PDBsum entry 2v5q

Transferase

PDB id: 2v5q

Contents

Protein chains

285 a.a. *

130 a.a. *

Waters ×406

* Residue conservation analysis

PDB id:

2v5q

Name:

Transferase

Title:

Crystal structure of wild-type plk-1 kinase domain in complex with a selective darpin

Structure:

Serine/threonine-protein kinase plk1. Chain: a, b. Fragment: kinase domain, residues 33-345. Synonym: plk-1, serine/threonine-protein kinase 13, stpk13, polo-like kinase 1, human polo-like kinase 1. Engineered: yes. Other_details: construct 4. Design ankyrin repeat protein. Chain: c, d.

Source:

Homo sapiens. Human. Organism_taxid: 9606. Expressed in: spodoptera frugiperda. Expression_system_taxid: 7108. Synthetic construct. Organism_taxid: 32630. Expressed in: escherichia coli. Expression_system_taxid: 562.

Resolution:

2.30Å R-factor: 0.184 R-free: 0.224

Authors:

T.M.Bandeiras,R.C.Hillig,P.M.Matias,U.Eberspaecher,J.Fanghaenel, M.Thomaz,S.Miranda,K.Crusius,V.Puetter,P.Amstutz,M.Gulotti- Georgieva,H.K.Binz,C.Holz,A.A.P.Schmitz,C.Lang,P.Donner,U.Egner, M.A.Carrondo,B.Mueller-Tiemann

Key ref:

T.M.Bandeiras et al. (2008). Structure of wild-type Plk-1 kinase domain in complex with a selective DARPin. Acta Crystallogr D Biol Crystallogr, 64, 339-353. PubMed id: 18391401 DOI: 10.1107/S0907444907068217

Date:

08-Jul-07 Release date: 01-Apr-08

Protein chains

P53350  (PLK1_HUMAN) -  Serine/threonine-protein kinase PLK1 from Homo sapiens

Seq: 603 a.a.

Struc: 285 a.a.

Protein chains

No UniProt id for this chain

Struc: 130 a.a.

Enzyme reactions

• Enzyme class: Chains A, B: E.C.2.7.11.21 - polo 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: 10.1107/S0907444907068217

Acta Crystallogr D Biol Crystallogr 64:339-353 (2008)

PubMed id: 18391401

Structure of wild-type Plk-1 kinase domain in complex with a selective DARPin.

T.M.Bandeiras, R.C.Hillig, P.M.Matias, U.Eberspaecher, J.Fanghänel, M.Thomaz, S.Miranda, K.Crusius, V.Pütter, P.Amstutz, M.Gulotti-Georgieva, H.K.Binz, C.Holz, A.A.Schmitz, C.Lang, P.Donner, U.Egner, M.A.Carrondo, B.Müller-Tiemann.

ABSTRACT

As a key regulator of mitosis, the Ser/Thr protein polo-like kinase-1 (Plk-1) is a well validated drug target in cancer therapy. In order to enable structure-guided drug design, determination of the crystal structure of the kinase domain of Plk-1 was attempted. Using a multi-parallel cloning and expression approach, a set of length variants were identified which could be expressed in large amounts from insect cells and which could be purified to high purity. However, all attempts to crystallize these constructs failed. Crystals were ultimately obtained by generating designed ankyrin-repeat proteins (DARPins) selective for Plk-1 and using them for cocrystallization. Here, the first crystal structure of the kinase domain of wild-type apo Plk-1, in complex with DARPin 3H10, is presented, underlining the power of selective DARPins as crystallization tools. The structure was refined to 2.3 A resolution and shows the active conformation of Plk-1. It broadens the basis for modelling and cocrystallization studies for drug design. The binding epitope of 3H10 is rich in arginine, glutamine and lysine residues, suggesting that the DARPin enabled crystallization by masking a surface patch which is unfavourable for crystal contact formation. Based on the packing observed in the crystal, a truncated DARPin variant was designed which showed improved binding characteristics.

Selected figure(s)

Figure 1.
Figure 1 Plk-1 construct design and examples of multi-parallel expression tests. The domain structure of human Plk-1 is shown, with P indicating the phosphorylation site Thr210. Below, an initial construct and a set of 20 length variants (#1-#20) is shown. Residue numbers for the N- and C-terminal amino acids are indicated.

Figure 7.
Figure 7 Comparison of Plk-1-3H10 with further kinase-ankyrin-repeat (AR) protein complexes. (a)-(c) Tube diagrams showing the overall folds of kinases in complex with AR proteins: (a) Plk-1-DARPin 3H10; (b) Cdk6-P19INK4D inhibitor complex (Russo et al., 1998[Russo, A. A., Tong, L., Lee, J. O., Jeffrey, P. D. & Pavletich, N. P. (1998). Nature (London), 395, 237-243.]; chains A and B from PDB entry 1bi8 ); (c) APH-AR inhibitor complex (Kohl et al., 2005[Kohl, A., Amstutz, P., Parizek, P., Binz, H. K., Briand, C., Capitani, G., Forrer, P., Plückthun, A. & Grütter, M. G. (2005). Structure, 13, 1131-1141.]; chains A and B from PDB entry 2bkk ). In all panels the kinase is coloured gold and the AR protein is coloured cyan. The ADP in APH is shown in ball-and-stick representation (C, N, O and P atoms are coloured grey, blue, red and green, respectively) and is a reference for the ATP site. This figure was prepared with DINO (Philippsen, 2002[Philippsen, A. (2002). DINO. http://www.dino3d.org .]).

The above figures are reprinted by permission from the IUCr: Acta Crystallogr D Biol Crystallogr (2008, 64, 339-353) copyright 2008.

Figures were selected by an automated process.