Dynamically committed, uncommitted, and quenched states encoded in kinase A revealed by NMR spectroscopy

Larry R. Mastersona,1, Lei Shib,1, Emily Metcalfeb, Jiali Gaob, Susan S. Taylorc,2, and Gianluigi Vegliaa,b,2

aDepartments of Biochemistry, Molecular Biology, and Biophysics, and bChemistry, University of Minnesota, Minneapolis, MN 55455-0431; and cDepartment of Chemistry and Biochemistry, University of San Diego, San Diego CA 92093-0654

Contributed by Susan S. Taylor, February 18, 2011 (sent for review October 19, 2010)

Protein kinase A (PKA) is a ubiquitous phosphoryl that of the catalytic cycle (3): open (apo form), intermediate (binary mediates hundreds of cell signaling events. During turnover, its form), and closed (ternary complex). NMR dynamic measure- catalytic subunit (PKA-C) interconverts between three major con- ments (7) linked the major conformational states along the reac- formational states (open, intermediate, and closed) that are dyna- tion coordinates, showing the critical role of the enzyme’s internal mically and allosterically activated by nucleotide binding. We show dynamics (the equilibrium fluctuations that allow the exploration that the structural transitions between these conformational states of the free energy landscape) for function. Speci- are minimal and allosteric dynamics encode the motions from one fically, the rates of conformational fluctuations are correlated state to the next. NMR and molecular dynamics simulations define with the transition from closed to open conformations. These the energy landscape of PKA-C, with the substrate allowing the en- fluctuations are synchronous with the enzyme rate-limiting step zyme to adopt a broad distribution of conformations (dynamically (product release), underscoring the prominent role of conforma- committed state) and the inhibitors (high magnesium and pseudo- tional dynamics in substrate recognition and catalysis (7). substrate) locking it into discrete minima (dynamically quenched Understanding how PKA-C interacts with both substrates and state), thereby reducing the motions that allow turnover. These inhibitors from both a structural and dynamic perspective will

results unveil the role of internal dynamics in both kinase function define general criteria for activation and deactivation of protein BIOPHYSICS AND

and regulation. kinases, with obvious repercussion in the design of new drugs. COMPUTATIONAL BIOLOGY Here, we examined the effects of inhibitors on the conforma- allostery ∣ cooperativity ∣ phospholamban ∣ substrate recognition ∣ tional dynamics of the enzyme: a competitive peptide inhibitor intrinsically disordered corresponding to the inhibitory region of PKI (PKI5–24) (8) and magnesium, which under high concentrations behaves kinetically osttranslational phosphorylation is among the most common as a noncompetitive inhibitor of PKA-C (9, 10). The combination 2þ Pmechanisms of cell signaling both in eukaryotes and prokar- of high Mg concentrations with PKI has implications for the yotes (1). Phosphorylation is orchestrated by kinases, which are cellular control of PKA-C activity to arrest transcription during involved in many vital cellular functions including metabolism, mitosis (11, 12). By comparing the results with those obtained in growth, and cell differentiation, and they target substrates loca- the presence of a peptide substrate (7) which competes with lized in several compartments, including cytoplasm, mitochon- PKI5–24 at the binding groove of PKA-C, we found that both 2þ dria, plasma membrane, sarcoplasmic reticulum membrane, PKI5–24 and excess Mg restrict the enzyme dynamics on a fast nucleus, , and actin filaments (2). The human ki- (picosecond to nanosecond) and slow (microsecond to millise- nome (the collection of all human protein kinases) accounts for cond) NMR timescale without drastically changing the conforma- approximately 2% of the entire genome and therefore encom- tion of the ternary complex. Inhibitor binding modifies the energy passes the largest family of enzymes (3). landscape by restricting the motions of the enzyme backbone In addition to differential expression in various cellular sites, (13). These findings unveil a relatively unexplored role of mag- kinases are activated or deactivated and localized by cofactors nesium in protein kinase regulation and establish a paradigm for and ancillary regulatory proteins to achieve precise control over the design of protein kinase inhibitors. time and space. Protein kinase A (PKA) is considered the pro- totype for the protein kinase family (4). PKA exists as an inactive Results heterotetrameric assembly with two catalytic subunits (PKA-C) Thermodynamics of Binding and Enzyme Stability. To compare bound to a dimer of regulatory (R) subunits. PKA-C is unleashed the effects of binding substrates and inhibitors to PKA-C, we upon β-adrenergic stimulation, which disassembles the heterote- synthesized two peptides: the first peptide corresponding to the tramer. The R subunits are responsible for the localization of cytoplasmic domain of phospholamban (PLN1–20), an endogen- PKA-C via interactions with A kinase anchoring proteins to ous inhibitor for the sarcoplasmic reticulum Ca-ATPase and achieve spatial control (5). native substrate of PKA-C in cardiac muscle (14); and the second The heat stable protein kinase inhibitor (PKI) also controls peptide corresponding to PKI5–24 (Fig. S1). Crystallographic data PKA activity and localization (5). This small protein comprises and enzyme kinetic assays indicate that these peptides compete a high-affinity pseudosubstrate region that binds competitively for the same binding site in PKA-C (4, 7). Enzyme thermostability to the substrate binding groove, as well as a nuclear export signal, directing PKA to locations outside the nucleus (6). Binding of Author contributions: L.R.M., S.S.T., and G.V. designed research; L.R.M., L.S., and E.M. PKI to PKA-C forms a complex in which the enzyme is poised performed research; L.R.M., J.G., S.S.T., and G.V. analyzed data; and L.R.M., L.S., E.M., for catalysis (correct positioning of atoms), but remains locked S.S.T., and G.V. wrote the paper. due to the lack of a hydroxyl group acceptor (3). The authors declare no conflict of interest. The bean-shaped fold of PKA-C is highly conserved (4), with 1L.R.M. and L.S. contributed equally to this work. two lobes (small and large) undergoing structural rearrangements 2To whom correspondence may be addressed. E-mail: [email protected] or staylor@ during the substrate recognition and product release steps of ucsd.edu. catalysis (3). Three major conformational states of PKA-C have This article contains supporting information online at www.pnas.org/lookup/suppl/ been identified by X-ray crystallography along various stages doi:10.1073/pnas.1102701108/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1102701108 PNAS Early Edition ∣ 1of6 Downloaded by guest on September 28, 2021 2þ and binding thermodynamics experiments were carried out at low and high Mg (ΔTm ∼ 4 °C), indicating a synergistic effect of and high concentrations of MgCl2. At low concentrations of stabilizing the enzyme when both inhibitors are present. 2þ MgCl2, PKA-C binds one cation, the primary Mg ion, essential Taken together, the calorimetry and thermostability measure- for enzyme activity (9). At high concentrations of MgCl2,PKA-C ments indicate that PKI5–24 binds to PKA-C with an opposite 2þ binds a secondary Mg ion, which inhibits catalytic activity in a enthalpic/entropic balance than PLN1–20. Whereas substrate noncompetitive manner (9). binding is entropically driven and confers little thermostability, To measure the dissociation constants for PKA-C bound to inhibitor binding is enthalpically driven and significantly en- substrate or inhibitor peptides, we employed isothermal titration hances the thermostability of the enzyme. calorimetry (ITC). The ITC measurements reveal that PKI5–24 binds to PKA-C 25 times more tightly than PLN1–20 (Fig. 1A and Chemical Shift Perturbations Map the Transitions from Open to Closed Table S1). This difference in affinity is amplified by ADP to 40 State. The residue-specific changes in the amide backbone of times, reflecting the strong cooperative effect between PKI and PKA-C upon binding AMP-PNP, and followed by substrate or 1 15 nucleotide (15, 16). Remarkably, PKI5–24 and PLN1–20 have inhibitor peptides at low magnesium were monitored by H∕ N- different thermodynamics of binding (Fig. 1A). PLN1–20 binding transverse relaxation-optimized spectroscopy (TROSY)-hetero- to PKA-C is dominated by a favorable overall entropy to the nuclear single quantum coherence NMR spectroscopy. As de- binding free energy (ΔG). The presence of ADP increases the monstrated with the seven-residue peptide, Kemptide (17), the favorable entropy of binding, resulting in an enhanced binding overall trend in chemical shift changes from these titrations affinity, revealing a positive cooperativity similar to that of correlate with the displacement of Cα atoms observed by X-ray Kemptide (17). In contrast, the binding of PKI5–24 to PKA-C is crystallography (Fig. S2 A–C). However, the chemical shift per- enthalpically driven, overcoming an unfavorable entropic contri- turbations were generally small (hΔδi ∼ 0.04 ppm), with AMP- bution to ΔG. Although the B factors from crystal structures of PNP binding accounting for the majority of the differences PKA-C complexes containing either PLN1–20 or PKI5–24 suggest (Fig. 1C and Fig. S2 D–G). Both PLN1–20 and PKI5–24 binding more favorable enthalpy for binding the inhibitor (more favor- to the binary complex gave similar Δδ relative to the apo state able intermolecular interactions), the structures alone are not (hΔδi ∼ 0.04 ppm; Fig. 1 C and D). Binding of a secondary 2þ adequate enough to predict these opposite thermodynamic driv- Mg ion to the PKA-C∕AMP-PNP∕PKI5–24 complex resulted ing forces to binding. The presence of ADP reduces the unfavor- in no appreciable effects in the enzyme fingerprint as demon- able entropy of binding PKI5–24, leading to a higher binding strated by the correlation plot in Fig. 1E. These data indicate that affinity and greater positive cooperativity (approximately 800 (i) once the nucleotide is bound to the enzyme, only minimal times). structural changes occur for the transitions from intermediate Thermostability measurements revealed that PKI5–24 con- to closed state, and (ii) the conformation of the enzyme bound ferred significantly greater stability to PKA-C than the substrate. to the peptide inhibitor closely resembles the substrate bound Thermal melting of PKA-C bound to nucleotide, substrate, and conformation. However, small but significant chemical shift inhibitor was monitored with CD spectroscopy (Fig. 1B) at low differences (Δδ ∼ 0.01–0.04 ppm) are present in catalytically and high Mg2þ concentrations. Apo-PKA-C melted at 47 °C, important regions of the enzyme (Fig. 1 F and G), such as the whereas under low Mg2þ, the nonhydrolyzable nucleotide, ade- glycine-rich, activation, DFG (Asp184-Phe185-Gly186), and peptide nosine 5′-(β,γ-imido)triphosphate (AMP-PNP), shifted the melt- positioning loops, which are conserved throughout all protein ing temperature (ΔTm) by approximately 1 °C and the ternary kinases (3). Strikingly, chemical shift changes were linear from complex with PKI5–24 shifted ΔTm by approximately 2 °C, consis- apo-PKA-C to binary and ternary complexes with substrate or tent with previous reports using adenosine 5'-[γ-thio]triphosphate inhibitor peptide, and finally to the ternary complex with inhibitor (18, 19). However, unlike the increased stability for PKI5–24, peptide at high magnesium (Fig. 1F). This linearity between addition of PLN1–20 to the binary complex had a negligible effect the different complexes indicates that the enzyme undergoes fast 2þ on ΔTm. High Mg shifted ΔTm by approximately 1 °C for both exchange between the major conformations and that the popula- the nucleotide bound form and ternary complex with PLN1–20.In tions are shifted by ligand binding. Nucleotide binding shifts the contrast, ΔTm was shifted the furthest in the presence of PKI5–24 conformational ensemble from the open to the intermediate

Fig. 1. Thermodynamic and NMR analysis of PKA-C. (A) Thermodynamics of PKA-C binding to substrate and inhibitor, with or without ADP. The binding of PLN1–20 is dominated by favorable entropy, whereas PKI5–24 is enthalpy driven, overcoming an entropic penalty. (B) Melting measurements showed that PKI5–24 þ2 confers the greatest thermostability to PKA-C as represented by the shift in T m (relative to apo-PKA-C, ΔT m). High Mg confers slightly higher stability to each complex. (C–E) Correlation of chemical shift perturbations (Δδ) in PKA-C between the different forms. The majority of perturbation occurs upon nucleotide binding (C), whereas formation of the ternary complexes were quite similar to one another (D and E). (F) Linearity of chemical shifts between the apo form and 2þ the ternary form (AMP-PNP∕PKI5–24 with high Mg ) is observed, indicating that the enzyme opens and closes on a fast timescale. (G) Enzymes views from the active site surface formed by the small and large lobes.

2of6 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1102701108 Masterson et al. Downloaded by guest on September 28, 2021 state. Subsequent binding of substrate or inhibitor skews the population toward the closed states. Although there is a clear distinction between the apo and nucleotide bound forms, the dif- ferences in chemical shifts between various ternary complexes are minimal (Fig. 1F). The latter indicates that both substrate and inhibitors shift the population toward closed states, which are structurally quite similar.

Inhibitor Binding Quenches the Dynamics in the Enzyme Backbone. The combination of thermodynamics and chemical shift pertur- bations show that the ternary complexes formed by the enzyme with substrate or inhibitor differ in thermostability, but not significantly in structure. To probe the internal dynamics of the enzyme, we used nuclear spin relaxation measurements for fast, picosecond to nanosecond [T1, T2, and heteronuclear (H-X) R NOE], and slow, microsecond to millisecond ( ex), dynamics on the NMR timescale for each complex (20). Our data show that 2þ the PKA-C∕AMP-PNP∕PKI5–24 complex with two Mg ions bound is the most compact with the fastest overall tumbling rate (T1∕T2 ratios) and least flexible (increased in H-X NOEs and T Fig. 2. Backbone dynamics of PKA-C in different ternary complexes. Map- longer 2 values; Fig. 2, Fig. S3 A and B, and Table S2). This find- ping of (A) fast and (B) slow backbone dynamics show that, upon inhibition 2þ ing is in agreement with previous fluorescence anisotropy studies with PKI5–24 and with high Mg , a decrease of picosecond to millisecond (21, 22). More importantly, a comparison of the ternary com- dynamics occurs throughout the backbone. For the comparison, the pre- 2þ plexes with one Mg ion reveals that the PKA-C∕AMP-PNP∕ viously published dynamics of PKA-C with the substrate PLN1–20 is shown (7). PKI5–24 complex has substantially decreased picosecond to nano- second dynamics with respect to the PKA-C∕AMP-PNP∕PLN1–20 The PCA results show the directionality and amplitude of protein complex (Fig. 2 and Table S2). This decrease becomes more motions, in which the first several principal components are 2þ correlated with large conformational changes. PCA analysis apparent under high Mg concentrations. The quenched fast BIOPHYSICS AND ∕ ∕ has been widely to interpret conformational variations observed

dynamics for the PKA-C AMP-PNP PKI5–24 complex indicates COMPUTATIONAL BIOLOGY – a decrease in conformational entropy of the enzyme. Although experimentally (24 26). Moreover, PCA analysis provides a rea- quenched dynamics cannot be directly compared to the macro- sonable method to extrapolate a relatively short MD trajectory scopic methods of thermodynamics, a decrease in conformational to provide a qualitative description of motions occurring over entropy agrees qualitatively with the trend of unfavorable overall a longer timescale (27), such as the opening and closing events entropy of binding and the enhanced enzyme thermostability. probed by NMR spectroscopy (7). The analysis of inverse peak heights at temperatures ranging We calculated the PCA for the PKA-C complex with PLN1–20, between 22–33 °C suggests a marked decrease in conforma- and found that the first two components account for ap- tional exchange (i.e., slow dynamics on the NMR timescale) for proximately 60% of variance in coordinates during the MD simu- PKA-C upon inhibitor binding (Fig. S3 C–E). We quantified lations (Fig. S4D). The first principal component (PC1) describes the microsecond to millisecond conformational exchange rates the opening and closing of the two lobes of the enzyme, whereas R the second component (PC2) describes shearing between the ( ex) across the enzyme backbone and found diminished values for the ternary complex with PKI5–24 compared to the ternary lobes (Fig. 3A and Fig. S4 E and F). To probe the opening and complex with substrate (7) (see Materials and Methods, Fig. 2, closing of the active site cleft (4, 7), we also monitored the intera- 2þ 53 186 and Table S2). Addition of the inhibitory Mg ion to the ternary tomic distance between Ser and Gly (dS53-G186) (3) during complex with PKI5–24 resulted in nearly absent microsecond to MD trajectories (Fig. 3A) with respect to PC1. This 2D plot millisecond dynamics (Fig. 2). Quenched dynamics are evident (Fig. 3B) describes relative motion between the two lobes and for regions surrounding the conserved loops at the active site the opening and closing of the active site. As points of reference, of PKA-C. the crystallographic structures were plotted on this graph. The comparison of the conformational ensemble sampled by the PKA-C Energy Landscape. To define the energy landscape and inter- MD simulations and the X-ray structures representative of the pret the dynamics for each form of PKA-C along stages of the three conformational states are reported in Table S4. catalytic cycle, we carried out molecular dynamics (MD) simula- The apoenzyme (orange trace) samples a broad distribution of tions of PKA-C in water using the apo, binary (nucleotide bound), conformations identified by X-ray crystallography (Fig. 3B), 2þ and ternary complexes (containing PLN1–20 or PKI5–24, see SI whereas those sampled by the binary form with one Mg have Materials and Methods for experimental setup and data analysis). distributions similar to open and intermediate conformations. In each case, the calculations were carried out in the presence of Addition of the substrate (ternary complex, blue trace) does not either one or two bound Mg2þ ions to model the low and high change PC1 observed for the binary form, but changes the distri- 2þ Mg conditions used experimentally (3). Rmsd plots vs. time bution of dS53-G186 toward the closed state. In contrast, the dis- showed that PKA-C structure reaches a fairly stable minimum tribution of conformations for the ternary complex with PKI5–24 after approximately 20 ns (Fig. S4A), whereas root mean square (red trace) is shifted, where PC1 encompasses conformations si- fluctuations (rmsf) confirmed that the most dynamic regions of milar to intermediate and open states. However, the dS53-G186 dis- the enzyme reside in the proximity of the catalytically important tance distribution indicates that the enzyme’s active site is not loops (3, 7) (glycine-rich, activation, DFG, and peptide position- completely open. This distribution between conformations is in ing loops), as well as some of the structural elements such as the agreement with the NMR data showing residual dynamics for 2þ B helix, H helix, F helix, with significant fluctuations detected for the PKA-C∕AMP-PNP∕PKI5–24 complex at low Mg concen- the N and C termini (Fig. S4 B and C). trations. The conformational interconversion of the enzyme through Calculations with the second Mg2þ ion resolves the degeneracy different states identified by X-ray crystallography (Table S3) of the conformational states and defines localized minima were monitored using principal component analysis (PCA) (23). (Fig. 3B): The binary form of the enzyme populates inter-

Masterson et al. PNAS Early Edition ∣ 3of6 Downloaded by guest on September 28, 2021 mediate conformational states, the ternary complex with the con- select these different states (Fig. 4). The apoenzyme is character- formational space defined by the corresponding substrate has ized by a dynamically uncommitted state—it can explore the dS53-G186 values clustered near the intermediate and closed states, energy landscape to access open and closed conformations, but and, strikingly, the ternary complex with inhibitor overlaps with does not transition between these conformations on a timescale the crystallographic structures (Fig. 3B, Bottom Right). relevant for catalysis (7). The dynamically committed states of the The results from MD simulations corroborate the NMR and protein kinase occupy conformational space in which the active thermodynamics data, providing a framework to interpret the site cleft opens and closes on a timescale optimal for turnover. experimental results in terms of the energy landscape. Although These fluctuations are induced by nucleotide binding, which acts the linchpin role of Mg2þ ions in both stabilization of the pyro- as a dynamic and allosteric activator (7), coupling the small and phosphate group of ATP and local rigidification of the glycine- large lobes, completing the catalytic spine of the enzyme (29, 30), rich loop has been previously reported (28), our simulations and preparing the active site for substrate binding. This event dif- reveal that the effect of Mg2þ binding is a global phenomenon, ferentiates protein kinases from kinases, such as affecting the overall dynamics of the enzyme and trapping it in adenylate kinase, because the assembly of the spine architecture inert states. is a prerequisite for protein kinase activation (29, 30). Substrate bound PKA-C retains sufficient conformational motions, main- Discussion taining the enzyme in a dynamically committed state for product The transitions between open and closed states in PKA-C are dri- release and turnover (7). Although the dynamics are on a slower ven by internal dynamics, which plays a major role for substrate timescale than the chemical step (phosphoryl transfer), they recognition and turnover (7). The nucleotide acts as a dynamic position and prepare the substrate for catalysis. In contrast, and allosteric effector, causing a population shift of PKA-C from dynamically quenched states of inhibited PKA-C are character- open to closed states and selecting for dynamically committed ized by narrow, deep wells with high-energy barriers between states—which are defined as nearly isoenergetic conformations conformations and hinder opening/closing of the active cleft. 2þ compatible with catalysis that interconvert rapidly. The rates of The two inhibitors analyzed here (PKI5–24 and excess Mg ) the structural fluctuations between these states are sensitive to raise the energy barriers of the conformational landscape, gener- nucleotide binding, which increases the substrate binding coop- ating discrete minima with quenched dynamics. This phenomen- eratively via a conformational selection mechanism (7). More on may explain why, in general, crystal structures of protein importantly, the rates of the enzyme conformational fluctuations kinases have been more accessible in their inhibited forms while are synchronous with the enzyme rate-limiting step (i.e., ADP substrate bound forms have been elusive to such analyses. release), underscoring the prominent role of conformational More importantly, these results support a possible role of dynamics in substrate recognition and catalysis (7). The unique magnesium for regulation and localization of PKA-C in the cell. aspect about the current study is the link between restricted Reports indicate that Mg2þ concentrations can change depend- conformational dynamics and enzyme inhibition, establishing a ing on the cell compartment (31) and phase of the paradigm for controlling protein kinase activity. (11, 32). Specifically, nuclear concentrations of Mg2þ increase Based on our previous data (7, 17) and this work, we propose substantially during mitosis (11). This increase triggers binding that the energy landscape of PKA-C comprises dynamically of PKA-C to PKI with high affinity (10), causing PKA-C to be uncommitted, committed, and quenched states. Ligand binding exported from the nucleus, and arresting transcription (12). Such (via nucleotide, substrates, or inhibitors) drives the enzyme to a mechanism of inhibition and localization exploits the enhanced affinity of PKI in the presence of high Mg2þ (10). Interestingly, enhanced binding affinity in the presence of excess Mg2þ was also reported for the type I (but not the type II) regulatory subunit of PKA (10). Because the regulatory subunits are loca- lized differently within the cell where Mg2þ concentrations may vary (33, 34), gradients in Mg2þ concentration would offer an- other form of localization and control. Therefore, Mg2þ can potentially act as a rheostat for the strength of PKA regulation, dictate its compartmental localization, and influence the overall effect on biological function. Allosteric enzymes often exist in ensembles of conformations, where catalytic efficiency is achieved by excited states able to cross inherently low-energy barriers between major conforma- tions (35–44). Although the dynamics measured by NMR do not influence the chemical step of catalysis directly (45), it has been shown that intrinsic protein dynamics is the driving force for crossing potential energy barriers with ligands, such as cofac- tors or substrates, activating or deactivating enzyme dynamics to modulate biological function (35, 41, 46–55). Although our findings are limited to the system studied here, the mechanism of inhibition via quenched dynamics may play a role in other systems. In fact, recent studies on dihydrofolate reductase (56), triosephosphate isomerase (57), ribonuclease A (58), HIV-1 reverse transcriptase (59), and imidazole glycerol Fig. 3. Comparison of MD simulations for PKA-C. (A) Global motions sug- phosphate synthase (60) show that slow microsecond to millise- gested by PCA analysis of MD trajectories correspond to opening and closing cond motions are severely dampened when these enzymes are of the active site (PC1), which compared well with the distances between inhibited (56, 59, 60) or when catalytically hindering mutations residues S53 and G186 in crystal structures of open (1CMK), intermediate (1BX6), and closed (1ATP) conformations. (B) A map of the interatomic are introduced (57, 58). distances vs. the PC1 from MD simulations indicate that PKA-C accessed Our results demonstrate that the inhibition of PKA-C is the major crystallographic conformations frequently, except in the presence marked by changes in intrinsic dynamics. These findings are of inhibitors. timely, as they suggest a mode by which inhibitors could be

4of6 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1102701108 Masterson et al. Downloaded by guest on September 28, 2021 designed to modulate PKA activity. Protein kinase inhibition is an important therapeutic avenue for treating diseases (61–63). Although many therapeutic agents targeting protein kinases are competitors for the nucleotide binding site, there is a growing interest in developing inhibitors that bind at remote sites (i.e., Abl kinase), working through an allosteric mechanism of inhibi- tion (63, 64). Although we have previously described an allosteric network of communication in PKA-C modulated by conforma- tional dynamics (7, 17), the current results reveal a role of this network in the enzyme inhibition. Gaining control of PKA-C through competitive or allosteric modulation of dynamics is an exciting possibility for further research. Materials and Methods Protein Expression and Purification. PKA-C was expressed and purified from Escherichia coli according to procedures previously published (17, 65). The enzyme concentration was determined spectrophotometrically by A280 A ¼ 1 2 ( 0.1% . ) and its activity was tested using the standard substrate Kemp- tide. Peptide synthesis of PKI5–24 was performed on a microwave synthesizer and purified using reverse-phase high-pressure liquid chromatography.

ITC Measurements. ITC data were acquired on a microcalorimeter (MicroCal Inc.). Stock solutions of PKA-C, PLN1–20, and PKI5–24 were dissolved in 20 mM phosphate buffer (pH 6.5) containing 180 mM KCl and 4 mM MgCl2, and degassed. Titrations were conducted at 27 °C using 0.1 mM PKA-C in the absence or presence of 6 mM AMP-PNP and with a stock of synthetic peptide (1.8 mM). The samples were stirred at 410 rpm. Twenty injections were sepa- rated by 300 s of equilibration (5 μL for the first, followed by 10 μLforeach of the remaining). A one-site binding model was assumed and the data were fit using MicroCal Origin software (version 5.0). Fig. 4. The energy landscape of PKA-C is modulated by ligand BIOPHYSICS AND

binding. The apo state is dynamically uncommitted, having dynamics which COMPUTATIONAL BIOLOGY Circular Dichroism. Concentrated stocks of PKA-C were diluted to 5 μMinCD are not tuned to turnover. Nucleotide binding induces motions which are ′ synchronized to turnover (dynamically committed) and are persistent in buffer [10 mM piperazine-N-N -bis(2-ethanesulfonic acid), pH 7.0, 150 mM 2þ NaCl], in the presence of 0.48 (low Mg2þ, 1∶1.2 Mg2þ:nucleotide) or the ternary complex with substrate. However, PKI5–24, or excess Mg and 2þ 2þ PKI5–24, induces favorable enthalpy which lowers the energy of one or more 3.0 mM MgCl2 (high Mg , 5∶1 Mg :nucleotide), 0.60 mM AMP-PNP (selected samples), and 15 μM peptide (selected samples). Samples were conformational states and raises the energy barriers in the conformational incubated over a range of 25–75 °C at 1 °C∕ min in a rectangular quartz landscape. This phenomenon hinders conformational fluctuations, inhibits turnover, and results in a dynamically quenched enzyme. cuvette in a Jasco J-815 spectropolarimeter. Spectra were acquired at 222 nm following an equilibration time of 10 s. A blank consisted of all Verification of R was done by measuring inverse peak heights (^I)at reaction components except PKA-C and was subtracted from each spectrum. ex decreasing temperatures (73), as described in SI Text. Resonances that The data were fitted to a two-state sigmoidal unfolding model using Origin experienced conformational exchange diminished in peak intensity more 8.0 (Microcal) using T m as the midpoint. Errors were derived from nonlinear significantly with decreasing temperatures (73) (Fig. S3 C–E). least squares fitting of the data. MD Simulations. MD simulations were set up using CHARMM c36a1 and run Acquisition of NMR Data. NMR samples consisted of approximately 500 μM 2þ with NAMD using the crystal structures described in the SI Materials and PKA-C, dissolved in buffer. Low (1∶1.2 Mg :nucleotide) or high (5∶1 þ 2þ 2þ 2þ Methods. All structures were solvated in a TIP3 water box with K and Mg :nucleotide) Mg conditions were done under 10 or 60 mM Mg ,re- − Cl added as counter ions to reach an ionic strength of approximately spectively. Experiments were carried out on a Varian instrument operating 1 150 mM. Following an initial equilibration, 75 ns MD simulations for each at 800.29 MHz H Larmor frequency at 33 °C. The data were processed system were performed at constant temperature and pressure. Rmsd, rmsf, with NMRPipe (66) and analyzed with SPARKY (67). Relaxation experiments and PCA for all simulations were performed as described in the SI Materials were described previously (68), with TROSY-detection (69) and a spectral 1 15 and Methods. width of 10,500 Hz (2,200 Hz) for the H( N) dimension. R1ρ measurements used a 1,500 Hz spin-lock field strength centered at the 15N carrier fre- ACKNOWLEDGMENTS. We are grateful for the fruitful discussions about data quency. R1ρ values were converted into R2 as described (70). interpretation with A. Kornev and G. Melacini. This work was supported R was measured as described previously (71). Briefly, the detection of α, ex by the National Institutes of Health (NIH) (GM072701 and HL080081 to β I , or longitudinal two-spin order magnetization ( zz) during a Hahn echo G.V.; T32DE007288 to L.R.M.; GM46736 to J.G.; and GM19301 to S.S.T.; 2∕J ¼ 10 8 period ( NH . ms) was used in the following relationship (72): University of Minnesota Graduate School Doctoral Dissertation Fellowship R ≈ C ðρ ÞþC ðρ Þ; [1] to L.S.,) and the American Heart Association (09PRE2080017 to E.M.). NMR ex zz ln zz β ln β data were collected at the National Magnetic Resonance Facility at Madi- C ¼ð2τÞ−1 C ¼ðhκi − 1Þð4τÞ−1 κ ¼ 1 − 2 ρ ∕ ρ ρ ¼ I ∕I son [NIH: P41RR02301, P41GM66326, RR02781, and RR08438; National where zz , β , ln zz ln β, zz zz α, Science Foundation (NSF): DMB-8415048, OIA-9977486, and BIR-9214394] ρ ¼ I ∕I and β β α. Experiments were recorded in triplicate and in an interleaved and the University of Minnesota NMR Facility (NSF BIR-961477). This work manner to obtain Izz, Iα, and Iβ. The value hκi was obtained from the trimmed was carried out using hardware and software provided by the University of mean of all 15N resonances that did not exhibit chemical exchange. Minnesota Supercomputing Institute.

1. Walsh DA, Van Patten SM (1994) Multiple pathway signal transduction by the 6. Dalton GD, Dewey WL (2006) Protein kinase inhibitor peptide (PKI): A family of cAMP-dependent protein kinase. FASEB J 8:1227–1236. endogenous neuropeptides that modulate neuronal cAMP-dependent protein 2. Shabb JB (2001) Physiological substrates of cAMP-dependent protein kinase. Chem Rev kinase function. Neuropeptides 40:23–34. – 101:2381 2411. 7. Masterson LR, et al. (2010) Dynamics connect substrate recognition to catalysis in 3. Johnson DA, Akamine P, Radzio-Andzelm E, Madhusudan M, Taylor SS (2001) protein kinase A. Nat Chem Biol 6:821–828. Dynamics of cAMP-dependent protein kinase. Chem Rev 101:2243–2270. 8. Glass DB, Cheng HC, Kemp BE, Walsh DA (1986) Differential and common recognition 4. Taylor SS, et al. (2004) PKA: A portrait of protein kinase dynamics. Biochim Biophys Acta 1697:259–269. of the catalytic sites of the cGMP-dependent and cAMP-dependent protein kinases by 5. Walsh DA, Van Patten SM (1994) Multiple pathway signal transduction by the inhibitory peptides derived from the heat-stable inhibitor protein. J Biol Chem cAMP-dependent protein kinase. FASEB J 8:1227–1236. 261:12166–12171.

Masterson et al. PNAS Early Edition ∣ 5of6 Downloaded by guest on September 28, 2021 9. Cook PF, Neville ME, Vrana KE, Hartl FT, Roskoshi R (1982) Adenosine cyclic 3′5′-mono- 40. Mittag T, Kay LE, Forman-Kay JD (2010) Protein dynamics and conformational disorder phosphate dependent protein kinase: Kinetic mechanism for the bovine skeletal in molecular recognition. J Mol Recognit 23(3):105–116. muscle catalytic subunit. Biochemistry 21:5794–5799. 41. Wright PE, Dyson HJ (2009) Linking folding and binding. Curr Opin Struct Biol 10. Zimmermann B, Schweinsberg S, Drewianka S, Herberg FW (2008) Effect of metal ions 19:31–38. on high-affinity binding of pseudosubstrate inhibitors to PKA. Biochem J 413:93–101. 42. Smock RG, Gierasch LM (2009) Sending signals dynamically. Science 324:198–203. 11. Strick R, Strissel PL, Gavrilov K, Levi-Setti R (2001) Cation-chromatin binding as shown 43. Tzeng SR, Kalodimos CG (2009) Dynamic activation of an allosteric regulatory protein. by ion microscopy is essential for the structural integrity of chromosomes. J Cell Biol Nature 462:368–372. 155:899–910. 44. Ma B, Nussinov R (2010) Enzyme dynamics point to stepwise conformational selection 12. Stoykova AS, Dabeva MD, Dimova RN, Hadjiolov AA (1985) Ribosome biogenesis and in catalysis. Curr Opin Chem Biol 14:652–659. nucleolar ultrastructure in neuronal and oligodendroglial rat brain cells. J Neurochem 45. Pisliakov AV, Cao J, Kamerlin SC, Warshel A (2009) Enzyme millisecond conformational 45:1667–1676. dynamics do not catalyze the chemical step. Proc Natl Acad Sci USA 106:17359–17364. 13. Mauldin RV, Carroll MJ, Lee AL (2009) Dynamic dysfunction in dihydrofolate reductase 46. Freire E (1999) The propagation of binding interactions to remote sites in proteins: results from drug binding: Modulation of dynamics within a structural state. Analysis of the binding of the monoclonal antibody D1.3 to lysozyme. Proc Natl Acad Structure 17:386–394. Sci USA 96:10118–10122. 14. Traaseth NJ, et al. (2008) Structural and dynamic basis of phospholamban and 47. Kumar S, Ma B, Tsai CJ, Sinha N, Nussinov R (2000) Folding and binding cascades: sarcolipin inhibition of ca(2+)-ATPase. Biochemistry 47:3–13. Dynamic landscapes and population shifts. Protein Sci 9:10–19. 15. Whitehouse S, Walsh DA (1983) Mg X ATP2-dependent interaction of the inhibitor 48. Hammes GG (2002) Multiple conformational changes in enzyme catalysis. Biochemis- protein of the cAMP-dependent protein kinase with the catalytic subunit. J Biol Chem try 41:8221–8228. 258:3682–3692. 49. Kern DZ, Zuiderweg ER (2003) The role of dynamics in allosteric regulation. Curr Opin 16. Lew J, Coruh N, Tsigelny I, Garrod S, Taylor SS (1997) Synergistic binding of nucleotides Struct Biol 13:748–757. and inhibitors to cAMP-dependent protein kinase examined by acrylodan fluores- 50. Pufall MA, et al. (2005) Variable control of ets-1 DNA binding by multiple phosphates cence spectroscopy. J Biol Chem 272:1507–1513. in an unstructured region. Science 309:142–145. 17. Masterson LR, Mascioni A, Traaseth NJ, Taylor SS, Veglia G (2008) Allosteric coopera- 51. Swain JF, Gierasch LM (2006) The changing landscape of protein allostery. Curr Opin tivity in protein kinase A. Proc Natl Acad Sci USA, 105 pp:506–511. Struct Biol 16:102–108. 18. Herberg FW, Doyle ML, Cox S, Taylor SS (1999) Dissection of the nucleotide and 52. Popovych N, Sun S, Ebright RH, Kalodimos CG (2006) Dynamically driven protein metal-phosphate binding sites in cAMP-dependent protein kinase. Biochemistry allostery. Nat Struct Mol Biol 13:831–838. 38:6352–6360. 53. Li P, Martins IR, Amarasinghe GK, Rosen MK (2008) Internal dynamics control activa- 19. Yang J, et al. (2005) Allosteric network of cAMP-dependent protein kinase revealed tion and activity of the autoinhibited vav DH domain. Nat Struct Mol Biol 15:613–618. by mutation of Tyr204 in the P þ 1 loop. J Mol Biol 346:191–201. 54. Boehr DD, Nussinov R, Wright PE (2009) The role of dynamic conformational ensem- 20. Palmer AG, 3rd (2001) Nmr probes of molecular dynamics: Overview and comparison bles in biomolecular recognition. Nat Chem Biol 5:789–796. with other techniques. Annu Rev Biophys Biomol Struct 30:129–155. 55. Das R, et al. (2009) Dynamically driven ligand selectivity in cyclic nucleotide binding 21. Gangal M, et al. (1998) Backbone flexibility of five sites on the catalytic subunit of domains. J Biol Chem 284:23682–23696. cAMP-dependent protein kinase in the open and closed conformations. Biochemistry 56. Mauldin RV, Lee AL (2010) Nuclear magnetic resonance study of the role of M42 in 37:13728–13735. the solution dynamics of Escherichia coli dihydrofolate reductase. Biochemistry 22. Li F, Juliano C, Gorfain E, Taylor SS, Johnson DA (2002) Evidence for an internal entropy 49:1606–1615. contributin to phosphoryl transfer: A study of domain clossure, backbone flexibility, 57. Berlow RB, Igumenova TI, Loria JP (2007) Value of a hydrogen bond in triosephosphate and the catalytic cycle of cAMP-dependent protein kinase. J Mol Biol 315:459–469. isomerase loop motion. Biochemistry 46:6001–6010. 23. Karplus M, Kushick JN (1981) Method for estimating the configurational entropy of 58. Doucet N, Watt ED, Loria JP (2009) The flexibility of a distant loop modulates active macromolecules. Macromolecules 14:325–332. site motion and product release in ribonuclease A. Biochemistry 48:7160–7168. 24. Miyashita O, Onuchic JN, Wolynes PG (2003) Nonlinear elasticity, proteinquakes, and 59. Seckler JM, Barkley MD, Wintrode PL (2011) Allosteric suppression of HIV-1 reverse the energy landscapes of functional transitions in proteins. Proc Natl Acad Sci USA transcriptase structural dynamics upon inhibitor binding. Biophys J 100:144–153. 100:12570–12575. 60. Lipchock JM, Loria JP (2010) Nanometer propagation of millisecond motions in V-type 25. Maragakis P, Karplus M (2005) Large amplitude conformational change in proteins allostery. Structure 18:1596–1607. explored with a plastic network model: Adenylate kinase. J Mol Biol 352:807–822. 61. Taylor SS, Radzio-Andzelm E (1997) Protein kinase inhibition: Natural and synthetic 26. Henzler-Wildman KA, et al. (2007) A hierarchy of timescales in protein dynamics is variations on a theme. Curr Opin Chem Biol 1:219–226. linked to enzyme catalysis. Nature 450:913–916. 62. Cohen P (2002) Protein kinases—the major drug targets of the twenty-first century? 27. Cheng X, Ivanov I, Wang H, Sine SM, McCammon JA (2007) Nanosecond-timescale Nat Rev Drug Discov 1:309–315. conformational dynamics of the human alpha7 nicotinic acetylcholine receptor. 63. Engh RA, Bossemeyer D (2002) Structural aspects of protein kinase control-role of Biophys J 93:2622–2634. conformational flexibility. Pharmacol Ther 93:99–111. 28. Khavrutskii IV, Grant B, Taylor SS, McCammon JA (2009) A transition path ensemble 64. Zhang J, et al. (2010) Targeting bcr-abl by combining allosteric with ATP-binding-site study reveals a linchpin role for mg(2+) during rate-limiting ADP release from protein inhibitors. Nature 463:501–506. kinase A. Biochemistry 48:11532–11545. 65. Malmendal A, Halpain S, Chazin WJ (2003) Nascent structure in the kinase anchoring 29. Kornev AP, Taylor SS, Ten Eyck LF (2008) A helix scaffold for the assembly of active domain of -associated protein 2. Biochem Biophys Res Commun protein kinases. Proc Natl Acad Sci USA 105:14377–14382. 301:136–142. 30. Kornev AP, Taylor SS (2009) Defining the conserved internal architecture of a protein 66. Delaglio F, et al. (1995) NMRPipe: A multidimensional spectral processing system based kinase. Biochim Biophys Acta 1804:440–444. on UNIX pipes. J Biomol NMR 6:277–293. 31. Fatholahi M, LaNoue K, Romani A, Scarpa A (2000) Relationship between total 67. Goddard TD, Kneller DG (1999) SPARKY 3. (University of California, San Francisco). and free cellular mg(2+) during metabolic stimulation of rat cardiac myocytes and 68. Farrow NA, et al. (1994) Backbone dynamics of a free and phosphopeptide-complexed perfused hearts. Arch Biochem Biophys 374:395–401. src homology 2 domain studied by 15N NMR relaxation. Biochemistry 33:5984–6003. 32. Murphy E (2000) Mysteries of magnesium homeostasis. Circ Res 86:245–248. 69. Pervushin K, Riek R, Wider G, Wuthrich K (1997) Attenuated T2 relaxation by mutual 33. Mauban JR, O’Donnell M, Warrier S, Manni S, Bond M (2009) AKAP-scaffolding cancellation of dipole-dipole coupling and chemical shift anisotropy indicates an proteins and regulation of cardiac physiology. Physiology (Bethesda) 24:78–87. avenue to NMR structures of very large biological macromolecules in solution. Proc 34. Alto N, Carlisle Michel JJ, Dodge KL, Langeberg LK, Scott JD (2002) Intracellular Natl Acad Sci USA 94:12366–12371. targeting of protein kinases and phosphatases. Diabetes 51(Suppl 3):385S–388. 70. Tjandra N, Wingfield P, Stahl S, Bax A (1996) Anisotropic rotational diffusion of 35. Hammes-Schiffer S, Benkovic SJ (2006) Relating protein motion to catalysis. Annu Rev perdeuterated HIV protease from 15N NMR relaxation measurements at two mag- Biochem 75:519–541. netic fields. J Biomol NMR 8:273–284. 36. Boehr DD, McElheny D, Dyson HJ, Wright PE (2006) The dynamic energy landscape of 71. Wang C, Rance M, Palmer AG, 3rd (2003) Mapping chemical exchange in proteins dihydrofolate reductase catalysis. Science 313:1638–1642. with MW > 50 kD. J Am Chem Soc 125:8968–8969. 37. Frederick KK, Marlow MS, Valentine KG, Wand AJ (2007) Conformational entropy in 72. Tatulian SA, Jones LR, Reddy LG, Stokes DL, Tamm LK (1995) Secondary structure molecular recognition by proteins. Nature 448:325–329. and orientation of phospholamban reconstituted in supported bilayers from polarized 38. Yao X, Rosen MK, Gardner KH (2008) Estimation of the available free energy in a attenuated total reflection FTIR spectroscopy. Biochemistry 34:4448–4456. LOV2-J alpha photoswitch. Nat Chem Biol 4:491–497. 73. Fenwick MK, Oswald RE (2008) NMR spectroscopy of the ligand-binding core of 39. Gsponer J, et al. (2008) A coupled equilibrium shift mechanism in calmodulin- ionotropic glutamate receptor 2 bound to 5-substituted willardiine partial agonists. mediated signal transduction. Structure 16:736–746. J Mol Biol 378:673–685.

6of6 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1102701108 Masterson et al. Downloaded by guest on September 28, 2021