PDBsum entry 2w5h

Transferase

PDB id: 2w5h

Contents

Protein chain

265 a.a. *

Waters ×120

* Residue conservation analysis

PDB id:

2w5h

Name:

Transferase

Title:

Human nek2 kinase apo

Structure:

Serine/threonine-protein kinase nek2. Chain: a. Fragment: kinase domain, residues 1-271. Synonym: nima-related protein kinase 2, nima-like protein kinase 1, hspk 21, nek2. Engineered: yes. Mutation: yes

Source:

Homo sapiens. Human. Organism_taxid: 9606. Expressed in: escherichia coli. Expression_system_taxid: 469008.

Resolution:

2.33Å R-factor: 0.197 R-free: 0.254

Authors:

R.Bayliss

Key ref:

I.Westwood et al. (2009). Insights into the conformational variability and regulation of human Nek2 kinase. J Mol Biol, 386, 476-485. PubMed id: 19124027 DOI: 10.1016/j.jmb.2008.12.033

Date:

10-Dec-08 Release date: 23-Dec-08

Protein chain

P51955  (NEK2_HUMAN) -  Serine/threonine-protein kinase Nek2 from Homo sapiens

Seq: 445 a.a.

Struc: 265 a.a.*

* PDB and UniProt seqs differ at 9 residue positions (black crosses)

Enzyme reactions

• Enzyme class: E.C.2.7.11.1 - non-specific serine/threonine protein kinase.
• Reaction:
  1. L-seryl-[protein] + ATP = O-phospho-L-seryl-[protein] + ADP + H⁺
  2. L-threonyl-[protein] + ATP = O-phospho-L-threonyl-[protein] + ADP + H⁺

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

reference

DOI no: 10.1016/j.jmb.2008.12.033

J Mol Biol 386:476-485 (2009)

PubMed id: 19124027

Insights into the conformational variability and regulation of human Nek2 kinase.

I.Westwood, D.M.Cheary, J.E.Baxter, M.W.Richards, R.L.van Montfort, A.M.Fry, R.Bayliss.

ABSTRACT

The Nek family of serine/threonine kinases regulates centrosome and cilia function; in addition, several of its members are potential targets for drug discovery. Nek2 is dimeric, is cell cycle regulated and functions in the separation of centrosomes at G2/M. Here, we report the crystal structures of wild-type human Nek2 kinase domain bound to ADP at 1.55-A resolution and T175A mutant in apo form as well as that bound to a non-hydrolyzable ATP analog. These show that regions of the Nek2 structure around the nucleotide-binding site can adopt several different but well-defined conformations. None of the conformations was the same as that observed for the previously reported inhibitor-bound structure, and the two nucleotides stabilized two conformations. The structures suggest mechanisms for the auto-inhibition of Nek2 that we have tested by mutagenesis. Comparison of the structures with Aurora-A and Cdk2 gives insight into the structural mechanism of Nek2 activation. The production of specific inhibitors that target individual kinases of the human genome is an urgent challenge in drug discovery, and Nek2 is especially promising as a cancer target. We not only identify potential challenges to the task of producing Nek2 inhibitors but also propose that the conformational variability provides an opportunity for the design of Nek2 selective inhibitors because one of the conformations may provide a unique target.

Selected figure(s)

Figure 1.
Fig. 1. Electron density maps at the DFG and HRD motifs of Nek2. (a–c) 2mF[o] − DF[c] (gray) and mF[o] − DF[c] (green, red) SigmaA-weighted electron density maps contoured at 1.0σ, 2.5σ and − 2.5σ around (a) Nek2-T175A^ATPγS, (b) Nek2^ADP and (c) Nek2-T175A^Apo, respectively. Carbon atoms are shown in yellow, orange and pale pink in (a), (b) and (c), respectively. Oxygen atoms are shown in red, whereas nitrogen atoms are shown in blue. The same color scheme for stick representation is used in subsequent figures.

Figure 2.
Fig. 2. The DFG motif and activation loop adopt different conformations dependent on the bound ligand. (a) Superposition of Nek2-T175A^ATPγS (yellow), Nek2^ADP (orange) and Nek2-T175A^SU (green) protein structures shown as a ribbon in two orientations related by a 90° rotation about the y-axis. (b) Stereoview of Nek2-T175A^ATPγS (yellow carbon atoms) and Nek2^ADP (orange carbon atoms) superposition at the DFG motif. (c) Stereoview of Nek2-T175A^ATPγS (yellow carbon atoms) and Nek2-T175A^SU (green carbon atoms) superposition at the DFG motif. (d) Stereoview of Nek2-T175A^ATPγS (yellow carbon atoms) and Nek2-T175A^Apo (light pink carbon atoms) superposition at the DFG motif. (e) Schematic of the secondary structures adopted by the four Nek2 structures and the amino acid sequence surrounding the DFG motif in Nek2 and Aurora-A. (f) Superposition of three Nek2 conformations of the DFG motif together with the likely position adopted in the fully active conformation based on the Aurora-A/TPX2 structure (magenta). The orientation is that of panels (b) to (d) viewed from the bottom left to the top right.

The above figures are reprinted from an Open Access publication published by Elsevier: J Mol Biol (2009, 386, 476-485) copyright 2009.

Figures were selected by the author.

Author's comment

Nek2 is a cell-cycle regulated, serine/threonine protein kinase that regulates the separation of centrosomes in G2/M. We have crystallised the catalytic domain on Nek2 in an apo-form suitable for soaking of ligands, and determined the structure of apo-, ADP-bound and ATP-analogue bound forms. The DFG-motif of Nek2 is able to adopt multiple, ordered conformations dependent on the ligand bound. This presents a challenge to the development of Nek2-selective inhibitors.