Structure Determination of the Transactivation Domain of p53 in Complex with S100A4 Using Annexin A2 as a Crystallization Chaperone

Abstract

In spite of the fact that structure solving methods are constantly improving, the biggest challenge of protein crystallography remains the production of well diffracting single protein crystals. Full understanding the environmental factors that influence crystal packing would be an enormous task, therefore crystallographers are still forced to work "blindly" trying as many crystallizing conditions and mutations, designed to improve crystal packing, in the sequence of the target protein as possible. Numerous times the random attempts simply fail even when using crystallization robots or recent techniques to determine the optimal mutations. As an alternative option in these cases, crystallization chaperones can be used. These proteins have a unique property, namely they easily form protein crystals, which can be exploited by using them as a heterologous fusion partner to promote crystal contact formation. Today, the most frequently used crystallization chaperone is the maltose-binding protein (MBP) and crystallographers are in need of other options. Our previous results showed the outstanding crystallization properties of a non-EF hand calcium-binding protein annexin A2 (ANXA2). Here, we compared ANXA2 with the wild type MBP and found that ANXA2 is just as good, if not a better crystallization chaperone. Using ANXA2 for this purpose, we were able to solve the atomic resolution structure of a challenging crystallization target, the transactivation domain (TAD) of p53 in complex with S100A4, an EF hand calcium-binding protein associated with metastatic tumors. The full-length TAD forms an asymmetric fuzzy complex with S100A4 and could interfere with its function.

Functionalization of the BCL6 BTB domain into a noncovalent crystallization chaperone

“…The main bottleneck in this technique is the production of well-ordered crystals that produce high-quality diffraction. Strategies to aid crystallization include the use of chaperones (Bukowska & Grü tter, 2013;Ecsé di et al, 2020), either in complex with the protein of interest or as a fusion partner, and the use of engineered lattices that accommodate the 'guest' protein. Lattices of defined dimensions should impose threedimensional order on the captured guest molecule, serving both to aid crystallization and to act as a phasing tool for structure determination.…”

Tumor‐Suppressor p53TAD1–60 Forms a Fuzzy Complex with Metastasis‐Associated S100A4: Structural Insights and Dynamics by an NMR/MD Approach

“…In order to be able to crystallize the "fuzzy" system, several modifications were carried out: a shortened p53TAD was covalently liked to a truncated S100A4Δ8 construct also fused to a non-EF hand Ca 2 + binding annexin A2 which was utilized as a crystallization chaperon. [26] In the crystal structure three short segments showed helical conformation: T18-L25, S37-D42 and P47-W53, whereas the V31-L35 segment could not be resolved in the electron density, thus indicating high conformational variability in this region. Residues belonging to L22-P27 of the first helix as well as S46 and I50 have lower B-factors than their surroundings, thus they seem to be the primary interaction sites.…”

“…Despite all these, only limited structural information is available. The latest effort by X‐ray crystallography was successful only if a shorter p53TAD 17–56 segment was covalently bound to the N terminus of a C‐terminally truncated S100A4Δ8 in the presence of a crystallization chaperon protein also covalently linked to the complex [26] . In the present work we modeled the solution structure of this complex using full length p53TAD 1–60 and wild‐type S100A4.…”

“…fusion protein 2-62 NMR [20] 6T58 S100A4Δ8 17-56 X-ray diffraction [26] termini remain unperturbed indicating that these segments are not directly bound to S100A4. SSP scores, determined from the H α , C α , C β chemical shift values, are used to obtain structural information ( Figure 1C).…”

“…The PreSMos can play important roles upon binding by frequently exhibiting a disorder-to-order transition. [13] A large number of structural studies were conducted with the full length TAD or selected TAD regions in complex with various proteins [14][15][16][17][18][19][20][21][22][23][24][25][26][27][28][29] (collected in Table 1 with the corresponding structures deposited in the Protein Data Bank with PDB ID). In all enumerated cases the disorder-to-order transition of TAD1 and/or TAD2 was detected, where the implicated subdomains formed stable helical structures upon binding to target proteins.…”