Pin1 is a two-domain cell regulator that isomerizes peptidyl-prolines. The catalytic domain (PPIase) and the other ligand-binding domain (WW) sample extended and compact conformations. Ligand binding changes the equilibrium of the interdomain conformations through an interdomain allosteric mechanism. We have described ligand-specific conformational changes that occur upon binding of pCDC25c and FFpSPR. pCDC25c binding doubles the population of the extended states compared to the virtually identical populations of the apo and FFpSPR-bound forms. pCDC25c binding to the WW domain triggers conformational changes to propagate via the interdomain interface to the catalytic site, while FFpSPR binding displaces a helix in the PPIase that leads to repositioning of the PPIase catalytic loop.

Here, we deposit the entire magnetic resonance-based CYANA structure calculation protocols of Pin1 two-state structural ensembles of apo, FFpSPR-bound and pCDC25c-bound form that allowed us to determine the coupling of intra- and interdomain structural sampling Pin1.

For assignment, scalar coupling, relaxation, and NOESY experiments, all samples contained ¹⁵N,¹³C-labeled Pin1 in NMR buffer at pH 6.5 with 3% D₂O (low D₂O to reduce H-D exchange). While the apo sample contained ~2 mM Pin1, the amount of protein had to be reduced for the ligand-bound samples due to low concentration of FFpSPR, and to reduce aggregation with pCDC25c. Therefore, the FFpSPR-saturated sample contained 600 μM Pin1 and 3.6 mM FFpSPR and the pCDC25c-saturated sample contained 750 μM Pin1 and 4 mM pCDC25c. Apo Pin1 was assigned as described in (Alexandra Born, Janne Soetbeer, Frauke Breitgoff, Morkos A. Henen, Nikolaos Sgourakis, Yevhen Polyhach, Parker J. Nichols, Dean Strotz, Gunnar Jeschke, Beat Vögeli, Reconstruction of coupled intra- and interdomain protein motion from nuclear and electron magnetic resonance, 2021, J Am Chem Soc, 143, 16055-16067). Using ¹⁵N-HSQC, ¹³C-resolved aliphatic and aromatic CT-HSQC, and a ¹⁵N/¹³C-resolved [¹H-¹H] NOESY experiment, we assigned the chemical shifts of the FFpSPR- and pCDC25c-bound Pin1 based on the apo Pin1 assignment (Alexandra Born, Parker J. Nichols, Morkos A. Henen, Celestine N. Chi, Dean Strotz, Peter Bayer, Shin-Ichi Tate, Jeffrey Peng, Beat Vögeli, Backbone and side-chain chemical shift assignments of full length, apo, human Pin1, a phosphoprotein regulator with interdomain allostery, 2019, Biomol NMR Assign, 13, 85-89). While chemical shift changes occur, the NOESY towers remain relatively intact and characteristic of each atom. The chemical shifts of FFpSPR-bound and pCDC25c-bound Pin1 have been deposited in the BMRB under accession codes 51043 [https://dx.doi.org/10.13018/BMR51043] and 51034 [https://dx.doi.org/10.13018/BMR51034], respectively. NOE buildup series were run as previously described¹ with mixing times of 24, 32, 40, 48, and 56 ms. Scalar coupling (³J_HN-Ha, ³J_Ha-Hβ ³⁴, and ³J_N-Cγ) and relaxation (R₁ and R_1ρ for determination of tumbling times) experiments were also recorded on these ligand-bound samples as described (Alexandra Born, Janne Soetbeer, Frauke Breitgoff, Morkos A. Henen, Nikolaos Sgourakis, Yevhen Polyhach, Parker J. Nichols, Dean Strotz, Gunnar Jeschke, Beat Vögeli, Reconstruction of coupled intra- and interdomain protein motion from nuclear and electron magnetic resonance, 2021, J Am Chem Soc, 143, 16055-16067).

The same cysteine-mutant constructs for PRE and DEER that were developed to measure the interdomain orientation of apo Pin1 were also used for ligand-bound measurements. The samples were expressed, purified, and spin-labeled as previously published (Alexandra Born, Janne Soetbeer, Frauke Breitgoff, Morkos A. Henen, Nikolaos Sgourakis, Yevhen Polyhach, Parker J. Nichols, Dean Strotz, Gunnar Jeschke, Beat Vögeli, Reconstruction of coupled intra- and interdomain protein motion from nuclear and electron magnetic resonance, 2021, J Am Chem Soc, 143, 16055-16067). For PRE, the ligands were added to be 8x the concentration of Pin1. R₂ PRE rates were measured on the ¹⁵N-labeled MTSL-conjugated (paramagnetic) and quenched (diamagnetic) samples M15C, N90C, S98C, and Q131C that all also contained C57A and C113D mutations. The relaxation enhancement due to the paramagnetic labels (R₂^sp) were obtained from the R₂ difference between the paramagnetic and diamagnetic samples. The DEER samples M15C-N90C, M15C-S98C, M15C-Q131C, and N90C-Q131C were all measured with 8x ligand. All PRE and DEER constructs maintained catalytic activity.

            Data fitting and analysis for the ligand-bound samples were performed as previously published for apo Pin1, using TopSpin (Bruker) version 7 and VNMRJ version 4.2 Revision A (Agilent), CCPnmr version 2.4.2 (CCP), and NMRpipe Version 10.9 (NIST IBBR), see (Alexandra Born, Janne Soetbeer, Frauke Breitgoff, Morkos A. Henen, Nikolaos Sgourakis, Yevhen Polyhach, Parker J. Nichols, Dean Strotz, Gunnar Jeschke, Beat Vögeli, Reconstruction of coupled intra- and interdomain protein motion from nuclear and electron magnetic resonance, 2021, J Am Chem Soc, 143, 16055-16067).

We combined all PRE, eNOE, residual dipolar couplings (RDCs) and scalar coupling restraints and calculated multi-state ensembles of Pin1. To account for the time- and ensemble-averaged nature of the probes, these restraints must be fulfilled by the average of the back-calculated contributions from each individual state. To allow large spatial sampling between the two domains, we 1) calculated the structure of the WW domain and then 2) froze the WW angles and used them as an input to determine the PPIase structure and interdomain positions (Alexandra Born, Morkos A. Henen, Parker J. Nichols, Beat Vögeli, On the use of residual dipolar couplings in multi-state structure calculation of two-domain proteins, 2021, Magn Reson Lett, 2, 61-68). In contrast to apo Pin1 (PDB ID: 7SA5 [http://doi.org/10.2210/pdb7SA5/pdb]), we omit RDC restraints from our ligand-bound ensembles as we saw that RDCs had a negligible effect on our resulting apo Pin1 ensemble. This has the advantage that we can exclude potential interactions between ligands and alignment media, or that the relative domain orientations are impacted by the induced alignment. The PDB IDs for FFpSPR- and pCDC25c-bound two-state ensembles are 7SUQ [http://doi.org/10.2210/pdb7SUQ/pdb] and 7SUR [http://doi.org/10.2210/pdb7SUR/pdb], respectively.

            The CYANA target function (TF, proportional to the sum of squared violations) was high for the single state structures (~300 Ų for all three ensembles) but decreased significantly upon addition of a second state to fulfill all the structure restraints. Note that the single-state structure is more akin to an averaged structure. All three Pin1 single-state structures resulted in a compact conformation as all the interdomain NOEs needed to be fulfilled. When a second state was allowed, an extended and a compact state were generated with all the interdomain NOEs fulfilled in the compact state.

While the interdomain NOEs and PREs were sufficient in orienting the two domains in apo Pin1 to agree with the DEER distribution, the position of the extended states did not fulfill longer distances from the DEER distance distributions with the ligand-bound ensembles. Therefore, we calculated population- and fluctuation-averaged distances between the Cbs of the spin-labeled residues based on the DEER distance distributions and used them as restraints in our structure calculations. A tolerance of ±5 Å was added to these effective distances to use as upper and lower limit restraints. While we optimized and lowered the PRE distance restraint weight to 0.01 (relative to the NOE weight of 1), we kept a weight of 1 for these DEER effective distance restraints as there were only 3 distances added for the calculation, and these restraints did not appreciably increase the target function. Importantly, the addition of the DEER effective distances adjusted the interdomain positions but did not change the intradomain structural correlations. When we calculated a ligand-bound two-state ensemble with the interdomain NOE, PRE, and averaged DEER restraints (see Supplementary Information), we obtained a satisfactory match to the experimental DEER distributions, with only a slight under-representation of small populations of longer distances.

CYANA, version 3.98 (http://www.cyana.org/wiki/index.php/Main_Page)

References:

• Güntert, P. & Buchner, L. Combined automated NOE assignment and structure calculation with CYANA. J. Biomol. NMR 62, 453-471 (2015)
• Schmidt, E. & Güntert, P. A new algorithm for reliable and general NMR resonance assignment. J. Am. Chem. Soc. 134, 12817–12829 (2012)