The αC-β4 loop controls the allosteric cooperativity between nucleotide and substrate in the catalytic subunit of protein kinase A

Allosteric cooperativity between ATP and substrates is a prominent characteristic of the cAMP-dependent catalytic (C) subunit of protein kinase A (PKA). Not only this long-range synergistic action is involved in substrate recognition and fidelity, but it is likely to regulate PKA association with regulatory subunits and other binding partners. To date, a complete understanding of the molecular determinants for this intramolecular mechanism is still lacking. Here, we used an integrated NMR-restrained molecular dynamics simulations and a Markov Model to characterize the free energy landscape and conformational transitions of the catalytic subunit of protein kinase A (PKA-C). We found that the apo-enzyme populates a broad free energy basin featuring a conformational ensemble of the active state of PKA-C (ground state) and other basins with lower populations (excited states). The first excited state corresponds to a previously characterized inactive state of PKA-C with the αC helix swinging outward. The second excited state displays a disrupted hydrophobic packing around the regulatory (R) spine, with a flipped configuration of the F100 and F102 residues at the tip of the αC-β4 loop. To experimentally validate the second excited state, we mutated F100 into alanine and used NMR spectroscopy to characterize the binding thermodynamics and structural response of ATP and a prototypical peptide substrate. While the activity of PKA-CF100A toward a prototypical peptide substrate is unaltered and the enzyme retains its affinity for ATP and substrate, this mutation rearranges the αC-β4 loop conformation interrupting the allosteric coupling between nucleotide and substrate. The highly conserved αC-β4 loop emerges as a pivotal element able to modulate the synergistic binding between nucleotide and substrate and may affect PKA signalosome. These results may explain how insertion mutations within this motif affect drug sensitivity in other homologous kinases.


INTRODUCTION
A distinct property of PKA-C is the binding cooperativity between ATP and substrate. 12 During 84 the catalytic cycle, the kinase recognizes and binds substrates with positive binding cooperativity 85 between ATP and unphosphorylated substrates, whereas a negative binding cooperativity be-86 tween ADP and phosphorylated substrate characterizes the exit complex. 13 The biological im-87 portance of binding cooperativity has been emphasized by our recent studies on disease-driven 88 mutations of PKA-C, 14-16 which all feature disrupted cooperativity between nucleotides and protein 89 kinase inhibitor (PKI) or typical substrates. 14-16 Since the recognition sequence of the substrate is 90 highly homologous to the regulatory subunits, 4 a loss of cooperativity of binding may affect not 91 only substrate binding fidelity but also the regulation by the R subunit. However, the molecular 92 determinants for the binding cooperativity between ATP and substrate and its role in PKA signal-93 osome remain elusive to date. ganization of the PKA-C core, with the R-spine (gold), C-spine (blue), shell residues (cyan), and 98 the C-4 loop (hot pink) that locks into E helix. 99 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted September 15, 2023. ; https://doi.org/10.1101/2023.09.12.557419 doi: bioRxiv preprint under the conditions used in this work. The enhanced sampling was achieved through bias-ex-125 change metadynamics along different collective variables (CVs) to boost the conformational plas-126 ticity of the enzyme (Figure 2 -figure supplement 1). 22 Back-calculation of the CS using the 127 Sparta+ software 23 shows that the CS restraints improved the agreement between back-calcu-128 lated and experimental CS. Specifically, we obtained an overall improvement of ~0.2 ppm on the 129 amide N atom and ~0.1 ppm on the remainder backbone atoms (Figure 2 -figure supplement  130 2). The bias-exchange metadynamics allowed for each replica to span a broader conformational 131 space relative to classical simulations (Figure 2 -figure supplement 3). The deposition of 132 Gaussian biases required by the metadynamics approach converged after 300 ns, where the 133 fluctuations along the first 3 CVs were less than 1 kcal/mol ( in the ternary form (Figure 2A ). The full free energy landscape was then reconstructed by sam-137 pling an extra 100 ns production phase with reduced biases along each CV. The free energy 138 landscape shows how the population of the conformers is modulated by ligands within the NMR 139 detection limit of sparsely populated states (~ 0.5% or G < 3.2 kcal/mol). 140 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted September 15, 2023. ; https://doi.org/10.1101/2023.09.12.557419 doi: bioRxiv preprint of PKA-C in the apo, ATP-bound, and ATP and the model substrate PKI bound forms. PC1 and 147 PC2 are projected from the first three CVs. The vertices represent conformational states. In the 148 apo form, multiple states have comparable free energy with G < 5 kcal/mol, whereas in the 149 binary form, fewer states have G < 5 kcal/mol, whereas for the ternary form only a major ground 150 state is populated. 151 According to these simulations, apo PKA-C populates preferentially a ground state and five 152 readily accessible low-populated excited states ( Figure 2B, Figure 2supplementary table 1). 153 The nucleotide-bound PKA-C (binary form) features a similar ground state and a broad higher 154 energy basin ( Figure 2C, Figure 2supplementary table 1). Finally, the ternary complex oc-155 cupies a narrow dominant ground state ( Figure 2D, Figure 2supplementary table 1). This 156 free energy landscape obtained from the RAM simulations is consistent with the qualitative picture 157 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted September 15, 2023. ; https://doi.org/10.1101/2023.09.12.557419 doi: bioRxiv preprint previously inferred from our NMR spin relaxation experiments, 24 while providing a detailed struc-158 tural characterization of the excited states. 159 MSM reveals the conformational transitions of PKA-C from ground to high free energy 160 (excited) states. To explore the conformational transitions of the kinase upon ligand binding, we 161 performed additional unbiased sampling to build a Markov State Model (MSM). MSMs are com-162 monly used to describe the dynamic transitions of macromolecules in terms of (1) the probabilities 163 of occupation of a specific set of states and (2) the transition probabilities of moving between 164 these states. In practice, a MSM is typically created by combining thousands of short unbiased 165 simulations. 25,26 Following this strategy, we performed several short simulations (10 -20 ns) 166 using thousands of the low free energy conformations (G < 3.2 kcal/mol) chosen randomly from 167 the three forms of PKA-C as starting structures. The conformational ensembles were clustered 168 into microstates and seeded to start a second round of adaptive sampling (see Methods). The 169 iterative process was repeated three times to assure convergence and yielded a total of 100 s 170 trajectories for both the apo and binary forms, whereas for the less dynamic ternary complex, we 171 collected trajectories of 60 s. Once we reached a sufficient sampling, we built a MSM including 172 L95, V104, L106, M118, M120, Y164, and F185 to investigate the dynamic transitions of the hy-173 drophobic R spine and shell residues (Figure 3figure supplement 1). These residues are ideal 174 reporters of the dynamic processes governing the activation/deactivation of the kinase. 27 To com-175 pare the free energy landscape of different complexes, we first projected the conformational en-176 sembles of three forms and existing crystal structures along the first two time-lagged independent 177 components (tICs) of the apo form, which were obtained by a time-lagged independent compo-178 nent analysis (tICA) analysis (see Methods). These tICs represent the directions of the slowest 179 motion of the kinase and visualize the conformational transitions of V104, L95, and F185 ( Figure  180 sents the ground state (GS) and represents the conformations of the kinase captured by essen-182 tially all crystal structures ( Figure 3A). Additionally, there are two distinct excited states: the first 183 excited state (ES1) features a disrupted hydrophobic packing of L95, V104, and F185. This con-184 formation features an inactive state with an orientation of the C-helix typical of the inhibited 185 states as found for the PKA-C bound to regulatory subunits RI and RII. The second excited 186 state (ES2), to our knowledge, was never captured in crystal structures. The ES2 state displays 187 a flipped configuration of the V104 side chain and a rearrangement of the C-4 loop, with the 188 dihedral angles of F100 and F102 adopting a gauche + configuration. This orientation of the F100 189 and F102 aromatic rings breaks the hydrophobic packing of the C-4 loop with the C-lobe, caus-190 ing steric contacts between F100 and V104 ( Figure 3A ). In contrast, the active GS ensemble 191 features a trans configuration of the F100 and F102 side chains that stabilizes the hydrophobic 192 interactions with E-and J-helices in the C-lobe ( Figure 3A ). Upon binding ATP, the confor-193 mational space span by the kinase becomes narrower, and the conformers populate mostly the 194 GS, with a small fraction in the ES1 state ( Figure 3B). This is consistent with the role of the 195 nucleotide as an allosteric effector, enhancing the affinity of the enzyme for the substrate. In the 196 ternary form (ATP and PKI-bound), PKA-C populates only the GS consistent with the competent 197 conformation observed in the first ternary complex structure. In this case, the C-4 loop of the 198 enzyme is locked in a well-defined configuration, as shown in Figure 3A. 199 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made  In the apo form, the C-4 loop is quite dynamic due to transient hydrophobic interactions 211 between F100 and V104 as well as W222 and the APE motif (A206 and P207) ( Figure 4A). The 212 binding of both nucleotide and PKI increases the rigidity of residues near F100 and V104 such as 213 V103 and F185 ( Figure 4A). In addition, several electrostatic interactions essential for catalysis 214 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made  (D166-N171, K168-T201, and Y204-E230), which are transient in the apo PKA-C, become more 215 persistent ( Figure 4B). 216 The MSM makes it possible to use kinetic Monte Carlo sampling to characterize the slow tran-217 sition between different states. 28 In the apo form, the GS features F100 and F102 in trans, a 218 configuration that stabilizes the interactions with the E and J helices and, together with the 219 nucleotide, locks the C-4 loop to elicit an active kinase conformation ( Figure 3A). The GS to 220 ES1 transition features the disruption of the K72-E91 salt bridge, whereas the GS to ES2 transi-221 tion involves a 120 o flip of the F100 aromatic group that interacts with V104, a conformation found 222 only in the uncommitted apo enzyme ( Figure 3A). The GS to ES2 transition involves a concerted 223 disruption of the D166-N171 and K168-T201 electrostatic interactions, essential for catalysis. Also, 224 this event destabilizes the packing between W222 and the APE motif (A206 and P207) required 225 for substrate recognition ( Figure 3C). All these conformational transitions suggest that ES1 and 226 ES2 represent destabilized states of the kinase. 227 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made  (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted September 15, 2023. ; https://doi.org/10.1101/2023.09.12.557419 doi: bioRxiv preprint The F100A mutation disrupts the allosteric network of the kinase. The above analysis 273 suggests that F100 located at the C-4 loop is critical for the GS to ES1 transition path of the 274 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted September 15, 2023. ; https://doi.org/10.1101/2023.09.12.557419 doi: bioRxiv preprint kinase. Therefore, we modeled the F100A mutant and analyzed its dynamic trajectories. First, we 275 performed a short equilibration using classical MD simulations starting from the coordinates of the 276 X-ray structure of the ternary complex (PDB ID: 4WB5). During 1 μs of MD simulations, the C-277 4 loop of the F100A mutant undergoes a significant motion as manifested by the increased val-278 ues of the backbone rmsd and the conformational transition (flipping) of the F102 side chain (Fig-279 ure 6A). This region in the WT PKA-C adopts a stable -turn in the WT, with a persistent H-bond 280 between the backbone oxygen of F100 and the amide hydrogen of L103. In the simulation of 281 F100A, H-bond is formed more frequently between A100 and F102, and this region adopts a -282 turn conformation. Such local rearrangement disrupts the hydrogen bond between N99 and Y156 283 of E, altering the anchoring of the C-4 to the E helix ( Figure 6B). (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted September 15, 2023. ; https://doi.org/10.1101/2023.09.12.557419 doi: bioRxiv preprint In the F100A mutant, the local changes caused by the C-4 loop structural transitions propagate 292 and alter the response of the nucleotide binding as shown on the rmsd changes for key hydro-293 phobic motifs such as the R-and C-spines and shell residues ( Figure 7A). While the nucleotide 294 binding decreases the average RMSD for these hydrophobic motifs for WT PKA-C, it stabilizes 295 only the C-spine and fails to drive the R-spine and shell residues to an intermediate state compe-296 tent for substrate binding ( Figure 7B). The latter can be attributed to the perturbation of hydro-297 phobic packing of L95, L106 of R-spine, and V104 of the shell residues close to the mutation site. 298 Using the lowest principal components, we also analyzed the global dynamic response to ATP 299 binding. Not only does F100A change the breathing mode of the two lobes (PC1), but it also alters 300 the shearing motion (PC2) of the kinase, emphasizing the importance of this allosteric mutation 301 on the internal communication across the hydrophobic core ( Figure 7C-E). 302 303 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted September 15, 2023. ; https://doi.org/10.1101/2023.09.12.557419 doi: bioRxiv preprint  Table 3). We found that PKA-C WT and PKAC F100A have similar binding affinities for ATPN (Kd = 341 83 ± 8 M and 73 ± 2 M, respectively). However, in the apo form F100A showed a 3-fold higher 342 binding affinity for the pseudosubstrate relative to PKA-C WT (Kd = 5 ± 1 M and 17 ± 2 M, re-343 spectively -Supplementary table 3). Upon saturation with ATPN, PKA-C F100A displayed a 12-344 fold reduction in binding affinity for PKI5-24, resulting in a  of ~3, a value significantly lower than 345 the wild-type ( greater than 100). 346 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted September 15, 2023.  (Figure 10figure supplement 1). A similar trend is 368 observed for PKA-C F100A , though the probability densities are broader, indicating that the amide 369 resonances follow a less coordinated response. 34 Also, the maximum probability density for the 370 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted September 15, 2023. ; https://doi.org/10.1101/2023.09.12.557419 doi: bioRxiv preprint closed state shows that the ternary complex of the mutant is slightly more open than the corre-371 sponding wild-type (Figure 10figure supplement 1). Overall, the shape of the probability dis-372 tributions of the amide chemical shifts suggests that several residues do not respond in a coordi-373 nated manner and that PKI5-24 binding shifts the conformation of the kinase toward a partially 374 closed state, which may explain the loss in binding cooperativity as previously observed. 14-16 375 376 A distinct feature of PKA-C is the binding cooperativity between nucleotide and substrate that 426 originate from the allosteric coupling between the nucleotide-binding pocket and the interfacial 427 region between the two lobes that harbors the substrate binding cleft. Structural and dynamic 428 NMR data suggested that a well-tuned coupling between the two lobes of the kinase is required 429 for efficient substrate binding. Also, additional NMR and functional studies showed that mutations 430 in the activation loop linked to Cushing's syndrome reduce drastically substrate binding affinity, 431 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted September 15, 2023. ; https://doi.org/10.1101/2023.09.12.557419 doi: bioRxiv preprint and more importantly, reduce the communication between the ligand binding pockets. 14-16 Inter-432 estingly, a mutation (E31V) distal from the active site and linked to the progression of Cushing's 433 syndrome has a similar effect, suggesting a possible allosteric modulation of the kinase substrate 434 recognition. 16 NMR chemical shift perturbation data suggested that these mutations are con-435 nected to allosteric nodes that, once perturbed, radiate their effects in the periphery of the enzyme 436 and prevent an efficient dynamic coupling between the two lobes of the enzyme. Interestingly, 437 these mutations do not prevent Kemptide phosphorylation. Rather, they cause a loss of substrate 438 fidelity with consequent aberrant phosphorylation of downstream substrates. 38-43 Additionally, 439 thermodynamics and recent NMR studies using different nucleotides and inhibitors demonstrated 440 that it is possible to control substrate binding affinity by changing the chemistry of the ligand at 441 the ATP binding pocket. Altogether, these studies suggest that phosphorylation reaction and bind-442 ing synergy between ATP and substrates may be controlled independently. 443 Our NMR data combined with RAM simulations and MSM enabled us to comprehensively map 444 the free energy landscape of PKA-C in various forms. We found that the active kinase unleashed 445 from the regulatory subunits occupies a broad energy basin (GS) that corresponds to the confor-446 mation of the ternary structure of PKA-C with ATP and pseudosubstrate (PKI5-24) that exemplifies 447 a catalytic competent state, poised for phosphoryl transfer. 44 We also found two orthogonal con-448 formationally excited states ES1 and ES2. While the ES1 state corresponds to the inactive kinase 449 conformations, the ES2 state was never observed in the crystallized structures. Our previous 450 CEST NMR measurements suggested the presence of a sparsely populated state that, at that 451 time, we were unable to characterize structurally. These new simulations and MSM show that the 452 transition from GS to ES2 is due to a disruption of hydrophobic packing, featuring a conformational 453 rearrangement for the C-4 loop, which causes a partial disruption of the hydrophobic R-spine. 454 These structural changes interrupt the allosteric coupling between the two lobes, as shown by 455 mutual information analysis. A single mutation (F100A), suggested by our simulations, promotes 456 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted September 15, 2023. (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made

Replica-averaged metadynamics (RAM) simulations 486
I. System setup. We used the crystal structure of the wild type PKA-C (PDB ID: 1ATP) as the 487 template and added the missing residues 1-14 at the N terminus. The protonation state of histidine 488 residues followed our previous settings. 51 The protein was solvated in a rhombic dodecahedron 489 solvent box with the tree-point charge TIP3P model 52 for water extended approximately 10 Å away 490 from the surface of the proteins. Counter ions (K + and Cl -) were added to ensure electrostatic 491 neutrality corresponding to an ionic concentration of ~ 150 mM. All protein covalent bonds were 492 constrained with the LINCS algorithm. 53 and long-range electrostatic interactions are treated with 493 the particle-mesh Ewald method with a real-space cut-off of 10 Å. 54 Parallel simulations on the 494 apo form, the binary form with one Mg 2+ ion and one ATP, and the ternary form with two Mg 2+ ions, 495 one ATP and one PKI5-24 are performed simultaneously using GROMACS 4.6 55 with 496 CHARMM36a1 force field. 56 For the two mutants, F100A and V104G, the corresponding residues 497 were mutated through the mutagenesis wizard of PYMOL. 498 II. Standard MD simulations. Each system was minimized using the steepest decent algorithm to 499 remove the bad contacts, and then gradually heated to 300 K at a constant volume over 1 ns, 500 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted September 15, 2023. ; https://doi.org/10.1101/2023.09.12.557419 doi: bioRxiv preprint using harmonic restraints with a force constant 1000 kJ/(mol *Å 2 ) on heavy atoms of both proteins 501 and nucleotides. Over the following 12 ns of simulations at constant pressure (1 atm) and tem-502 perature (300 K), the restraints were gradually released. The systems were equilibrated for an 503 additional 20 ns without positional restraints. A Parrinello-Rahman barostat 57 was used to keep 504 the pressure constant, while a V-rescale thermostat 58 with a time step of 2 fs was used to keep 505 the temperature constant. Each system was simulated for 1.05 µs, with snapshots recorded every 506 20 ps. 507 VII. Adaptive sampling. The first round of adaptive sampling started from the snapshots of low-548 energy microstates obtained from the previous step, i.e., 1200 structures for the apo form, 400 549 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted September 15, 2023. ; https://doi.org/10.1101/2023.09.12.557419 doi: bioRxiv preprint structures for the binary form and 200 structures for the ternary form. The initial velocities were 550 randomly generated to satisfy the Maxwell distribution at 300K. For the apo form, a 10 ns simula-551 tion was performed for each run, whereas for the binary, each simulation lasted 30 ns, resulting 552 in a total of 12 μs trajectories for both the apo and binary forms. To obtain converged free energy 553 landscape, a total of three rounds of adaptive sampling was started from the 400 microstates that 554 was obtained by K-mean clustering of all snapshots of previous ensembles. Therefore, a total of 555 100 µs trajectories and 100,0000 snapshots (100 ps per frame) were collected for both the apo 556 and binary form after three rounds of adaptive sampling, and a total of 60 µs trajectories were 557 collected for the ternary form. 558

VIII. Markov state model (MSM) and time-lagged independent component analysis (tICA). The 559
Cartesian coordinates of key hydrophobic residues, include R-spine residues, L95, L106, Y164 560 and F185, and the shell residues, V104, M118 and M120, were chosen as the metrics to charac-561 terize the conformational transition of the hydrophobic core of PKA-C. Specifically, each snapshot 562 was first aligned to the same reference structure by superimposition of E (residues 140-160) 563 and F helices (residues 217-233), and represented by the deviation of Cartesian coordinates of 564 the key residues. The representation in this metric space was further reduced to 10-dimension 565 vectors using time-lagged independent component analysis (tICA) 63 at a lag time of 1 ns. All the 566 snapshots were clustered into 400 microstates with K-mean clustering. A MSM was built upon 567 the transition counts between these microstates. 568 IX Kinetic Monte Carlo trajectory of PKA-C in different forms. Long trajectories were generated 569 using a kinetic Monte Carlo method based on the MSM transition probability matrix of the three 570 forms of PKA-C. Specifically, the discrete jumps between the 100 microstates were sampled for 571 60 us. And then random conformations were chosen for each state from all the available snap-572 shots. Time series of various order parameters were analyzed subsequently. 573

Protein expression and purification 574
. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted September 15, 2023. ; https://doi.org/10.1101/2023.09.12.557419 doi: bioRxiv preprint The recombinant human C subunit of cAMP-dependent protein kinase with the Phe to Ala mu-575 tation in position 100 (PKA-C F100A ) was generated from the human PKA-C wild-type using Quik-576 Change Lightning mutagenesis kit (Agilent genomics). The key resource table lists the PCR pri-577 mers used to modify the pET-28a expression vector encoding the wild-type human PKA-C gene 578 (PRKACAuniprot P17612) 14-16 . The unlabeled and uniformly 15 N-labeled PKA-C F100A mutant 579 was expressed and purified following the same protocols used for the wild-type protein. 64  20 mM KH2PO4 at pH6.5. 65 The purified protein isoforms were then stored in phosphate buffer 598 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted September 15, 2023. ; https://doi.org/10.1101/2023.09.12.557419 doi: bioRxiv preprint containing 10 mM dithiothreitol (DTT), 10 mM MgCl2, and 1 mM NaN3 at 4 °C. The protein purity 599 was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). 600

Isotherm titration calorimetry (ITC) measurements 609
PKA-C F100A was dialyzed into 20 mM MOPS, 90 mM KCl, 10 mM DTT, 10 mM MgCl2, and 1 mM 610 NaN3 (pH 6.5) and concentrated using conical spin concentrator (10 KDa membrane cut-off, Mil-611 lipore) to a solution at 80-100 μM, as confirmed by A280 = 55,475 M −1 cm −1 . Approximately 300 μL 612 of protein was used for each experiment, with 50 μL of 2 mM ATPγN and/or 1 mM PKI5-24 in the 613 titrant syringe. All measurements were performed at 300 K in triplicates with a low-volume 614 NanoITC (TA Instruments). The binding was assumed to be 1:1, and curves were analyzed with 615 the NanoAnalyze software (TA Instruments) using the Wiseman isotherm 32 616 where d [MX] is the change in total complex relative to the change in total protein concentration, 618 d[Xtot] is dependent on r (the ratio of Kd relative to the total protein concentration), and Rm (the 619 ratio between total ligand and total protein concentration). The heat of dilution of the ligand into 620 the buffer was considered for all experiments and subtracted. 621 The free energy of binding was determined from: 622 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made where R is the universal gas constant and T is the temperature at measurement (300 K). The 624 entropic contribution to binding was calculated using: 625 The degree of cooperativity (σ) was calculated as: 627 where Kd apo is the dissociation constant of PKI5-24 binding to the apo-enzyme, and Kd Nucleotide is the 629 corresponding dissociation constant for PKI5-24 binding to the nucleotide-bound the kinase. Community CHESCA analysis is a chemical shift-based correlation map between functional com-677 munities within the kinase. Each community is a group of residues 37 associated with a function 678 or regulatory mechanism. To represent community-based CHESCA analysis, we lowered the cor-679 relation cut-off such that Rcutoff > 0.8. 680 Suppose communities X and Y have nA and nB number of assigned residues respectively, the 681 correlation score between A and B is defined as, 682 where RiJ is the CHESCA correlation coefficient between residue i (belongs to community A) and 684 residue j (belongs to community B). Rcutoff is the correlation value cutoff. RA,B can take values from 685 0 (no correlation between residues in A and B) to 1 (all residues in A has correlation > cutoff with 686 all residues in B). 687 To whom correspondence should be addressed. Email: vegli001@umn.edu. 706 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made  (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made  (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made  (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made  To best separate ES from GS, snapshots with tIC1 < 1.2 were clustered as ES, whereas those 919 with tIC1 > 0.2 were clustered as GS. (B,C) Representative structure of ES (B) reveals different 920 hydrophobic packing from that of GS (C), highlighted by the distinction at Leu103, Val104, Ile150, 921 Leu172, and Ile180, where all show slow chemical exchanges in CPMG experiment of the apo 922 PKA-C. 923 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted September 15, 2023. ; https://doi.org/10.1101/2023.09.12.557419 doi: bioRxiv preprint CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted September 15, 2023. ; https://doi.org/10.1101/2023.09.12.557419 doi: bioRxiv preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted September 15, 2023. ; https://doi.org/10.1101/2023.09.12.557419 doi: bioRxiv preprint