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.

Figure 1

Urea equilibrium unfolding of wild-type (WT) and mutant p53 core domain represented as fraction of unfolded protein vs. concentration of denaturant. Unfolding was monitored in 50 mM sodium phosphate (pH 7.2) and 5 mM DTT at 10°C by fluorescence using an excitation wavelength of 280 nm and normalized emission at 356 nm (11). The protein concentration was 2 μM. The fitting of the experimental data to a two-state transition equation (11) was performed by the program kaleidagraph, and the best fit is indicated by solid lines.

Figure 2

Binding curves of the wild-type and quadruple mutant p53 core domain to the gadd45 double-stranded DNA. The experiments were performed at 20°C in 50 mM Tris (pH 7.2) with 5 mM DTT. The protein concentration ranged from 0.03 to 1 μM whereas that of the double-stranded oligonucleotide was 1 μM. The experimental data were fitted by the program kaleidagraph, and the best fit is indicated by solid and dotted lines. The dissociation constant K_D was calculated as the midpoint of the transition curve.

Figure 3

Diagram of p53 DNA binding domain produced by molscript (22) showing only the position of the four mutations used to design the quadruple mutant.