Conformational Excitation and Non-Equilibrium Transition Facilitate

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Biophysical Chemistry, Biomolecules, and Biomaterials; Surfactants and Membranes

Conformational Excitation and Non-Equilibrium Transition Facilitate Enzymatic Reactions: Application to Pin1 Peptidyl-Prolyl Isomerase Toshifumi Mori, and Shinji Saito J. Phys. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acs.jpclett.8b03607 • Publication Date (Web): 04 Jan 2019 Downloaded from http://pubs.acs.org on January 10, 2019

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The Journal of Physical Chemistry Letters

Conformational Excitation and Non-Equilibrium Transition Facilitate Enzymatic Reactions: Application to Pin1 Peptidyl-Prolyl Isomerase Toshifumi Mori∗ and Shinji Saito∗ Institute for Molecular Science, Myodaiji, Okazaki, Aichi, 444-8585, Japan, and School of Physical Sciences, The Graduate University for Advanced Studies, Okazaki, Aichi 444-8585, Japan E-mail: [email protected]; [email protected]

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Abstract Conformational flexibility of protein is essential for enzyme catalysis. Yet, how proteins conformational rearrangements and dynamics contribute to catalysis remains highly controversial. To unravel proteins role in catalysis, it is inevitable to understand the static and dynamic mechanisms simultaneously. To this end, here the Pin1catalyzed isomerization reaction is studied from the two perspectives. The static view indicates that the hydrogen bonds involving Pin1 rearrange in a tightly coupled manner with isomerization. In sharp contrast, the isomerization dynamics are found to be very rapid; protein’s slow conformational rearrangements thus cannot occur simultaneously with isomerization, and the reaction proceeds in a non-equilibrium manner. The distinctive protein conformations necessary to stabilize the transition state are prepared a priori, i.e. as conformational excited states. The present result suggests that enzymatic reaction is not a simple thermal activation from equilibrium directly to the transition state, thus adds a novel perspective to Pauling’s view.

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Enzymes catalyze chemical reactions in a highly efficient manner. The catalytic cycle involves ligand binding, chemical reaction, and product release steps; yet, in many reactions, the chemical reaction step has been of particular interest, and the transition state theory has played a central role in studying the microscopic mechanism of enzyme catalysis. 1–3 Thus, free energy barriers and kinetic rates of the catalyzed chemical reactions have been studied extensively, yet the dynamic aspect, e.g. how the reactions actually proceed, have gained less attention. Recently, conformational heterogeneity of proteins has become of great interest, 4–11 and the dynamic nature of protein conformations has further been realized to be important for function. 10–16 In addition, single-molecule experiments further suggest that individual rates of enzymatic reactions can be also heterogeneous. 17 Yet, how the conformational changes of proteins play a role in enzyme catalysis remains unclear, e.g. whether protein dynamics contribute to catalysis have been highly controversial. 18–27 In order to advance the microscopic understanding of enzyme catalysis, it is thus essential to elucidate the dynamics of catalytic reactions, and bridge the gap between the view of thermal activation along the free energy projected onto a reaction coordinate and that of reactive transition dynamics. In this regard, here we study the cis-trans isomerization reaction catalyzed by Pin1 based on the free energy of isomerization and dynamic calculations. Pin1 is a peptidyl-prolyl isomerase (PPIase) that selectively catalyzes the isomerization of the peptidyl-prolyl bond in the phosphorylated Ser/Thr (pSer/pThr)-Pro motif. Pin1 is known to facilitate protein folding, and also plays an important role in cellular signaling 28 and gene expression 29 by correlating phosphorylation with isomerization-induced conformational changes. Dysfunctioning of Pin1 thus leads to varisous diseases including cancer 30 and protein folding diseases such as Parkinson’s and Alzheimer’s. 31,32 Pin1 consists of catalytic and WW domains; while the two domains are shown to correlate when Pin1 function, 33,34 the isomerization activity is considered to be maintained solely at the catalytic domain. 35 Pin1 reduces the free energy barrier for isomerization by ∼7 kcal/mol without any bond formation or cleavage. 35–37 Yet,

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the molecular basis for the isomerization reactions remains elusive, 38–41 and little is known about the reaction dynamics. The system thus is an ideal showcase to study the conformational transitions during the isomerizations using extensive molecular dynamics simulations. In this work, we study the isomerization reaction of a model ligand, Ace-Ala-pSer-ProPhe-Nme 36,37 (hereafter denoted as residues 164 to 170), catalyzed by Pin1 as well as without the enzyme from the free energy and the transition dynamics perspectives. By comparing the catalytic reaction mechanism from the two approaches in molecular detail, we are able to reveal the reaction mechanism and the fundamental role of protein conformational flexibility and dynamics in enzyme catalysis. First we calculate the free energy profiles for the catalyzed and uncatalyzed isomerization reactions. The improper angle ζ between pSer167 and Pro168 (Fig. 1(a)) is used as a reaction coordinate, and replica exchange umbrella sampling (REUS) and umbrella sampling (US) simulations are performed for the catalyzed and uncatalyzed reactions, respectively. The details of the simulations are provided in Supporting Information. Fig. 1(c) shows the free energies of isomerization as a function of the improper angle ζ, defined in Fig. 1(a). The free energy barrier is found to decrease by ∼5.0 kcal/mol by Pin1, consistent with previous studies. 35–37 We also see a slight shift in the location of the transition state between the two reactions, which may be due to steric distortion caused by the protein environment (see below). To discuss how each residue contributes to stabilizing the cis-trans isomerization, the hydrogen bond (HB) contacts about the ligand are calculated along the REUS trajectories. Here, hydrogen bond contact, qij , is defined as 1 − [dij (t)/d0 ]6 qij (t) = 1 − [dij (t)/d0 ]12

(1)

where dij (t) = |rij (t) − r0 | with rij (t) being the distance between the hydrogen and oxygen atoms, and d0 and r0 are set to 1.0 ˚ A and 2.0 ˚ A, respectively. Fig. 1(d) summarizes

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Figure 1: Pin1-catalyzed isomerization mechanism based on the free energy of isomerization. Representative transition state snapshots of (a) the ligand and the residues directly interacting with the carbonyl oxygen of pSer167 (shown in red ball) and (b) the residues about the ligand that rearrange hydrogen bond (HB) contacts during isomerization along ζ. (c) Free energy profiles along ζ, where ζ is defined in panel (a). (d) The mean of the HB contacts from the REUS trajectories. The hydrogen atoms are shown in (a) and (b) only when they are involved in the HB contacts summarized in (d). Black, orange, and green colors in (a), (b), and (d) represent the ligand-ligand, ligand-protein, and protein-protein HBs. As the phosphoryl group of pSer167 has three symmeteric oxygen atoms (denoted as (“OP”), the one closest to Arg68(H) is used in calculating the distance between pSer167(OP) and Arg68(H).

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the mean of representative qij s along ζ. In accord with previous studies, 36,37 Fig. 1(d) shows that the carbonyl oxygen of pSer167 (pSer167(O)), responsible for the isomerization, forms HBs with the Pin1 residues Cys113(Hγ ), His59(H 1), His157(H 1), Ser154(Hγ ), and Gln131(H 2) as ζ changes from 0◦ to 180◦ (cis- to trans- states). These residues thus guide the rotation about ζ. The HBs not involving pSer167(O), e.g. pSer167(OP)-Arg68(H ) and Phe169(O)-Arg68(Hη 22), are also found to contribute to catalysis. The intra-ligand HB Ala166(O)-Phe169(H) appears frequently at the transition state as well as in the transside. 37 We further find that intra-protein HBs also rearrange as changes in ζ occur; in particular, the HBs consisting the 310 helix, e.g. Cys113(O)-Ala116(H), become unstable in the cis-side, whereas the Ser154(Oγ )-Gln131(H 2) HB is formed only occasionally in the trans-side. By examining the structure, we find that the weakening of these HBs occurs as pSer167(O) interacts with the HB donors, thereby stabilizes the ligand binding. These results indicate that various protein-protein as well as ligand-protein HBs are tuned during the isomerization about ζ, suggesting the importance of Pin1’s conformational rearrangement along the minimum free energy path. To reveal how these conformational changes actually occur during the isomerization transition events, we next explore the transition dynamics in detail. Transition path sampling is carried out to obtain more than 1800 transition trajectories each for the catalyzed and uncatalyzed reactions. In particular, various initial transition trajectories are prepared from the REUS and US results sampled at ζ ∼ 90◦ , and the shooting algorithm 42 is applied to sample the trajectories from a broad range of ζ including the cis- and trans-basins (Fig. S2). Further details are provided in Supporting Information. Since the sign of the overall velocity can be assigned arbitrarily in each initial configurations, in the following analyses the transition trajectories are aligned so as to start from the cis-state and end at the trans-state. The time origin is set to the time of the (median of) transition event(s), i.e. the time the border ζ = 90◦ is crossed, in each trajectory. Fig. 2(a) shows the transition path time (TPT) distributions for the catalyzed and

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uncatalyzed reactions. Here, the transition region is set to 30◦ ≤ ζ ≤ 150◦ . The transitions are found to occur very rapidly, i.e. the mean of the TPTs for the catalyzed and uncatalyzed reactions are 2.5±2.3 and 3.2±1.7 ps, respectively. This indicates that Pin1 not only reduces the free energy barrier for isomerization, but also indeed accelerates the transition dynamics. To see which atoms move during the transition events, the root-mean-square displacements (RMSDs) of the heavy atoms in the ligand are analyzed. Here, the displacement is calculated as a deviation from the conformations at t = 0 ps within each trajectory. Each snapshot is aligned using the heavy atoms of the protein and ligand, and the RMSDs are averaged over trajectories. The results at t = ±10 ps are shown in Figs. 2(b) and (c), and the RMSDs of all the heavy atoms in the protein and ligand from -10 ≤ t ≤ 10 ps are also shown in Fig. S3. We can clearly see that changes are localized at pSer167(O) and the proline ring of Pro118 in the catalyzed reactions, whereas the displacements are distributed over many residues in the uncatalyzed case. This indicates that the ligand is “locked” by Pin1 during the transition events. Interestingly, the broader distribution of successful shooting in the catalyzed case (compare Fig. S2(c) and (d)) also implies that the catalytic environment of Pin1 is rigid and can facilitate the isomerization transitions more efficiently.

Figure 2: Transition dynamics show rapid changes along ζ. (a) Transition path time distributions for the catalyzed and uncatalyzed reactions, and the root-mean-square displacement (RMSD) of the heavy atoms in the ligand, measured from the transition event (t = 0ps) for the (b) catalyzed and (c) uncatalyzed reactions. To analyze how the ligand-protein interactions change during the isomerization events, the HB contacts averaged over the transition trajectories are examined as a function of time

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in Fig. 3. Note that HBs with respect to the time from transition events are shown in Fig. 3, whereas those as a function of ζ are in Fig. 1(d). Many of the HBs found in the transition state of the free energy profile (ζ ∼90◦ in Fig. 1(c)) are indeed present at t = 0 ps. However, in contrast to the rapid rearrangements of these HBs as ζ changes along the minimum free energy path (in Fig. 1(d)), the HBs at t = 0 ps in Fig. 3 are found to persist even after ζ has relaxed to the cis- or trans-state, e.g. 10 ps from the transition events. For instance, the pSer167(O)-Cys113(Hγ ) and pSer167(O)-Hie59(H 1) HBs are still formed in the cis-state at -10ps, and do not disappear at t = −1 ns. Similarly, the pSer167(O)Gln131(H 2) and pSer167(O)-Arg68(Hη 22) HBs are found in the trans-state even at t = 1 ns. Yet, we note that the pSer167(O)-Ser154(Hγ ) HB is hardly formed during the transition events. The intra-protein HBs, Ser115(Oγ )-Hie59(H 2) and Ser154(Oγ )-Gln131(H 2), are also formed throughout the transition events, whereas these HBs quickly disappear in the cis- and trans-sides of the minimum free energy path, respectively (Fig. 1(c)). These results highlight that the transition pathway is remarkably different from the minimum free energy path; the transition thus occurs in a non-equilibrium and transient manner, and cannot be considered as a simple thermal activation process from the reactant equilibrium.

Figure 3: Changes of hydrogen bond contacts during catalyzed transitions. The pairs analyzed in Fig. 1(d) are shown as a function of time from the transition event, and each contact is given as an average over transition trajectories at different times. The long-lived HBs in Fig. 3 are in sharp contrast to the rapid transitions along ζ (Fig. 2), indicating that these HBs are formed a priori in the reactant state before changes along 8


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ζ occur. Furthermore, as these HBs are not characteristic in the cis- and trans- equilibria, they are expected to exist in a minor state, i.e. conformational excited state. To see how these excited conformations are set up, we calculate the response function of the HB contacts as a function of time from the transition events, (±)

Cij (t) =

hqij (t)i − hqij (±∞)i hqij (0)i − hqij (±∞)i

(2)

Here, qij (t) and qij (±∞) are the HB contacts (defined in Eq. (1)) at time t from the transition event and at cis- (−∞) or trans- (+∞) equilibrium, respectively, h· · · i represent the average over trajectories, and the superscript ± describes which state is used as a reference. The REUS trajectories with the restraint centered at ζ = 0◦ and ζ = 180◦ are used for the (±)

cis- and trans-equilibria (see also Fig. 1(c)). Cij (t) describes how qij (t) in the transition (−)

(+)

trajectory ensemble deviates from cis- and trans- equilibria (Cij (−∞) = Cij (∞) = 0) (±)

as it approaches the transition event (Cij (0) = 1). The results relative to cis- and transequilibria are summarized in Figs. 4(a) and (b), respectively. The pairs shown here are typical examples which frequently form HBs during transitions but interact only occasionally (±)

or rarely in each equilibrium. The lifetimes of the slow relaxations of Cij (t) are estimated (±) by fitting Cij (t) with a stretched exponential function, f (t) = a × exp −(t/τ )β , and the results are given in Fig. S4. Fig. 4(a) shows how the HBs are built towards the transition event from the cis-side. The intra-protein HBs, Cys113(O)-Ala116(H) and Ser115(Oγ )-His59(H 2), are found to change slowly, with the lifetime of > 2µs (Fig. S4(a)). As Cys113(O)-Ala116(H) is part of the 310 helix that is mainly unfolded in the cis-equilibrium, the folding of 310 helix is thus considered as a character of the conformational excited state. These slow HB rearrangements further induce the ligand-protein HB, pSer167(O)-Cys113(Hγ), which can be seen in the slowly changing component from -100 to -1 ns. The ligand-protein HBs critical for catalysis are then prepared during -1 ns to -10 ps; as the isomerization about ζ starts at ∼-2 ps, dras-

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Figure 4: Response function of hydrogen bond (HB) contacts as a function of time from the transition events. The cis- and trans-equilibria are used as a reference in (a) and (b), respectively. The HB contacts considered are shown in colored lines on the left panel, and the corresponding response function plots are given on the right plot. The timescales for the slow decays estimated using a stretched exponential function, and are summarized in Fig. S4. tic changes in HBs of pSer167(O)-Cys113(Hγ) and pSer167(O)-His157(H1) further occur as pSer167(O) moves rapidly (Fig. 2(b)). The intra-ligand HB, Ala166(O)-Phe169(H), is weakly formed at ∼-1 ns, and becomes solid as other ligand-protein HBs are set up before the change in ζ occurs. These results thus indicate that the slow folding of the 310 helix in the cis-state set up the pSer167(O)-Cys113(Hγ) HB via tightening the catalytic core with the Ser115(Oγ)-His59(H2) HB. As this conformational excited state further induces the HBs about pSer167(O) on the sub-nanosecond timescale, this intermediate state plays an important role in preparing the catalytic environment for the isomerization reaction from the cis-side. On the contrary, Fig. 4(b) shows that multiple HBs are set up more than a few ns prior to the transition event from the trans-side. The intra-protein HB, Ser154(Oγ)-Gln131(H21), is built with a lifetime of 131 ns, and this HB enhances the chance of forming the ligandprotein HB pSer167(O)-Gln131(H22). The ligand-protein HBs, pSer167(OP)-Arg68(H) and pSer167(O)-Arg68(Hη22), are also found to rearrange slowly in ∼390 and ∼300 ns, repectively (Fig. S4(b)). While the side chain of Arg68 takes multiple conformations in 10


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the trans-state equilibrium (Fig. S5), a proper repositioning of the Arg68 side chain is thus important for catalysis. We also see that the response function of pSer167(O)-Arg68(Hη22) and pSer167(OP)-Gln131(H22) become negative; this indicates that these HBs are weaker at the “transition state” than in trans-equilibrium, and the large negative value from 1 ns to ∼3 ps implies that these HBs are tight during this period. From 1 ns to 10 ps prior to the transition event, the ligand is thus “locked” at the catalytic core with the pSer167(O)-Arg68(Hη22) and pSer167(OP)-Gln131(H22) HBs, and Arg68 starts to interact with Phe169 via the Phe169(O)-Arg68(Hη22) HB; the isomerization about ζ then starts at ∼ 2 ps with sharp rise in the pSer167(O)-His157(H1) HB. These results thus indicate that the conformational excited state on the trans-side may be characterized with the interactions between the loops involving Ser154 and Gln131 as well as the repositioning of Arg68, both contributing to tightening the ligand-protein interaction and inducing further ligand-protein HBs. We note that most of the fittings required β to be much smaller than 1, i.e. 0.5∼0.65 (Fig. S4), which are common values seen in systems showing dynamic heterogeneity. This strongly indicates that these conformational changes occur in a highly heterogeneous manner, e.g. not a simple two-step process and may proceed via multiple pathways. While determining a single coordinate suitable for separating the conformational excited states and equilibria is thus difficult due to the highly heterogeneous behavior of Pin1’s conformational transitions, the slowness of the critical HBs shown here strongly indicates that conformational excited states exist, and further play an important role in connecting the slow HB rearrangements and rapid transitions about ζ in the enzyme catalysis of Pin1 (Fig. 5). In summary, here the molecular mechanism of the Pin1-catalyzed isomerization reaction is studied to reveal how the enzyme conformations adopt during catalysis. The free energy analysis shows that the protein-protein as well as the ligand-protein interactions rearrange along the minimum free energy path, indicating that protein conformations simulatneously adopt to the position of the isomerizing atom (pSer167(O)) and changes about ζ during the

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Figure 5: Scheme of the catalytic cis-trans isomerization mechanism in Pin1. Conformational excited states are visited during conformational fluctuation in the equilibrium, and only from these “preferential” conformations the isomerization reaction about ζ takes place rapidly. The representative excited conformations of the ligand are given in atom-dependent colors, those of Pin1 at the cis- and trans- equilibria are in yellow and orange, respectively, and typical structures in the equilibria are in gray. isomerization reaction. On the contrary, the transition dynamics reveal that the isomerization transitions occur locally; only pSer167(O) and the proline ring of Pro168 show notable movement during transitions, indicating that the catalytic core is rigid. As a result, HBs not involving pSer167(O) are found to remain mostly unchanged while the change in ζ occurs, and the conformations right before/after the isomerization is apparently different from those in equilibrium. This indicates that the HBs necessary for catalysis need to be set up a priori, i.e. as a conformational excited state. To understand how these excited states are setup in equilibrium, the HB lifetimes are analyzed from the response functions. We find that the HB rearrangements from equilibrium occur over a wide range of timescales, as slow as ∼100 ns for loop-loop tightening and even >2 µs for the 310 helix folding. These conformational changes are found to “lock” the residues near the catalytic center and strengthen the ligand-protein interactions, e.g. pSer167(O)Cys113(Hγ ) and pSer167(O)-Arg68(Hη 22), thereby facilitate isomerization transitions about ζ. Such heterogeneous conformations about the 310 helix and the loops involving Gln131 and Ser154 have been observed in the crystal structures and in NMR relaxation dispersion measurements. 43 We note that the latter result have measured the overall reaction rate which includes the rarely-occuring cis-trans isomerization, resulting in a timescale of ∼ms; in order

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to separate the conformational excitation and isomerization steps, a ligand in the cis- or trans-locked form may be used to suppress the isomerization events. 44 Furthermore, Pin1 also appears to involve slow dynamics on the ∼ms timescale even in the absence of a ligand; 43 it is thus conceivable that the timescale required to reach the conformational excited state from equilibrium is much longer than µs. As a result of the two independent processes, i.e. transition to the conformational excited state from equilibrium fluctuation and subsequent isomerization reaction, which is also a rare event, the overall reaction rate of Pin1-catalyzed isomerization occurs on the order of ∼ms. We stress that the HBs analyzed here are frequently found in the transition state, but are formed only occasionally. The chances of simultaneously finding these HBs in equilibrium are very low, thus are set up only in the conformational excited state. The rearrangement of the HBs along the transition pathways are also quite different from those along the minimum free energy path, and the protein environment is not in equilibrium with the ligand; thus the transitions occur in a non-equilibrium fashion. The excited conformational excited states of enzymes, which mimic the transition state of the catalytic reaction,thus may also play important roles in other enzymatic and catalytic reactions. Experimentally, these excited state may be explored in NMR dispersion measurements which have been used to study the excited states, i.e. states with minor populations. Computationally, this indicates the need to explore protein conformations not only about the reactant minimum for finding the optimal transition pathways and transition states. Importantly, the current finding indicates that, as the chemical reaction step can be separated from the protein conformational rearrangement step, the use of computationally expensive quantum mechanical calculation may be safely avoided in exploring the enzyme conformational rearrangements. This may lead to designing computational protocols for studying enzymatic reaction mechanisms more efficiently, and also help reveal how protein conformational flexibility plays a role in other enzyme catalysis. Finally, the timescale separation, i.e. slow conformational rearrangements of the enzyme

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and rapid (chemical) reaction transitions, are likely to occur more generally, i.e. not only in enzymatic reactions but also in slow reaction/structural changes in condcensed phases. For example, the rapid structural changes from excited conformations are reminiscent of jump motions in HB rearrangements 45,46 and supercooled liquids. 47–49 In particular, a recent work has revealed an important role of specific HB rearrangement that facilitate rapid structural changes in supercooled water. 50 Furthremore, the present result reveals that the catalytic transitions proceed in a non-equilibrium manner from the conformational excited state, thus is not a simple thermal activation from the reactant equilibrium to the transition state. Therefore, this study adds a novel perspective to the Pauling’s view on the enzymatic reactions 1 in which the reactions proceed thermally from reactants to stabilized transition state and products.

Acknowledgement This work was supported by Grant-in-Aid for Scientific Research (JP18K05049 to T.M. and JP16H02254 to S.S.) from JSPS. The calculations are partially carried out at the Research Center for Computational Sciences in Okazaki.

Supporting Information Available Computational details of the system setup, free energy calculation, and transition path sampling for the catalyzed and uncatalyzed reactions. Figures for the free energy profiles of isomerization, distribution of shooting attempts in transition path sampling, root-meansquare displacements of atoms during transitions, response function of the hydrogen bond contacts with fitting parameters, and distribution of χ angles in equilibria. is available free of charge via the Internet at http://pubs.acs.org/.

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Conformational Excitation and Non-Equilibrium Transition Facilitate

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