states, adopting distinct conformations in different kinases when they are inactive (unphosphorylated activation loop), a fact that has recently been exploited to great medical benefit  and which makes our hNek6 mutant an interesting target to be studied.
Our hNek6(S206A) model generated by SWISS-MODEL  shows a short region of α-helix composed of twelve residues (G192LGRFFSSETTA203) following the conserved DLG motif, with high score (Figure 3). A helical structure following the DFG/DLG motif is also present in hNek2(T175A) structures (PDB: 2JAV, 2W5H and 2W5B) [26, 27] and in other kinase families, such as inactive forms of both the EGFR kinase  and Src/Hck . Therefore, although the activation loop is missing in the electron density map of hNek7, a short helical structure is possibly present in hNek6(S206A), which was predicted in the model generated by SWISS-MODEL .
Human Nek6 is a monomeric mostly globular, though slightly elongated protein in solution, as revealed by SAXS
Analytical size-exclusion chromatography reveals variations in human Nek6 conformation dependent on its phosphorylation status
Recombinant hNek6 Stokes radii (Rs) determined by analytical size-exclusion chromatography.
14.9 ± 0.1
3.5 ± 0.2
15.8 ± 0.1
2.9 ± 0.0
16.9 ± 0.1
2.2 ± 0.0
18.2 ± 0.0
1.4 ± 0.1
16.4 ± 0.3
2.6 ± 0.1
17.1 ± 0.0
2.1 ± 0.0
16.7 ± 0.2
2.4 ± 0.0
17.1 ± 0.0
2.1 ± 0.0
17.6 ± 0.3
1.8 ± 0.1
Recombinant hNek6 weight average molecular masses (Mw) determined by SEC-MALS and melting temperatures (Tm) during thermal shift denaturation.
38.4 ± 0.6
39.5 ± 0.1
37.8 ± 2.2
40.8 ± 0.1
38.1 ± 2.1
38.0 ± 0.1
38.4 ± 3.3
36.8 ± 0.3
33.3 ± 1.6
41.0 ± 0.2
These results suggest that, although having the same molecular mass of ~38 kDa, wild-type hNek6 is purified from bacteria more phosphorylated than its mutant variant, mainly because of their different activation/autophosphorylation status, as described by Meirelles et al. , and these different phosphorylation degrees may cause changes in protein conformation and compactness, resulting in changes in their Stokes radii. This was better visualized for both proteins when dephosphorylated by lambda phosphatase, which promoted smaller radii and, possibly, more compact or less hydrated conformations. It seems that an increase in phosphorylation induces a structural change that increases the apparent size or shape of hNek6. In fact, in most kinases, the activation loop is phosphorylated when the kinase is active, which stabilizes it in an open and extended conformation that is permissive for substrate binding . This phosphorylated extended conformation may therefore contribute to the increase in hNek6 Stokes radius. All hNek6 variants were submitted to SEC-MALS twice, using two different buffers (the same one used for SAXS and another one containing 600 mM NaCl in order to avoid any unspecific binding to the gel filtration column resin), and the same Stokes radii for each protein were obtained in both measurements. Figure 6 shows the results from SEC-MALS using the buffer containing 600 mM NaCl, and Tables 2 and 3 show all the results obtained from both measurements.
Our data presents the first low resolution 3D structure of hNek6 protein in solution. SAXS experiments show that hNek6 is a monomer of a mostly globular, though slightly elongated shape, which was also confirmed by analytical SEC-MALS experiments. These also showed that hNek6 conformation is dependent on its activation/phosphorylation status, a higher phosphorylation degree corresponding to a bigger Stokes radius. Thermal denaturation shift assays revealed a slightly higher stability of wild-type hNek6 compared to the activation loop mutant hNek6(S206A).
In silico sequence analysis
Human Nek6 and Nek7 amino acid sequences were used as queries in five different secondary structure prediction databases: PredictProtein/Prof , PSIPRED , SSpro , SOPMA  and GOR4 . Comparison of their outputs resulted in a consensus of predicted secondary structure, where each amino acid was assigned a score ranging from 1 to 5. Our Nek7 consensus of predicted secondary structure was compared to the author-approved secondary structure in PDB (2WQM) as a measure to validate our analysis. We also performed disordered regions analysis for both protein sequences using nine different predictors: FoldIndex , GlobPlot Russell/Linding , PONDR VL-XT , DISpro , IUPred , DisEMBL Hot-loops, DisEMBL Remark-465, DisEMBL Loops/coils , and VSL2B . From this, a consensus of predicted disordered regions was generated with a consensus score ranging from 0 to 9, where a score above 4 represents disorder. Additionally, NetPhosK  and NetPhos  databases were used to predict phosphorylation sites for human Nek6 and Nek7. The conserved glycine-rich sequence, the HRD and DLG motifs, the conserved residues K74 (β3 strand) and E93 (αC helix), the putative nuclear export signal LGDLGL based on la Cour et al., 2004 , the putative WW domain binding motifs PY and pSP based on Ingham et al., 2005 , as long as the PPLP motif, experimentally described for hNek6 by Lee et al., 2007 , were also assigned to both protein sequences.
All plasmid constructions were developed accordingly to Meirelles et al., 2010 .
The hNek6 activation loop mutation S206A was introduced by PCR-based mutagenesis accordingly to Meirelles et al., 2010 .
Protein Expression and Purification
Soluble full-length hNek6 wild-type - 6xHis-hNek6wt - and mutant - 6xHis-hNek6(S206A) - or truncated hNek6 wild-type kinase domain - 6xHis-hNek6(Δ1-44) - fused to a 6xHis tag were expressed and purified accordingly to Meirelles et al., 2010 .
In order to obtain dephosphorylated wild-type and mutant hNek6, plasmids encoding either 6xHis-hNek6wt or 6xHis-hNek6(S206A) and λ phosphatase were transformed into E. coli BL21 (DE3/pRARE) cells that were induced and purified as described by Meirelles et al., 2010 . Lambda phosphatase cloned into pCDF-Duet (Novagen) was kindly provided by Dr. Richard Bayliss (Section of Structural Biology, Institute of Cancer Research, London, UK).
Circular dichroism (CD) spectra were recorded in a JASCO model J-810 CD spectropolarimeter equipped with Peltier-type system PFD 425S. Data were collected from 260 to 200 nm at 4°C using a quartz cuvette of 1 mm pathlength. Thirty-two spectra of purified 6xHis-hNek6wt at 5.8 μM, in 50 mM Phosphate buffer pH 7.5, 300 mM NaCl, were averaged and corrected from the baseline for buffer solvent contribution. Experimental data were analyzed using CDNN  and K2d  softwares.
Comparative/Homology Molecular Modeling
The comparative/homology molecular modeling and model validation were performed in a similar way to that described in Bodade et al., 2010 . Briefly, several comparative/homology modeling tools were used: I-TASSER [69–71], Geno3D , 3D-JIGSAW [73–75], SWISS-MODEL  and MODELLER 9v8 . The NCBI Basic Local Alignment Search Tool (BLAST, http://www.ncbi.nlm.nih.gov/BLAST/) was used to search the crystal structure of the closest homologue available in the Protein Data Bank (PDB, http://www.rcsb.org/pdb/). The input was the amino acid sequence of hNek6(S206A). The NCBI results revealed that the structure of hNek7, deposited under the code 2WQM in the PDB, was a very suitable template (identity score of 81% and E-value 3 × 10-141). This structure was used as a single template for the modeling approach. The overall stereochemical quality of the models was assessed by PROCHECK software . The quality of the models was also evaluated by PROSA [51, 52] and by the standard validation procedures included in the automated mode of the SWISS-MODEL server .
Small Angle X-Ray Scattering Analysis
The sample was first inspected by dynamic light scattering (DLS) to test for its monodispersity and then ultracentrifuged at 200.000 × g for 40 min at 4°C to remove any possible aggregates. The SAXS experiments were performed at the D02A-SAXS2 beam line at the LNLS, and data treatment and analyses were done following standard procedures similar to those described in Trindade et al., 2009 . Briefly, the measurements were performed at 4°C and the sample-to-detector distance was 902 mm, covering a scattering vector range of 0.015Å-1 < q <0.25 Å-1 (q is the magnitude of the q-vector defined by q = (4π/λ)sinθ and 2θ is the scattering angle) using a wavelength of λ = 1.488 Å. The measurements were performed using two different protein concentrations in HEPES buffer (50 mM HEPES pH 7.5, 5 mM sodium phosphate, 300 mM NaCl, 5% glycerol, 200 mM imidazole): 0.5 and 1.0 mg/mL. A 8 mg/ml BSA (66 kDa) solution in the same sample buffer was used as a standard sample to estimate the molecular mass of 6xHis-hNek6(S206A) making use of the ratio of the extrapolated values of the intensity at the origin, I(0) [78, 79]. The radius of gyration (Rg) was calculated from the Guinier approximation (valid for qRg < 1.3) [80–82] and also from the pair distance distribution function, p(r), which was obtained using the program GNOM . The maximum dimension (Dmax) of the molecule was obtained from the p(r) function. The Kratky plot (q2I(q) vs. q) [81, 82] was used to analyze the compactness of the protein conformation.
Low resolution SAXS-based modeling
The low resolution model of 6xHis-hNek6(S206A) was obtained from the SAXS data using a combination of ab initio calculation and rigid body modeling methods. Taking advantage of the homology model obtained, we used the program BUNCH  to model the protein. No symmetry restraints were used in the calculation. We would like to mention that no unique solution can be obtained from these calculations. For this reason, 10 independent calculations were run for each sample data. The multiple solutions were analyzed and the reliability and stability of the set of models were estimated. A pairwise comparison and the normalized spatial discrepancy (NSD) evaluation was performed using the DAMAVER program suite  complemented by the SUPCOMB  routine. Analyzing the NSD values (which describe the dissimilarity between pairs of models of the several calculations), the models with common features led to the selection of a representative, low resolution conformation for hNek6(S206A) protein. Models were displayed by the PyMOL program .
For comparison purposes, two other low resolution models were also obtained by using two different ab initio approaches: the dummy atoms method implemented in the program DAMMIN  and the dummy residues method implemented in GASBOR . The procedures were similar to those described in Trindade et al. .
We used Analytical S ize-E xclusion C hromatography coupled to M ulti-A ngle L ight S cattering (SEC-MALS) to estimate the hydrodynamic or Stokes radii (Rs) of recombinant hNek6wt, hNek6(S206A), hNek6(Δ1-44) and dephosphorylated hNek6wt and hNek6(S206A), all fused to a 6xHis tag. SEC was performed with an analytical Superdex 200 10/300 GL column using an ÄKTA FPLC system (GE Healthcare) equilibrated with two column volumes of 50 mM HEPES pH 7.5, 5 mM sodium phosphate, 600 mM NaCl, 5% glycerol, at a flow rate of 0.5 ml/min, at 20°C. Recombinant hNek6 variants at concentrations ranging from 0.2 to 0.7 mg/ml and a mixture of standard proteins with known Stokes radii (conalbumin: 3.64 nm, 3.2 mg/ml; ovalbumin: 3.05 nm, 4.2 mg/ml; carbonic anhydrase: 2.30 nm, 3.0 mg/ml; and ribonuclease: 1.64 nm, 3.4 mg/ml) (Sigma) were loaded onto the column and their elution profiles were monitored by absorbance at 280 nm. The Stokes radius of each hNek6 variant was estimated by a linear fit of the Stokes radii of the standard proteins versus the partition coefficient Kav[88, 89] as described by the equation: Kav = Ve - Vo/Vt - Vo , where Ve is the elution volume of the protein, Vo the void volume and Vt is the total volume of the column. The SEC was also coupled to a DAWN TREOS™ MALS instrument (Wyatt Technology). The on-line measurement of the intensity of the Rayleigh scattering as a function of the angle of the eluting peaks in SEC was used to determine the weight average molecular masses (Mw) of the eluted proteins , using the ASTRA™ (Wyatt Technologies) software. SEC-MALS measurements were performed using two different buffers: the first one described above for SEC and a second one also used in SAXS experiments (50 mM HEPES pH 7.5, 5 mM sodium phosphate, 300 mM NaCl, 5% glycerol, 200 mM imidazole). The chromatographic profile of the recombinant hNek6 variants were the same in both measurements and the mean and standard errors of their Mw and Mn were calculated.
Thermal Shift Assays
Thermal shift assays were performed based on a protocol devised by the Structural Genomics Consortium  using a real time PCR machine 7300 (Applied Biosystems). Proteins were buffered in 10 mM HEPES pH 7.5, 150 mM NaCl and assayed at a final concentration of 2.0 μM in 25 μL volume. SYPRO-Orange (Molecular Probes) was added as a fluorescence probe at a dilution of 1 in 1000. The emission filter for the SYPRO-Orange dye was set to 580 nm. Temperature was raised with a step of 1°C per 1.0 min from 10°C to 85°C and fluorescence readings were taken at each interval. OriginPro 8 software was used to fit data to the Boltzmann equation, y = LL+(UL-LL)/1+exp((Tm-x)/a), where LL and UL are the slopes of the native and denatured baselines, Tm is the apparent melting temperature and a describes the slope of the denaturation. Tm values were calculated by determination of the maximum of the first derivative.
Financially supported by: Fundação de Amparo à Pesquisa do Estado São Paulo, the Conselho Nacional de Pesquisa e Desenvolvimento and the LNLS. We thank Maria Eugenia R. Camargo for technical assistance, Rodrigo Martinez for the technical support at the SAXS2 beamline, and Dr. Richard Bayliss (Section of Structural Biology, Institute of Cancer Research, London, UK) for providing the pCDF-Duet lambda phosphatase construct.
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