Semirational design of active tumor suppressor p53 DNA binding domain with enhanced stability.


Abstract

We have designed a p53 DNA binding domain that has virtually the same binding affinity for the gadd45 promoter as does wild-type protein but is considerably more stable. The design strategy was based on molecular evolution of the protein domain. Naturally occurring amino acid substitutions were identified by comparing the sequences of p53 homologues from 23 species, introducing them into wild-type human p53, and measuring the changes in stability. The most stable substitutions were combined in a multiple mutant. The advantage of this strategy is that, by substituting with naturally occurring residues, the function is likely to be unimpaired. All point mutants bind the consensus DNA sequence. The changes in stability ranged from +1.27 (less stable Q165K) to -1.49 (more stable N239Y) kcal mol-1, respectively. The changes in free energy of unfolding on mutation are additive. Of interest, the two most stable mutants (N239Y and N268D) have been known to act as suppressors and restored the activity of two of the most common tumorigenic mutants. Of the 20 single mutants, 10 are cancer-associated, though their frequency of occurrence is extremely low: A129D, Q165K, Q167E, and D148E are less stable and M133L, V203A and N239Y are more stable whereas the rest are neutral. The quadruple mutant (M133LV203AN239YN268D), which is stabilized by 2.65 kcal mol-1 and Tm raised by 5.6 degreesC is of potential interest for trials in vivo.

Submission Details

ID: WjJC3h6P

Submitter: Shu-Ching Ou

Submission Date: Nov. 21, 2018, 2:44 p.m.

Version: 1

Publication Details
Nikolova PV;Henckel J;Lane DP;Fersht AR,Proc Natl Acad Sci U S A (1998) Semirational design of active tumor suppressor p53 DNA binding domain with enhanced stability. PMID:9843948
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
1A1U 1997-12-16T00:00:00+0000 0 SOLUTION STRUCTURE DETERMINATION OF A P53 MUTANT DIMERIZATION DOMAIN, NMR, MINIMIZED AVERAGE STRUCTURE
1AIE 1997-04-17T00:00:00+0000 1.5 P53 TETRAMERIZATION DOMAIN CRYSTAL STRUCTURE
1C26 1999-07-22T00:00:00+0000 1.7 CRYSTAL STRUCTURE OF P53 TETRAMERIZATION DOMAIN
1DT7 2000-01-11T00:00:00+0000 0 SOLUTION STRUCTURE OF THE C-TERMINAL NEGATIVE REGULATORY DOMAIN OF P53 IN A COMPLEX WITH CA2+-BOUND S100B(BB)
1GZH 2002-05-22T00:00:00+0000 2.6 Crystal structure of the BRCT domains of human 53BP1 bound to the p53 tumor supressor
1GZH 2002-05-22T00:00:00+0000 2.6 Crystal structure of the BRCT domains of human 53BP1 bound to the p53 tumor supressor
1H26 2002-07-31T00:00:00+0000 2.24 CDK2/CyclinA in complex with an 11-residue recruitment peptide from p53
1HS5 2000-12-22T00:00:00+0000 0 NMR SOLUTION STRUCTURE OF DESIGNED P53 DIMER
1JSP 2001-08-17T00:00:00+0000 0 NMR Structure of CBP Bromodomain in complex with p53 peptide
1KZY 2002-02-08T00:00:00+0000 2.5 Crystal Structure of the 53bp1 BRCT Region Complexed to Tumor Suppressor P53

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
93.1 Cellular tumor antigen p53 Q9TTA1 P53_TUPBE
95.7 Cellular tumor antigen p53 P56424 P53_MACMU
95.7 Cellular tumor antigen p53 P61260 P53_MACFU
95.7 Cellular tumor antigen p53 P56423 P53_MACFA
95.2 Cellular tumor antigen p53 P13481 P53_CHLAE
100.0 Cellular tumor antigen p53 P04637 P53_HUMAN