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Previous work using X-ray crystallography has investigated the structural basis of DNA recognition by p53 core-domain tetramers [1-3], and the functional loss of p53 hotspot mutants and their reactivation by suppressor mutations [4-5]. To uncover the structural basis of rescuing p53 mutants by MQ, X-ray crystallography was carried out at beamlines ID23-1, ID23-2, ID29 and ID30A-3, and an in-house facility, to obtain high-resolution structures of the core domains of mutant and wild-type p53 bound to MQ in their free state and/or bound to their DNA response elements. The wild-type and mutant p53DBD, including two DNA- contact mutants, R273H and R273C, and a structural mutant, R282W, were studied. Fourteen structures were determined, allowing for detailed analysis of the effects of MQ on mutant and wild-type p53 and their complexes with DNA.

The detailed structural analysis shows that out of the 10 native cysteines of the core domain, only five residues (C124, C182, C229, C275, C277), as well as the hotspot mutation product C273, were found modified in the various DNA-free structures, all located at the surface of the protein. (Figures 33a-c). MQ selectivity is higher in the DNA-bound state of p53; only three MQ-modified

cysteines (C124, C229 and C277) were observed in the p53-DNA-MQ structures of both mutant and wild-type p53 (Figures 33d-f); MQ-C124 and MQ-C229 support the inter-dimer interface and MQ- C277 stabilises the protein-DNA interface (Figure 34). In both DNA-free and DNA-bound p53, MQ binding to cysteines appears variable and dynamic, showing a large diversity in its interaction modes.

Fig. 34: Tetramer assembly of mutant p53DBD bound to DNA and MQ based on the structure of R282W-DNA-MQ (PDB ID 7B4E).

p53 monomers are shown in blue, cyan, light blue and green, DNA in grey and MQ in yellow. MQ bound to cysteines 124 and 229 support the interface between p53 dimers (AB and CD), whereas

MQ bound to C277 stabilises the protein-DNA interface.

Fig. 33: MQ bound to cysteines in the DNA-free and DNA-bound mutant p53DBD structures. a) MQ bound to C277 in one monomer of R273H-MQ structure. b) MQ bound to C273 in one monomer of R273C-MQ structure. c) Superposition of all monomers from R273C-MQ (I) and (II) structures (each incorporating four monomers in the asymmetric unit) shown in light green, onto a monomer from tetrameric wt-p53DBD bound to DNA (based on PDB 5MCV) shown in magenta and grey. This view demonstrates that MQ bound to C182, C229, C273, and C277 are compatible with DNA binding, but not C275. d) MQ bound to C124 in R282W-DNA-MQ structure. e) MQ bound to C229 in one monomer of R282W-DNA-MQ structure. f) MQ bound to C277 in R273H-DNA-MQ structure. Also shown cartoon representations of the DNA backbones of the MQ-modified (magenta) and the MQ-free (light brown) R273H-DNA complexes. The corresponding enantiomers of MQ (shown in green) are assigned as MQs and MQr. Neighbouring monomer is shown in brown in (a) and blue in (d) and (e).