Isolation of Temperature-sensitive p53 Mutations from a Comprehensive Missense Mutation Library

Abstract

Temperature-sensitive (ts) mutations have been used as a genetic and molecular tool to study the functions of many gene products. Each ts mutant protein may contain a temperature-dependent intramolecular mechanism such as ts conformational change. To identify key ts structural elements controlling the protein function, we screened ts p53 mutants from a comprehensive mutation library consisting of 2,314 p53 missense mutations for their sequence-specific transactivity through p53-binding sequences in Saccharomyces cerevisiae. We isolated 142 ts p53 mutants, including 131 unreported ts mutants. were ts hot spots. Among the 142 mutants, 54 were examined further in human osteosarcoma Saos-2 cells, and it was confirmed that 89% of the mutants were also ts in mammalian cells. The ts mutants represented distinct ts transactivities for the p53 binding sequences and a distinct epitope expression pattern for conformation-specific anti-p53 antibodies. These results indicated that the intramolecular ␤-sheet in the core DNA-binding domain of p53 was a key structural element controlling the protein function and provided a clue for finding a molecular mechanism that enables the rescue of the mutant p53 function.p53 tumor suppressor is a 393-amino acid transcription factor that activates the transcription of a number of downstream genes through p53 binding to two copies of the specific consensus DNA sequence 5Ј-RRRC(A/T)(T/A)GYYY-3Ј (in which R is a purine nucleoside and Y is a pyrimidine nucleoside) in their regulatory regions (1). These molecular switches are activated by post-translational modifications, including phosphorylation, acetylation, and prolyl isomerization (2-5) of p53 in response to genotoxic or non-genotoxic stresses. The resulting biological effects are cell cycle arrest, apoptosis, DNA repair, and angiogenesis (6 -10). A growing number of p53 downstream genes have been isolated, and p53 has been structurally and functionally divided into three portions, namely the NH 2 -terminal portion containing the transactivation domain, the central core portion corresponding to the DNA-binding domain, and the COOH-terminal portion containing the oligomerization domain. The evolution of the DNA-binding domain is highly conserved in p53 orthologues (11) and also in the conserved human homologues p63 and p73 (12, 13).The structure of the DNA-binding domain (residues 94 -312) was resolved by x-ray crystallography (14). The domain consists of two ␣-helixes (H1 and H2) and 11 ␤-strands (S1, S2, S2Ј, and S3-S10) that were interconnected by loops (long L1-L3 loops and other short loops). Two anti-parallel ␤-sheets containing four (S1, S3, S5, and S8) and five (S4, S6, S7, S9, and S10) ␤-strands make up a large ␤-sandwich that serves as a scaffold for a loop-sheet-helix (LSH) motif (L1, S2, S2Ј, S10, and H2) and two large loops (L2 and L3). The loop-sheet-helix consists of two separate regions as follows: (i) the L1 loop (residues 113-123) and the S2-S2Ј ␤-hairpin (residues 124 -135) that correspond to evolutionary conserved regio...

Changing the p53 master regulatory network: ELEMENTary, my dear Mr Watson

“…However, many p53 mutations do not eliminate transactivation capability (Kato et al, 2003;Hamroun et al, 2006). In an exhaustive yeast-based study that investigated nearly all possible single base substitution mutations, approximately 30% of the mutant proteins retained function towards at least one RE, consistent with other yeast-based studies (Kato et al, 2003;Shiraishi et al, 2004). These altered-function mutants differ from the 'gain-of-function' mutants described in human cells, which have lost sequence-specific transactivation capability.…”

Solution structure of p53 core domain: Structural basis for its instability

“…8, which is published as supporting information on the PNAS web site) had very low solvent accessibility ( Table 2). At least five of them (Thr-155, Ser-215, Thr-253, Thr-256, and Ser-269) are temperature-sensitive hot spots (14). Some of these residues had low protection factors in the H͞D experiments (e.g., Ser-240, Tyr-163) indicating that their backbone accessibility is higher than their side-chain accessibility.…”

Reactivation of mutant p53: molecular mechanisms and therapeutic potential

“…In addition, the isolated mutant core domain proteins R245S, R282W, V143A and others were shown to have residual (30-60%) DNA-binding activity at 201C (Bullock et al, 2000). The transcriptional transactivation activity of at least 10% of p53 mutants reported in p53 databases is temperature-sensitive (Shiraishi et al, 2004). Thus, many p53 mutants maintain the intrinsic ability to bind DNA.…”