Design and characterization of a hyperstable p16INK4a that restores Cdk4 binding activity when combined with oncogenic mutations.


Abstract

Cyclin-dependent kinase inhibitor p16(INK4a) is the founding member of the INK4 family of tumor suppressors capable of arresting mammalian cell division. Missense mutations in the p16(INK4a) gene (INK4a/CDKN2A/MTS1) are strongly linked to several types of human cancer. These mutations are evenly distributed throughout this small, ankyrin repeat protein and the majority of them disrupt the native secondary and/or tertiary structure, leading to protein unfolding, aggregation and loss of function. We report here the use of multiple stabilizing substitutions to increase the stability of p16(INK4a) and furthermore, to restore Cdk4 binding activity of several defective, cancer-related mutant proteins. Stabilizing substitutions were predicted using four different techniques. The three most effective substitutions were combined to create a hyperstable p16(INK4a) variant that is 1.4 kcal/mol more stable than wild-type. This engineered construct is monomeric in solution with wild-type-like secondary and tertiary structure and cyclin-dependent kinase 4 binding activity. Interestingly, these hyperstable substitutions, when combined with oncogenic mutations R24P, P81L or V126D, can significantly restore Cdk4 binding activity, despite the divergent features of each destabilizing mutation. Extensive biophysical studies indicate that the hyperstable substitutions enhance the binding activity of mutant p16 through several different mechanisms, including an increased amount of secondary structure and thermostability, reduction in exposed hydrophobic surface(s) and/or a reduced tendency to aggregate. This apparent global suppressor effect suggests that increasing the thermodynamic stability of p16 can be used as a general strategy to restore the biological activity to defective mutants of this important tumor suppressor protein. Study holds ProTherm entries: 15950, 15951, 15952, 15953, 15954, 15955, 15956, 15957, 15958, 15959, 15960, 15961, 15962, 15963, 15964, 15965 Extra Details: 1mM EDTA and 1mM DTT were added in the experiment p16INK4a; cyclin-dependent kinase inhibitor; cancer-related mutations; protein design; stability

Submission Details

ID: 9aRPLw5r

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:46 p.m.

Version: 1

Publication Details
Cammett TJ;Luo L;Peng ZY,J. Mol. Biol. (2003) Design and characterization of a hyperstable p16INK4a that restores Cdk4 binding activity when combined with oncogenic mutations. PMID:12614625
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
1DC2 1999-12-23 SOLUTION NMR STRUCTURE OF TUMOR SUPPRESSOR P16INK4A, 20 STRUCTURES
1A5E 1999-08-13 SOLUTION NMR STRUCTURE OF TUMOR SUPPRESSOR P16INK4A, 18 STRUCTURES
2A5E 1999-08-13 SOLUTION NMR STRUCTURE OF TUMOR SUPPRESSOR P16INK4A, RESTRAINED MINIMIZED MEAN STRUCTURE
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