Influence of Point Mutations on PR65 Conformational Adaptability: Insights from Nanoaperture Optical Tweezer Experiments and Molecular Simulations

PR65 is the HEAT-repeat scaffold subunit of the heterotrimeric protein phosphatase 2A (PP2A) and an archetypal tandem-repeat protein, forming a spring-like architecture. PR65 conformational mechanics play a crucial role in PP2A function by opening/closing the substrate-binding/catalysis interface. Using in-silico saturation mutagenesis we identified “hinge” residues of PR65, whose substitutions are predicted to restrict its conformational adaptability and thereby disrupt PP2A function. Molecular simulations revealed that a subset of hinge mutations stabilized the extended/open conformation, whereas another had the opposite effect. By trapping in nanoaperture optical tweezer, we characterized PR65 motion and showed that the former mutants exhibited higher corner frequencies and lower translational scattering, indicating a shift towards extended conformations, whereas the latter showed the opposite behavior. Thus, experiments confirm the conformations predicted computationally. The study highlights the utility of nanoaperture-based tweezers for exploring structure and dynamics, and the power of integrating this single-molecule method with in silico approaches.


Introduction
Maintaining cellular signaling and homeostasis is crucial for the proper functioning of living organisms, and dysregulation of these processes can result in the development of many diseases.A complex interplay between kinases and phosphatases contributes to signaling events and cellular homeostasis 1 .
Abnormal activation of kinases and inactivation of phosphatases can lead to pathological hyperphosphorylation, a key factor in the development of numerous diseases, including cancer and neurodegenerative disorders 2,3 .Much attention has been given to kinase inhibitors for the treatment of these diseases.Phosphatases, on the other hand, have been much less studied as drug targets [4][5][6][7][8][9] mainly due to the lack of druggable pockets near their active sites 10,11 .
One major class of phosphatases playing a central role in maintaining cellular homeostasis is the family of serine/threonine protein phosphatases 2A (PP2A) [12][13][14] .PP2A is frequently dysregulated in human diseases, making it an attractive target for therapeutic interventions 15 .It is a heterotrimer, composed of a scaffold (A) subunit, known as PR65, a catalytic (C) subunit, and one of many regulatory (B) subunits (Figure 1A).The A and C subunits form the core of PP2A.The speci city of PP2A is controlled by the choice of the regulatory subunit that binds the AC core, with over 40 different B subunits each determining the speci c substrate bound to PP2A 3 .The diverse array of B subunits allows PP2A to exert control over a majority of cellular signaling pathways.
PR65 serves as a structural scaffold that provides a platform for the assembly of the heterotrimer 16 .Among the three PP2A subunits, PR65 experiences the highest frequency of mutations, which have been implicated in altering PP2A activity [17][18][19] .Understanding the impact of PR65 point mutations on PP2A structure and function is essential to unraveling the mechanisms underlying various diseases and developing targeted therapeutic strategies 20 .PR65 is a tandem repeat (TR) protein consisting of 15 HEAT repeats, each comprising ~ 40-residue antiparallel helices.These repeats stack in a one-dimensional fashion, forming an elongated, horseshoe-like superhelical structure composed of outer and inner helices layers.Many TR proteins act as hubs in multiprotein complexes, whereby their conformational uctuations facilitate the function of the assembly [21][22][23] .In the case of PR65, its mechanics play a crucial role in regulating PP2A function; the collective motions of PP2A mediated by PR65, open and close the enzyme's substrate binding/catalysis interface.Maintaining the exibility of PR65 to sample alternative conformers is crucial, with the closed state being active and facilitating the formation of the PP2A complex with catalytic and regulatory subunits 24,25 .Although the effects of PR65 mutations on folding and binding energetics have been studied [17][18][19][20] , a systematic investigation of the modulation of PR65's conformational state, exibility, and adaptability by point mutations is lacking.In this study, we aim to ll this gap and explore the effects of point mutations on the structure and dynamics of PR65.We focus on a unique aspect of PR65 structural mechanics-the so-called 'hinge' sites that coordinate the global dynamics of the entire complex.Hinge regions usually play a key role in mediating the conformational mechanics of the proteins and enabling conformational changes that underlie functional transitions 26 .
By speci cally targeting mutations at the hinge sites, we aim to investigate how subtle alterations in these regions in uence the conformational space accessible to PR65.Our approach is to examine the changes in the conformations and dynamics of PR65 induced by introducing point mutations at those sites.We hypothesize that certain mutations may restrict the conformational adaptability of PR65, potentially impacting the formation of the PP2A complex and its activity.To assess the effect of point mutations, we integrated molecular dynamics (MD) simulations and elastic network model-based analysis 27 and experimentally tested the ndings using nanoaperture optical tweezer measurements 28 .
An outstanding challenge in biology is how to visualize protein structure at the single molecule level and on functionally relevant timescales without the use of labels, immobilization, or tethering, which can result in artifacts that perturb the system we are trying to observe 29 .Here we present nanoaperture optical tweezers.Optical tweezers have emerged as a powerful tool for probing the biophysics of proteins at the molecular level.The present study demonstrates the utility of the enhanced eld con nement and sensitivity provided by nanoaperture-based tweezers for studying the structure and dynamics of single proteins.Unlike conventional optical tweezer techniques, this approach allows for characterization of individual unmodi ed proteins in solution for extended durations and without the need for tethers or labels 30,31 .
Using site-directed saturation mutagenesis in-silico 32,33 , six PR65 mutants (Y168V, L197V, D315E, S323L, E375D, and F502W) because of their colocalization with the global hinge sites.Extensive MD simulations revealed that S323L, E375D, and F502W favored the adoption of relatively more extended conformations compared to the wild type (WT), while the conformational exibility enabling the uctuations between open and closed conformers was relatively suppressed.The Y168V and D315E mutants, in contrast, favored relatively more compact conformations with mixed effects on conformational dynamics.The F502W mutation helped to regulate the transitions between the compact and extended form in a more favorable way than the WT PR65.D315E, on the other hand, signi cantly perturbed the overall stability and dynamics, interfering with the ability of PR65 to adapt to conformational changes required for function.Concurrently, by trapping the mutants in the optical tweezer setup, we measured their motion and observed that the S323L, E375D, and F502W mutants shifted towards extended conformations, exhibited by higher corner frequencies and lower root-mean-square-deviations (RMSD) in their amplitudes of motions compared to the WT PR65.Since elongation typically leads to a higher polarizability 34 and therefore larger optical tweezer stiffness, these ndings appear to independently con rm the conformational variability for all six mutants.Overall, our study represents an integration of results from in-silico saturation mutagenesis, MD simulations, and optical tweezers experiments to characterize the conformational mechanics of PR65 and its mutants.The combination of these complementary approaches provides insights into the modulation of PR65 conformational exibility and adaptability, laying the foundation for further investigations and potential therapeutic interventions.It also offers an integrated protocol for exploring the structural and dynamic consequences of mutations, generalizable to other systems and highlights the utility of optical tweezers in molecular biophysics applications.

Results
In-silico Saturation Mutagenesis of PR65.The in-silico saturation mutagenesis study of PR65 was performed using a recently introduced structure-and dynamics-based machine learning methodology, implemented in the online accessible tool Rhapsody 32,37 (Figure S1).The approach allows for assessing the impact, neutral or pathogenic, of any substitution at any residue along the protein based on sequence (conservation and co-evolution), structure (accessible surface area), and dynamics (equilibrium uctuations, allosteric couplings, and mechanical behavior) of the protein.In parallel, we estimated the change in folding free energies associated with point mutations using ProTSPoM 33 (Figure S2A).ProTSPoM uses residue physicochemical and energetic properties in the folded state, environmental compatibility, and evolutionary information to predict the change in Gibbs free energy (DDG) of folding associated with point mutations.
Figure 1C-F presents the results for the apo structure of PR65.The diagram in panel C is color-coded by average pathogenicity score for each residue I, i.e., the probability of having a deleterious/pathogenic effect upon mutating the i th residue, averaged over all 19 amino acid substitutions at that position.The effects of the individual substitutions are described by the elements of the i th column in the saturation mutagenesis heat map (see Figure S1 for a PR65 segment).The scores vary from 0 (neutral, blue) to 1 (strongly pathogenic, red), with a cutoff of 0.65 determining the decision between neutral and deleterious.
Using the residue ranges de ned earlier 36 , we evaluated the pathogenicity scores of the residues within each of the 15 HEAT repeats.The results are presented in Figure 1D, organized by repeat number (ordinate) and corresponding residue positions (abscissa).The heat map shows that the counterparts of the repeat 1 residues P11 (in helix 1), L26, R27, S30, and L34 (in helix 2) in all repeats consistently exhibit high pathogenicity probabilities.These residues are indicated by blue arrows along the abscissa.Their resistance to tolerate mutations is consistent with their high degree of sequence conservation at those positions, usually occupied by hydrophobic residues (leucine, valine, and isoleucine) or by arginine.See the counterpart of this heat map corresponding to change in free energy of folding, ΔΔG, in Figure S2B.
Closer examination of HEAT repeat structural elements (loop 0, helix 1, loop 1, helix 2 and loop 2; Figure 1E) revealed the distinctive behavior of inter-and intra-repeat elements.In Figure 1F we present the average pathogenicity for these structural elements.The corresponding residue ranges are listed in Table S1 36 .Notably, the loops 0 and 2 linking successive repeats generally exhibit relatively high pathogenicity if mutated, suggesting the high sensitivity of PR65 if not inability to tolerate mutations at inter-repeat regions.See the peaks in Figure 1F between repeats 1-2, and those at the loop 0 or 2 between repeats 6-7, 8-9, 10-11, 11-12, and 12-13.The latter two represent kinks of single residues (G434 and G473, respectively), rather than loops, that presumably play a critical role.The counterpart of this analysis for ΔΔG is presented in Figure S3, which also draws attention to the critical role of S119 between repeats 3-

4.
This analysis therefore identi ed the inter-repeat loop residues to generally play a critical role in ensuring the overall stability and/or functional exibility of PR65.Closer analysis also identi ed speci c mutations at the regulatory and catalytic subunit interfaces of PR65 that would induce the strongest destabilization and pathogenicity.Table 1 lists these mutations, Figure S4 displays their location in the PP2A structure.
We note again the propensity of helix 2 residues among these critical sites.Selection of Hinge Site Mutations.Our goal in this study was to explore the possibility of altering the conformational state and exibility, and thereby function, of PR65 without completely destabilizing the scaffold or abolishing its function.Toward this goal, we turned our attention at residues predicted to play a key mechanical role as hinges/anchors during cooperative movements (global modes of motion) of PR65.We focused on 21 residues predicted by the Gaussian network model (GNM) 38,39 to participate in hinge regions modulating the softest six modes (Table S2) and explored how substitutions at those sites could alter the PR65 structure and dynamics.Figure 2A-D shows the shapes of GNM modes 1-4 (left) and corresponding diagrams color-coded by the direction (middle) and size (right) of residue displacements along those modes.
Based on the pathogenicity scores and ΔΔG values predicted for selected point mutations at those hinge sites (Table S2), we categorize the hinge site mutations into two broad groups, (a) pathogenic and (b) non-pathogenic depending on their pathogenicity scores.The former group also entails an increase in ΔG, indicating that these mutations would be destabilizing.The latter group, on the other hand, which is of interest as potential mutations that can alter the function while retaining the fold, is divided into three subgroups based on their DDG values: (b1) not destabilizing (negative ΔΔG), (b2) mildly destabilizing (ΔΔG < 1.25 kcal/mol), and (b3) highly destabilizing ΔΔG ≥ 1.25 kcal/mol, as mentioned in Table S2.
Experimental assessment of the thermodynamic stability of mutants.We previously used E. coli to express PR65 WT and mutants for folding studies 20 .To test the thermodynamic stabilities of the mutants in Table S2, we rst examined the mutations in group (a), which were predicted to adversely affect the protein or function (pathogenicity score > 0.65).As seen in Table S2, the group comprises of 12 mutations.Most of them are also predicted to be highly destabilizing (DDG ≥ 1.25 kcal/mol).We performed small-scale protein expression tests in E. coli on them and the results showed that 11 of these mutants had no expression or were insoluble, indicative of instability and thus consistent with the predicted impact of these mutations from the computational analysis.On the other hand, out of the six mutations predicted to be stabilizing and non-pathogenic (subgroup b1), three -D315E, S323L, E375Dwere expressed in good quantity.One mildly destabilizing and non-pathogenic mutation (subgroup b2), F502W, was also successfully expressed.Notably, two subgroup b3 mutations Y186V, and L197V (predicted to be neutral by Rhapsody but destabilizing by ProTSPoM), were also expressed in good yield.
We next performed large-scale expression of these six mutants and used thermal unfolding to qualitatively assess their thermodynamic stabilities as measured by melting temperature (the temperature at which the protein is 50% unfolded) (Table S3).All mutants had melting temperatures within 1 °C of the WT value (51.3 °C), indicating that the mutations had only very small effects on stability.Therefore, experiments were in general in accordance with predicted potential pathogenicity; and they were also consistent with the predicted mild-to-none effects of mutations on fold stability except for the b3 mutations.We therefore moved on to examine the impact of these hinge site point mutations on the structure, dynamics, and potentially function, of PR65 by molecular dynamics (MD) simulation and optical tweezer experiments.
MD simulations indicate that the mutants S323L, E375D, and F502W preferentially sample extended conformations.Simulations were initiated from the compact form of PR65, taken from the heterotrimeric PP2A 3 .The distributions of the end-to-end distances, de ned as the distance between the C α atoms of N29 and F577 (as in earlier work 36 ), are presented in Figure 3A and B-G (six mutants).In each case, the average histogram deduced from triplicate runs is shown in the left panel, and the individual histograms from each of the three runs are shown in the middle.The mean values and standard deviations (SD) are written in each case, and their averages over triplicate runs are reported in Table 2 columns 3 and 4. The panels on the right of Figure 3 show the time evolution of the end-to-end distance for each run.The triplicate runs conducted for each of the mutants S323L, E375D, and F502W also showed overlapping distributions of end-to-end distances despite small shifts (middle histograms in Figure 3E-G).
However, the main difference from the WT PR65 was the shifts in the end-to-end distances towards more extended states.The corresponding mean end-to-end distances (70.3, 68.4 and 70.4 Å, respectively; cumulative histograms on the left) are larger than that of the WT.Thereby, these mutations favor more extended conformations in comparison to the WT PR65.
In contrast, the mutants Y168V and L197V (Figure 3B-D) were observed to sample end-to-end distances comparable to that of the WT PR65, if not more compact forms.D315E was able to sample much more compact conformations (as low as 17.5Å) than WT and gave the lowest mean end-to-end distances.For Y168V, L197V, and D315E, the main effect of mutations seems to compromise the ability of the structure to uniformly sample the conformational space; instead, the individual runs tend to gravitate/drift towards different forms, as evidenced by the histograms (middle diagrams) that show only a partial overlap.This effect was particularly pronounced in the mutant D315E, where two of the runs sampled rather compact forms with new peaks appearing at end-to-end distance of 41.3 and 48.0 Å -; whereas the third run sampled an extended form (mean value of 72.7 Å) with no transition to the compact form (Figure 3D).
The interface between repeats 12 and 13 signi cantly contributes to the opening and closing of PR65. Figure 4 displays the root-mean-square-uctuations (RMSF) pro le of residues (average size of uctuations observed in triplicate trajectories).The regular patterns of the repeat units can be distinguished.Panel B displays the mutants colored by their RMSFs, in line with the shades in panel A. Examination of the RMSFs shows that the structure can be divided into three substructures: a middle section comprised of repeats 3-12 that shows small displacements (in blue), anked by two segments (Nterminal repeats 1-2 and C-terminal repeats 13-15) that move signi cantly in space (in green).
Further examination of these individual sections shows that their spatial displacements do not necessarily re ect their conformational exibilities.For example, even though the middle section is subject to minimal motions, it undergoes substantial internal rearrangements or deformations, as measured by the internal RMSDs (between 2.7 Å and 4.0 Å) evaluated for each mutant (by aligning with compact PR65 (PDB: 6NTS)).These rearrangements are presumably required to accommodate the local conformational uctuations with minimal effects on the anking regions.In contrast, the N-and Ctermini that signi cantly move in space show much smaller internal RMSDs indicative of en-bloc movements of the repeats.In particular, the C-terminal section undergoes such rigid overall reorientation with respect to the middle section, enabled by hinge-bending at the interface between repeats 12 and 13.The internal RMSDs are con ned in this case to 1.2-1.6Å.These rigid-body movements of the C-terminal section, combined with the conformational rearrangements of the remaining structure, enable the opening/closing of PR65 that may sample compact and extended forms, as illustrated in Figure 4. Notably, F502W, located at the inner helix (helix 2) of repeat 13 near this hinge region, signi cantly alters PR65 equilibrium dynamics in favor of more extended conformations, underscoring the mechanical signi cance of this particular site.
Compared to WT PR65, F502W exhibits an increased ability to transition between open and closed forms, whereas D315E exhibits a decreased ability.To further investigate the effect of these point mutations on PR65 structural dynamics, we evaluated the correlation cosines between the global movements observed during the simulations and the deformation vector representing the experimentally observed difference between the compact and extended PR65 structures.The global movements sampled in MD simulations were characterized by principal component analysis (PCA) 40 .The PCA was performed using the triplicate MD trajectories (total of 1.962 µs) generated for WT PR65 and each of the six mutants.First, we aligned the conformations observed in MD with the compact PR65 structure to exclude rotational and translational motions.Thus, PCA yields 3N-6 internal motions.The rst principal component (PC1) describes the most dominant mode of collective motion, which is also energetically favorable (soft mode), succeeded by PC2 and PC3.
Our goal was to assess whether the mutations impaired (or enhanced) the ability of PR65 to undergo its functionally required transitions between extended and compact forms.To this aim, we calculated the 3N-dimensional deformation vector pointing from the compact (PDB: 6NTS) to the extended (PDB: 1B3U) structures and examined if/how the PCs derived from MD simulations correlated with this structural change.As a quantitative measure of the ability of the PR65 mutants to effectuate these functional movements, we used the correlation cosine between the PCs (from simulations) and the deformation vector (from experiments) 16 .The heatmap in Figure 5 presents the results for all simulated systems.First, we note that the WT PR65's PC1 yields a correlation cosine of 0.756 with the experimentally observed structural change, indicating this mode's ability to predict 75.6% of the structural transition between the compact and extended PR65 structures.This is in accord with the previously reported intrinsic ability of the scaffold to accommodate, if not drive, these functional changes in structure.More important is to see to what extent the mutants exhibit similar abilities.In the cases of D315E and L197V mutants, PC1's ability to describe the conformational transition between the compact and extended forms decreased to 69.55% and 73.37% respectively, indicating a small loss in the ability of the mutant to accommodate these changes in structure.In contrast, Y168V, S323L, and E375D, yielded values of 78.23%, 77.88%, and 78.37%, respectively, suggesting that the PR65 global dynamics is robust to those point mutations.Strikingly, the PC1 of F502W stood out from the others by yielding a correlation cosine of 0.8844 between PR65's compact and extended forms, which is even higher than that of the WT.This result draws attention to the signi cance of F502 on PR65's functional dynamics (and enhancing it when replaced by a tryptophan), which may have important rami cations for redesign or alteration of catalytic activity.
We further evaluated the cumulative correlation cosines with subset of 2 modes (PC1-2 in Figure 5), in accord with our previous study 41 .The cumulative correlation cosines for F502W PC1-2 reaches 0.895, which is higher than that of WT (0.858).D315E results in lower cumulative correlation cosines (0.739 in PC1-2) than the WT.Therefore, our ndings suggest that the single point mutations F502W could impart alterations in protein dynamics that promote the ability of PR65 to undergo transitions between the compact and extended structures to accommodate trimeric assembly or diverse regulatory subunit binding.Conversely, D315E nudges the system towards a state that less favorably accommodates such transformations.These shifts could potentially impact the function of PR65.
Nanoaperture Optical Tweezer based characterization of the PR65 protein and its variants.Optical tweezers have been used widely to probe the biophysics of proteins at the single molecule level [42][43][44] .By using enhanced eld con nement and sensitivity, nanoaperture-based plasmonic tweezers have been adopted by several groups to study single proteins, protein complexes and their interactions without the need for (or potential impact from) tethers or labels 30,31,[45][46][47][48][49] .Here we trap the protein using double nanoholes 50 fabricated by a random colloidal lithography technique 28 .A 980 nm laser with a 1.3 NA 100× objective is focused on the aperture, with 22.5 mW of power incident on the aperture in a diffraction limited spot.The transmission through the aperture is monitored on an avalanche photodiode (using a 1.3 OD lter to prevent saturation).When trapped, the PR65 protein undergoes a characteristic step as it enters the trap, with an increase in the noise amplitude, as shown in Figure 6A.
Once in the trap, the Brownian motion of the particle results in increased "noise" in the photodiode signal (transmission through the aperture, T, normalized to the pre-trap level).A histogram of this noise sampled after the trapping event is given in Figure 6B.The stiffer the optical tweezer potential, the less motion that the particle undergoes 42 , so the amplitude of the noise is expected to be smaller (RMSD).The stiffness is proportional to polarizability of the particle, which is higher for longer particles, so less deviation is expected if the protein is extended by a point mutation.Another quantity that can be extracted from the detected transmission is the power spectral density shown in Figure 6C.This has a corner frequency proportional to the trap stiffness divided by the hydrodynamic drag on the protein 42 and so it is expected to show the opposite trend of increasing as the particle lengths (opposite to the RMSD).These two quantities for the point mutations are compared with the WT in Figure 6D-E and Table 2.
As noted above, S323L, E375D, and F502W shifted towards the extended conformation, and these showed the highest corner frequencies among all studied mutants as well as the WT PR65 (Figure 6D).They also showed the lowest RMSD (Figure 6D), however, E375D was larger than the WT.We stress that this RMSD is the result of Brownian motion of the protein in the optical potential, and therefore re ect both overall tumbling and global internal motions, different from the internal motions seen in MD simulations which occur at a much faster timescale, even though the end-to-end uctuations observed in MD and the extracted PCs re ect relatively slow events.Both of these ndings separately con rm the insilico prediction that the protein is extended in the presence of these three mutations S323L, E375D, and F502W.The mutants L197V, Y168V and D315E, on the other hand, showed the opposite trend in the experiments, and this con rms the in-silico predictions of a more compacted form (Figure 6F).Finally, we note that the most dramatic behavior departing from the WT (and other mutants) has been observed in the mutant D315E, which is also consistent with MD results where D315E is distinguished by the impact of the point mutations on its structure (Figure 4) and functional dynamics (Figure 5).

Discussion
PP2A is a heterotrimeric Serine/Threonine protein phosphatase that plays a signi cant role in maintaining cellular processes and signaling pathways and whose dysregulation has been linked to multiple cancers, Alzheimer's disease, and increased susceptibility to pathogen infections.In this study, we explored the effects of point mutations on the structure and dynamics of PR65, the scaffold subunit of the heterotrimeric PP2A complex.By targeting speci c mutations at the hinge sites of PR65, we aimed to investigate how subtle alterations in these regions can in uence the conformational space accessible to PR65 and potentially impact the assembly and function of the PP2A complex.Our in-silico analysis revealed that mutations at certain hinge sites, such as S323L, E375D and particularly F502W, stabilized relatively more extended conformations of PR65, whereas other mutations, e.g., Y168V and speci cally D315E, favored a more compact form.In addition, mutations F502W and D315E affected the global dynamics of PR65, though they did so in contrasting manners: F502W enhanced the ability of PR65 to accommodate its structural transition between its compact and extended forms, whereas D315E had the opposite effect.
We leveraged the power of optical tweezers to analyze the changes in conformational exibility of both the WT PR65 and its mutants at the single-molecule level.Unlike conventional optical tweezers that use labels and/or tethers, we used nanoplasmonic optical tweezers that allow for studying the unmodi ed protein at the single-molecule level.Comparison of the optical tweezer data obtained for PR65 mutants with those observed in the WT PR65, revealed trends consistent with the molecular simulations.The mutants S323L, E375D, and F502W showed the highest corner frequencies in the experimental measurements, indicating that these mutations resulted in higher trap stiffnesses and more extended conformations, and the opposite trend was observed for the other mutants.
This study illustrates how our recently developed in-silico saturation mutagenesis screen can identify critical residues where mutation is associated with decreased functionality while retaining their fold.The ndings contribute to a better understanding of the changes in the structure and dynamics of PR65 as a function of amino acid substitutions, thus providing new insights for rational modi cation or redesign of its function.Importantly, the multidisciplinary approach utilized in this study can be applied to investigate other proteins and facilitate the development of targeted therapies.Future exploration of the impact of point mutations on modulating the conformational space of PR65 may pave the way for the development of novel therapeutic strategies for diseases associated with PP2A dysfunction.Notably, the current integrated experimental and computational analysis helps dissect the role of individual residues in supporting the conformational mechanics of PR65.Building upon the established optical trapping approach, mmWave-THz dielectric spectroscopy emerges as a viable method for the real-time tracking of protein dipole movement and globular vibration modes.This technique offers the capability to investigate ultrafast dynamics implicated in extensive biomolecular conformational shifts on a signi cant scale, while maintaining a non-contact and non-intrusive nature.Thus, it presents a compelling pathway for delving into the intricacies of PR65 protein conformational alterations.By unraveling the complexities of PR65 and its role within the broader PP2A family, the study helps us move closer to unlocking the potential for targeted therapies and improved treatments for diseases linked to PP2A dysregulation.

Methods
System and MD simulations-The PR65 structure resolved for the trimeric PP2A, deposited in the Protein Data Bank (PDB: 6NTS) 3 was used as starting conformation in our simulations.PR65 is in its compact form in the trimer.The structure was simulated in TIP3 explicit water having 25 Å of water padding in all directions.Thus, providing at least 50 Å of water between PR65 and its periodic images.Ions were added to neutralize the systems and ion concentrations were set to 150 mM NACl.System size was ~ 164,147 atoms for WT PR65 simulations.All system preparations were performed in VMD and all MD simulations were performed in NAMD3 51 using the CHARMM36 all-atom additive protein force eld 52 .A time step of 2 fs was used in the simulations.Temperature was kept constant at 310 K via Langevin dynamics using a damping coe cient of 1 ps − 1 .The pressure was kept at 1 atm using the Langevin Nosé-Hoover method with an oscillation period of 100 fs and a damping time scale of 50 fs.A cut-off distance of 12 Å was applied for van der Waals interactions.To calculate long-range electrostatic interactions, the particlemesh Ewald method was used.PR65 mutants were generated by introducing single point mutations using the mutator plugin of VMD.PR65 mutants were simulated following the steps indicated above.
Two rounds of minimization and equilibration simulations were performed prior to each production run.First, the protein was maintained in a xed structure and the system was subjected to 10,000 energy minimization steps, which was followed by 1 ns of stabilization to equilibrate the solvent around the protein.Subsequently, a second round of minimization-equilibration was performed, where each system underwent an additional 10,000 step minimization, this time without any restrictions, which was subsequently followed by 4 ns of stabilization using harmonic constraints (of 1 kcal/mol/Å 2 ) on C αatoms only.Following these simulations, constraints were completely removed, and the system went through another round of 4 ns equilibration.Upon completion of this second round of minimizationequilibration, production runs were performed.
Conformations were sampled every 0.1 ns in the MD simulations and used in RMSD, RMSF, and PCA calculations.Thus, when combining the three sets of simulations for each of the WT and mutant systems, we obtained an ensemble of 19620 snapshots from a total of 3x654 ns = 1962 ns of MD simulations.Cumulatively MD simulations of 13.734 µs were evaluated.We performed all calculations using our custom analysis codes, executed in VMD and MATLAB, which also utilized some of their built-in functions.
In silico saturation mutagenesis-We used the 32,37 tool to carry out in-silico saturation mutagenesis on the apo form of PR65 (PDB: 1B3U).Rhapsody was used to predict the pathogenicity corresponding to all possible single point mutations at each residue position of PR65.We further used ProTSPoM 33 to predict the change in Gibbs free energy of folding associated with all possible point mutations for each residue position.
Hinge site detection-Hinge sites within a speci c ENM mode refer to regions that exhibit minimal displacements, if any, during that particular mode.Residues participating in these regions act as pivotal or anchor points, connecting substructures that move collectively around them and as such, they play a crucial mechanical role.In the GNM analysis, the hinge sites are identi ed as the zero-crossover points in the mode shapes generated for each mode 38,39 .The i th mode shape is obtained by plotting the elements of the i th eigenvector of the N x N connectivity/Kirchhoff matrix as a function of residue index for a protein of N residues 38,39 .In our study, we speci cally concentrated on the global hinges found in the soft (lowest frequency) GNM modes, e.g.modes 1-6 at the lower frequency range of the mode spectrum.
We used the calcHinges function of ProDy 53 with the default parameters and protocol to compute the hinge sites corresponding to these global modes.
Optical trapping experiments.To perform the optical trapping experiment, we rst make a microwell on a clean glass microscope slide of 150 µm thickness (Ted Pella, Inc.) using an imaging spacer (Secure Seal imaging spacer, Grace Bio-labs) to form an open chamber measuring 120 µm in depth and 9 mm in diameter.Using a this chamber was lled with 10 µL of the analyte, diluted to 20 times its original concentration, and a sample of gold-on-glass, on which double nanohole (DNH) apertures have been fabricated by colloidal lithography, was inverted over it, sealing off the chamber.The sample so prepared was placed on a sample stage between a 100× oil immersion objective and a 10× collection objective, with the side of the microscope cover slide in contact with the oil immersion objective.By turning the z-knob on a 3-stage piezo-controller used to move the sample mounted on the sample stage, a focal position was reached whereupon multiple bright spots, corresponding to apertures on the gold-onglass sample, show up on a computer screen connected to a CCD camera which captures light going through the apertures.The laser was then turned on to a very low drive current (18 mA, for example) and the x-, y-knobs of the piezo were used to move the gold-on-glass sample in a horizontal plane to select an aperture by aligning a white spot on the screen to the center of the laser's diffraction pattern on the same screen.A half-wave plate (Thorlabs, WPH05M-980) mounted on the laser path was used to check whether the selected aperture was a DNH by rotating the half-wave plate and observing the change in signal level on a computer screen connected to a USB-4771A data acquisition module from Thorlabs.For a DNH, the change in signal level should be between 30% and 50% 28 .If an aperture failed this test, a different one was selected by the same procedure and the test was repeated until a DNH was found.The laser power was then increased to 22.5 mW before the 100× objective (corresponding to a drive current of 79 mA by our calibration), and identify trapping, observed as a discrete jump and an increase in noise of at least 10% on the screen connected to the signal acquisition module (as in Fig. 7A above).The data, acquired at a 100 kHz sampling rate, was then exported to MATLAB for analysis.
Protein expression and puri Site-speci c mutations were introduced to the thrombin cleavable GST-PR65-H 6 fusion protein pRSETa plasmid using Quikchange Site-directed mutagenesis protocol (Agilent (UK) Ltd.).PR65 proteins (wild-type and mutants) were expressed in E. coli as described previously 54 .
Protein measurements.stability was measured using a thermal shift assay on a Bio-Rad CFX Connect qPCR instrument, whereby the unfolding is detected by uorescence of the hydrophobic dye Spyro Orange that binds to the unfolded state of the protein.The experiments were performed in clearbottom, half-volume, 96-well plates using nal well volume of 25 µL, PR65 concentration of 10 µM and Sypro Orange concentration of 0.025 µM.The samples were incubated at 25°C for 2 minutes before increasing the temperature by 0.5°C every 30 seconds up to 70°C.At each temperature, the uorescence intensity was measured using an excitation wavelength of 471 nm and an emission wavelength of 570 nm.Data were analyzed using the GraphPad Prism software.
PP2A and F577 are used as references to de ne the end-to-end extension 36 .(C) PR65 color-coded by average pathogenicity of residues as predicted by Rhapsody.The color ranges from blue (lowest probability of being deleterious; 0.12 here) to red (highest probability, 0.80).Note that mutations at the inner portions of the repeats generally tend to have more deleterious effects compared to the outer regions.(D) Heat map showing the average pathogenicity of residues (abscissa) within each of HEAT repeat (ordinate).Nonexistent residue positions are shown in black.(E) The loop0-helix1-loop1-helix2-loop2 motif of HEAT repeats, illustrated here for Repeat 3. Helix 1 is outer and helix 2 is inner.(F) Average pathogenicity probability for the helices and loops of each HEAT repeat.Consistent with panel A, helix 2 tends to exhibit higher sensitivity to mutations than helix 1; whereas the inter-repeat loops 0 and 2 exhibit higher pathogenicity than the intra-repeat loop 1.The color-coded secondary structure corresponding to the rst repeat is shown along the abscissa (bottom) of panel B. Note that not all repeats have the same helix and loop lengths.S2) were found to be expressed and soluble in signi cant amounts, enabling further investigation of their impact on PR65.The same results are presented for GNM modes 2-4 in the respective panels (B)-(D).
The mutants S323L, E375D and particularly F502W sample more extended conformations than WT PR65 End-to-end distributions and evolution for WT PR65.Left and middle panels show the distribution of end-to-end distances for the combined MD trajectory (blue) and each set of simulations separately (light blue, magenta, and orange), respectively.The mean ± standard deviation is indicated in each histogram.Location of peaks are indicated on the distributions.Dashed linesrepresent the end-toend distances observed in the crystallographically resolved compact (PDB: 6NTS) and extended (PDB: 1B3U) structures of PR65.The right panel shows the time evolution of the end-to-end distance.(B-G) Same as A for the indicated mutants.
heterotrimer, conformational and potential pathogenicity of its scaffolding subunit PR65.(A) Two different views of the PP2A trimer (Protein Data Bank (PDB): 6NTS 3 ) with PR65 shown in navy/mauve, and the catalytic and regulatory subunits in yellow and gray, respectively.The locations of six residues mutated in the present study are indicated by beads.(B) Compact (solid) and extended (green, transparent; PDB: 1B3U 35 ) conformations of PR65 in the trimer and in isolation, respectively.N29

Table 1 .
PR65 mutations distinguished by highly destabilizing and/or pathogenic effects

Table 2 .
Pathogenicity and conformational dynamics of the six selected mutations (76.3 Å), with a mean value of 66.2 Å.All three independent runs consistently exhibited similar distributions.