PDBsum entry 1mx2

Cell cycle inhibitor

PDB id: 1mx2

Contents

Protein chain

156 a.a. *

Waters ×192

* Residue conservation analysis

PDB id:

1mx2

Name:

Cell cycle inhibitor

Title:

Structure of f71n mutant of p18ink4c

Structure:

Cyclin-dependent kinase 6 inhibitor. Chain: a, b. Synonym: p18-ink6, cyclin-dependent kinase 4 inhibitor c, p18-ink4c. Engineered: yes. Mutation: yes

Source:

Homo sapiens. Human. Organism_taxid: 9606. Expressed in: escherichia coli. Expression_system_taxid: 562.

Resolution:

2.25Å R-factor: 0.214 R-free: 0.254

Authors:

R.Marmorstein,R.N.Venkataramani,T.K.Maclachlan,X.Chai,W.S.El-Deiery

Key ref:

R.N.Venkataramani et al. (2002). Structure-based design of p18INK4c proteins with increased thermodynamic stability and cell cycle inhibitory activity. J Biol Chem, 277, 48827-48833. PubMed id: 12370184 DOI: 10.1074/jbc.M208061200

Date:

01-Oct-02 Release date: 16-Oct-02

Protein chains

P42773  (CDN2C_HUMAN) -  Cyclin-dependent kinase 4 inhibitor C from Homo sapiens

Seq: 168 a.a.

Struc: 156 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

DOI no: 10.1074/jbc.M208061200

J Biol Chem 277:48827-48833 (2002)

PubMed id: 12370184

Structure-based design of p18INK4c proteins with increased thermodynamic stability and cell cycle inhibitory activity.

R.N.Venkataramani, T.K.MacLachlan, X.Chai, W.S.El-Deiry, R.Marmorstein.

ABSTRACT

p18(INK4c) is a member of the INK4 family of proteins that regulate the G(1) to S cell cycle transition by binding to and inhibiting the pRb kinase activity of cyclin-dependent kinases 4 and 6. The p16(INK4a) member of the INK4 protein family is altered in a variety of cancers and structure-function studies of the INK4 proteins reveal that the vast majority of missense tumor-derived p16(INK4a) mutations reduce protein thermodynamic stability. Based on this observation, we used p18(INK4c) as a model to test the proposal that INK4 proteins with increased stability might have enhanced cell cycle inhibitory activity. Structure-based mutagenesis was used to prepare p18(INK4c) mutant proteins with a predicted increase in stability. Using this approach, we report the generation of three mutant p18(INK4C) proteins, F71N, F82Q, and F92N, with increased stability toward thermal denaturation of which the F71N mutant also showed an increased stability to chemical denaturation. The x-ray crystal structures of the F71N, F82Q, and F92N p18INK4C mutant proteins were determined to reveal the structural basis for their increased stability properties. Significantly, the F71N mutant also showed enhanced CDK6 interaction and cell cycle inhibitory activity in vivo, as measured using co-immunoprecipitation and transient transfection assays, respectively. These studies show that a structure-based approach to increase the thermodynamic stability of INK4 proteins can be exploited to prepare more biologically active molecules with potential applications for the development of molecules to treat p16(INK4a)-mediated cancers.

Selected figure(s)

Figure 1.
Fig. 1. Residues of p18^INK4c targeted for mutations. The ankyrin repeat elements are shown in alternating red and orange, and the residues targeted for mutations are shown in red.

Figure 3.
Fig. 3. Structure of the F71N, F82Q, and F92N p18^INK4c mutants in comparison to the native structure of p18^INK4c protein. a, superposition of the mutant, F71N (in green) and the native protein (in gray) along with the simulated annealing omit map around the site of mutation contoured at 1.5 . The yellow sphere represents a water molecule. b, detailed interactions made in the F71N p18^INK4c mutant are shown in green with CPK coloring, while the structure of the native protein, which lacks these new interactions are shown in gray. The mutation results in a new hydrogen-bonding interaction with arginine 79, and a water (shown in yellow)-mediated hydrogen bond with aspartate 100. Glycines are shown as green or gray spheres. c, same as a except that the F82Q mutant is shown. d, detailed interactions made in the F82Q p18^INK4c mutant are shown in green with CPK coloring, while the structure of the native protein, which lacks these, new interactions are shown in gray. The mutation results in new hydrogen bonding interaction with the backbone NH of glycine 48 and a water (shown in yellow)-mediated hydrogen bond with arginine 117. Glycines are shown as green or gray spheres. e, same as a except that the F92N mutant is shown. f, detailed interactions made in the F92N p18^INK4c mutant are shown in green with CPK coloring, while the structure of the native protein, which lacks these, new interactions are shown in gray. The mutation results in a new water-mediated hydrogen bond with arginine 54. Glycines are shown as green or gray spheres. Molscript objects for the electron density was created using CONSCRIPT, and the figures were prepared with the programs MOLSCRIPT (37) and RASTER3D (38).

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2002, 277, 48827-48833) copyright 2002.

Figures were selected by an automated process.