The tumor suppressor p53 is a key target for cancer therapy, but its low expression levels, poor conformational stability, and high degree of disorder remain major challenges to its structural investigation. Here, we address these issues by fusing the N-terminal transactivation domain of p53 to an engineered spider silk domain termed NT*. Molecular dynamics simulations show that the disordered transactivation domain of p53 wraps around the NT* domain via a series of folding events, resulting in a globular structure.

Contained in this dataset is the ion mobility mass spectormetry and MD simulation analysis of the recombinantfusion protein between the NT*tag derived from spider silk, and the human tumor suppressor p53 N-terminal transactivation domain. The dataset contrains three parts:

1. Expression and purification of the fusion protein

2. Ion mobility MS analysis of the fusion protein

3. MD simulations of the fusion protein

1. Purification of NT*-TAD1-TAD2

The gene for MGH10-TEV-TAD1-TAD2 cloned into the pET26b+ expression vector was transformed into chemically competentE. coliBL21 (DE3) cells. Over-night cultures were inoculated 1:100 to Luria-Bertani medium containing 70 mg L^-1kanamycin. The cultures were grown at 30 °C to an OD₆₀₀of 0.9 before induction by 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG) and over-night expression at 20 °C. For each construct, 500 mL culture was harvested by centrifugation at 5000 × gfor 20 min. The pellet was resuspended in 20 mM Tris-HCl, pH 8.0 loading buffer (40 mL) and stored at -20 °C overnight. After thawing at room temperature, the cells were lysed in a cell disrupter (Constant Systems Ltd, Daventry, UK) at 30 kPsi followed by centrifugation at 27 000 × gfor 30 min. A Ni-Sepharose (GE Healthcare, Uppsala, Sweden) column was packed from 6 mL resin and equilibrated with loading buffer. The supernatant from centrifugation was loaded on the column followed by washes with loading buffer supplemented with 10 mM imidazole. The protein was eluted (1.5 mL fractions) with loading buffer supplemented with 300 mM imidazole. Fractions with high absorbance at 280 nm were pooled and transferred to a Spectra/Por® dialysis membrane (6-8 kDa molecular weight cut-off, Spectrum Laboratories, Rancho Dominguez, CA, USA) for overnight dialysis in 5 L loading buffer at 4 °C. Samples taken from steps during expression, lysis and purification were analyzed by SDS-PAGE using 4-20% Mini-PROTEAN® TGX™ polyacrylamide gels (Bio-rad Laboratories Inc., USA) stained with Coomassie Brilliant Blue dye.

2. Ion mobility mass spectrometry of NT*-TAD1-TAD2

Purified NT*-TAD1-TAD2 and beta-lactoglobulin (Sigma, MO) as calibrant were buffer-exchanged into 100 mM ammonium acetate, pH 7.0, using BioSpin6 columns (BioRad, CA). IM-MS spectra were acquired on a Waters Synapt G2 travelling wave ion mobility mass spectrometer (Waters, UK) equipped with an offline nanospray source. The capillary voltage was 1.5 kV, the source presure was 3.4 mbar, and the source temperature was 80 °C. The collision energy in the ion trap was 10 V. Wave height and wave velocity were 35 V and 700 m/s in the IMS cell and 248 m/s and 10 V in the transfer. IMS gas was nitrogen with a flow of 50 mL/min. Mass spectra were visualized using MassLynx 4.1. CCS calculations were performed based on the beta-lactoglobulin monomer and dimer using PULSAR.

3. Construction of the NT*-TAD1-TAD2-PRD model

The NMR structure of spider silk N-terminal domain (NT; PDB ID: 2LPJ ) was downloaded from the Protein Data Bank. The lowest energy state structure (Model 1) from the reported ensemble of twenty NT structures was selected and two in-silico mutations “D40K and K65D” were incorporated (NT*) as per the recombinant protein used in the current work. A 97 amino acid sequence comprising of TEV cleavage site (1ENLYFQS7) along with the transactivation domains (TAD1 and TAD2), and the proline rich domain (PRD) of p53 was considered for modelling. Extended state conformations of “TEV+TAD1 (residues 1-40)”, TAD2 (residues 41-70) and PRD (residues 71-90) polypeptide segments were generated separately with the LEaP module of AMBER18 [4]. The MDM2 binding site (18TFSDLWKLL26) located in TAD1 was assigned a helical backbone conformation. Modelling of NT* and the N-terminal domains from p53 was done in a sequential manner. TEV+TAD1 was first attached via a peptide bond with NT* and subjected to explicit solvent MD simulations (details mentioned below) to generate NT*-TAD1 structures. An end-state representative structure of this system was then attached to TAD2 and simulated to obtain “NT*-TAD1-TAD2” structure. PRD was finally added to the construct to model “NT*-TAD1-TAD2-PRD” structure.

Molecular dynamics simulations

The N- and C-terminus of “NT*-TAD1”, “NT*-TAD1-TAD2” and “NT*-TAD1-TAD2-PRD” models were all capped with ACE (acetyl) and NME (N-methyl) functional groups respectively. These models were placed in the centre of an octahedral box whose dimensions were fixed by maintaining a minimum distance of 8 Å between any protein atom and the box boundaries. TIP3P water model [1] was used to solvate the structure. The net charge of the systems were -12e (NT*-TAD1) and -21e (NT*-TAD1-TAD2 and NT*-TAD1-TAD2-PRD) which were neutralized by adding 12 Na+ and 21 Na+ counter ions respectively. All-atom molecular dynamics simulations were carried out using the PMEMD module through the AMBER18 suite of programs employing ff14SB force field parameters [2]. The systems were energy minimized using steepest descent followed by conjugate gradient schemes, heated to 300 K over 30 ps under NVT ensemble and equilibrated for 200 ps in NPT ensemble. The final production dynamics was run for 1 µs each under NPT conditions. NT*-TAD1 system was simulated in triplicates with different initial velocities. Simulation temperature was maintained at 300 K using Langevin dynamics [3] (collision frequency: 1.0 ps^-1), and the pressure was kept at 1 atm using weak-coupling [4] (relaxation time: 1ps). Periodic boundary conditions were applied on the box edges and Particle Mesh Ewald (PME) method [5] was used to compute long-range electrostatic interactions. SHAKE algorithm [6] was used to constrain all the bonds involving hydrogen atoms and the equation of motion was solved with an integration time step of 2fs. 

References

1. Jorgensen, W. L., et al (1983). Comparison of simple potential functions for simulating liquid water. J Chem Phys 79, 926-935.

2. Maier, J. A., et al (2015). ff14SB: Improving the Accuracy of Protein Side Chain and Backbone Parameters from ff99SB. J Chem Theory Comput 11, 3696-3713.

3. Loncharich, R. J., Brooks, B. R. & Pastor, R. W. (1992). Langevin dynamics of peptides: the frictional dependence of isomerization rates of N-acetylalanyl-N'-methylamide. Biopolymers 32, 523-535.

4. Berendsen, H. J. C., Postma, J. P. M., Van Gunsteren, W. F., DiNola, A. & Haak, J. R. (1984). Molecular Dynamics with coupling to an external bath. J. Chem. Phys. 81, 3684-3690.

5. Darden, T., York, D. & Pedersen, L. (1993). Particle mesh Ewald-an Nlog(N) method for Ewald sums in large systems. J. Chem. Phys. 98, 10089-10092.

6. Ryckaert, J.-P., Ciccotti, G. & Berendsen, H. J. C. (1977). Numerical Integration of the cartesian equations of motion of a system with constraints: Molecular dynamics of n-alkanes. J. Comput. Phys. 23, 327-341.

The ion mobility data is provided as PULSAR file (see T. M. Allison, E. Reading, I. Liko, A. J. Baldwin, A. Laganowsky and C. V. Robinson, Nat. Commun., 2015, 6, 8551.). PULSAR is available for free download from http://pulsar.chem.ox.ac.uk. The PULSAR file (NT_TAD1_TAD2_IMMS.pkl.gzip) contains native IM-MS spectra of the NT-TAD1-TAD2 fusion protein recorded at increasing collision voltages to induce unfolding. For CCS calculations, the attached calibration file containing ubiquitin and the beta lactoglobulin monomer and dimer (Ubiq_blactmono_blactdimer.ionsMS_calibration) can be loaded into PULSAR.

The MD end-point structure of NT-TAD1-TAD2-PRD is provided as PDB file (NT-TAD1-TAD2-PRD_endpoint.pdb). For comparison of the model and the IMMS data, the prion-rich domain (residues 61-93), which is not included in the fusion protein, has to be deleted from the PDB file.