PDBsum entry 1h24

Transferase

PDB id: 1h24

Contents

Protein chains

294 a.a. *

258 a.a. *

Ligands

PRO-VAL-LYS-ARG-ARG-LEU-ASP-LEU-GLU

Waters ×231

* Residue conservation analysis

References listed in PDB file

Key reference

Title

Specificity determinants of recruitment peptides bound to phospho-Cdk2/cyclin a.

Authors

E.D.Lowe, I.Tews, K.Y.Cheng, N.R.Brown, S.Gul, M.E.Noble, S.J.Gamblin, L.N.Johnson.

Ref.

Biochemistry, 2002, 41, 15625-15634. [DOI no: 10.1021/bi0268910]

PubMed id

12501191

Abstract

Progression through S phase of the eukaryotic cell cycle is regulated by the action of the cyclin dependent protein kinase 2 (CDK2) in association with cyclin A. CDK2/cyclin A phosphorylates numerous substrates. Substrate specificity often employs a dual recognition strategy in which the sequence flanking the phospho-acceptor site (Ser.Pro.X.Arg/Lys) is recognized by CDK2, while the cyclin A component of the complex contains a hydrophobic site that binds Arg/Lys.X.Leu ("RXL" or "KXL") substrate recruitment motifs. To determine additional sequence specificity motifs around the RXL sequence, we have performed X-ray crystallographic studies at 2.3 A resolution and isothermal calorimetry measurements on complexes of phospho-CDK2/cyclin A with a recruitment peptide derived from E2F1 and with shorter 11-mer peptides from p53, pRb, p27, E2F1, and p107. The results show that the cyclin recruitment site accommodates a second hydrophobic residue either immediately C-terminal or next adjacent to the leucine of the "RXL" motif and that this site makes important contributions to the recruitment peptide recognition. The arginine of the RXL motif contacts a glutamate, Glu220, on the cyclin. In those substrates that contain a KXL motif, no ionic interactions are observed with the lysine. The sequences N-terminal to the "RXL" motif of the individual peptides show no conservation, but nevertheless make common contacts to the cyclin through main chain interactions. Thus, the recruitment site is able to recognize diverse but conformationally constrained target sequences. The observations have implications for the further identification of physiological substrates of CDK2/cyclin A and the design of specific inhibitors.

Secondary reference #1

Title

The structural basis for specificity of substrate and recruitment peptides for cyclin-Dependent kinases.

Authors

N.R.Brown, M.E.Noble, J.A.Endicott, L.N.Johnson.

Ref.

Nat Cell Biol, 1999, 1, 438-443. [DOI no: 10.1038/15674]

PubMed id

10559988

Figure 1.
Figure 1. The location of the peptide-binding sites on phospho-CDK2 -cyclin A3 complex. CDK2 is shown in yellow with the C (PSTAIRE) helix in red and the activation segment in magenta. Cyclin A3 is shown in khaki. a, Conventional view of the CDK2 -cyclin A3 complex, also showing the peptide substrate (green ball-and-stick diagram) and the recruitment peptide (cyan ball-and-stick diagram). Note that the structures of the phospho-CDK2 -cyclin A3 -peptide complexes were determined separately. b, Dimeric phospho-CDK2 -cyclin A3 complexes. The phospho-CDK2 -cyclin A3 -peptide complexes in the crystal formed dimers, with the two complexes related by a non-crystallographic two-fold axis, in an arrangement similar to that of the phospho-CDK2 -cyclin A3 crystal (coordinates 1JST)5, which crystallized in a different space group to the peptide complexes. The contacts across the dimer interface are between the -sheet region of CDK2 (end of 2 and start of 3) and the groove between helices in cyclin A3 (helices 3 and 5). c, Surface representation of the substrate-peptide-binding pocket of CDK2, showing the site for proline at P+1 and the proximity of lysine at P+3 to cyclin A3. d, Surface representation of the recruitment-peptide complex. Diagrams generated in AESOP (M.E.M.N., unpublished observations).

Figure 3.
Figure 3. The recruitment-peptide-binding site of the phospho-CDK2 -cyclin A3 complex. a, Diagram showing residues RRLFGE from the recruitment peptide of p107 bound at the hydrophobic site on cyclin A3, making contacts with residues from successive turns of the 1 and 3 helices. The view, which is rotated ~90° from that shown in Fig. 2, shows the complete fold of the cyclin box. b, Similar view of the equivalent residues from the phospho-CDK2 -cyclin A3 -p27^KIP1 complex (coordinates IJSU from the PDB^25).

The above figures are reproduced from the cited reference with permission from Macmillan Publishers Ltd