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


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. Study holds ProTherm entries: 17721, 17722, 17723, 17724, 17725, 17726, 17727, 17728, 17729, 17730, 17731, 17732, 17733, 17734, 17735 Extra Details: INK4 family; thermodynamic stability; cell cycle inhibitory activity; structural basis

Submission Details

ID: NkH7fUBj

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:50 p.m.

Version: 1

Publication Details
Venkataramani RN;MacLachlan TK;Chai X;El-Deiry WS;Marmorstein R,J. Biol. Chem. (2002) Structure-based design of p18INK4c proteins with increased thermodynamic stability and cell cycle inhibitory activity. PMID:12370184
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
2A5E 1999-08-13 SOLUTION NMR STRUCTURE OF TUMOR SUPPRESSOR P16INK4A, RESTRAINED MINIMIZED MEAN STRUCTURE
1A5E 1999-08-13 SOLUTION NMR STRUCTURE OF TUMOR SUPPRESSOR P16INK4A, 18 STRUCTURES
1BU9 1999-09-13 SOLUTION STRUCTURE OF P18-INK4C, 21 STRUCTURES
1DC2 1999-12-23 SOLUTION NMR STRUCTURE OF TUMOR SUPPRESSOR P16INK4A, 20 STRUCTURES
1IHB 1998-12-02 1.95 CRYSTAL STRUCTURE OF P18-INK4C(INK6)
1MX4 2002-10-16 2.0 Structure of p18INK4c (F82Q)
1MX6 2002-10-16 2.0 Structure of p18INK4c (F92N)
1MX2 2002-10-16 2.25 Structure of F71N mutant of p18INK4c
1G3N 2001-01-10 2.9 STRUCTURE OF A P18(INK4C)-CDK6-K-CYCLIN TERNARY COMPLEX
1BI7 1999-01-13 3.4 MECHANISM OF G1 CYCLIN DEPENDENT KINASE INHIBITION FROM THE STRUCTURE OF THE CDK6-P16INK4A TUMOR SUPPRESSOR COMPLEX

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Cyclin-dependent kinase inhibitor 2A P42771 CDN2A_HUMAN
100.0 Cyclin-dependent kinase 4 inhibitor C P42773 CDN2C_HUMAN
93.5 Cyclin-dependent kinase 4 inhibitor C Q60772 CDN2C_MOUSE