http://pubs.acs.org/journal/acsodf Article Molecular Insights into the Regulation of 3‑Phosphoinositide- Dependent Protein Kinase 1: Modeling the Interaction between the Kinase and the Pleckstrin Homology Domains Mireia Garcia-Viloca, Jose Ramón Bayascas,* José M. Lluch, and À ngels González-Lafont* Cite This: ACS Omega 2022, 7, 25186−25199 Read Online ACCESS Metrics & More Article Recommendations * sı Supporting Information ABSTRACT: The 3-phosphoinositide-dependent protein kinase 1 (PDK1) K465E mutant kinase can still activate protein kinase B (PKB) at the membrane in a phosphatidylinositol-3,4,5- trisphosphate (PIP3, PtdIns(3,4,5)P3) independent manner. To understand this new PDK1 regulatory mechanism, docking and molecular dynamics calculations were performed for the first time to simulate the wild-type kinase domain−pleckstrin homology (PH) domain complex with PH- in and PH-out conformations. These simulations were then compared to the PH-in model of the KD−PH(mutant K465E) PDK1 complex. Additionally, three KD−PH complexes were simulated, including a substrate analogue bound to a hydrophobic pocket (denominated the PIF-pocket) substrate-docking site. We find that only the PH-out conformation, with the PH domain well- oriented to interact with the cellular membrane, is active for wild-type PDK1. In contrast, the active conformation of the PDK1 K465E mutant is PH-in, being ATP-stable at the active site while the PIF-pocket is more accessible to the peptide substrate. We corroborate that both the docking-site binding and the catalytic activity are in fact enhanced in knock-in mouse samples expressing the PDK1 K465E protein, enabling the phosphorylation of PKB in the absence of PIP3 binding. 1. INTRODUCTION migration, and dissemination of tumor cells.5 For this reason, PDK1 has become a target for cancer therapy.6−9 3-Phosphoinositide-dependent protein kinase 1 (PDK1) is a The modular architecture of PDK1 consists of a N-terminal key protein kinase that activates at least 23 members of the Ser or Thr kinase domain (KD) and a C-terminal pleckstrin AGC (cyclic AMP-dependent kinases, cyclic GMP-dependent homology (PH) domain that binds to PIP3, phosphatidylino- kinases, and protein kinase C) kinase family by phosphor- sitol-3,4-bisphosphate (PtdIns(3,4)P2, PIP2), and with lower ylation. These include isoforms of protein kinase B (PKB, also affinity to phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)- known as Akt), p70 ribosomal S6 kinase (S6K), serum and P2) at the plasma membrane. PDK1 possesses the intrinsic glucocorticoid-induced protein kinase (SGK), p90 ribosomal ability to autophosphorylate both its own T-loop at residue S6 kinase (RSK), and isoforms of protein kinase C (PKC).1 Ser241 within the KD and the Thr513 residue at the PH, These proteins are phosphorylated by PDK1 at highly which has been shown to be critical for PDK1 activation.10 For conserved Ser and Thr residues located at the T-loop (also this reason, PDK1 is considered a constitutively active kinase known as activation loop) of the kinase domain (KD). The in mammalian cells.11 Consequently, it was proposed first that mammalian target of rapamycin (mTOR) complexes phos- 3-phosphoinositide lipids were only necessary to activate phorylate a critical Ser or Thr residue within the hydrophobic substrates for phosphorylation by PDK1.12,13 However, there is motif site, a highly conserved domain that represents a now evidence for the acute activation of PDK1 itself, and many molecular signature for the AGC family members. Phosphor- efforts have been dedicated to understanding the mechanisms ylation of both the T-loop and the hydrophobic motif residues of PDK1 regulation as well as those of the kinases that is required for the maximal activation of those enzymes. The constitute its substrate-signaling network.14 cascade of kinases initiated by PDK1 serves to propagate the Among the AGC family, PDK1 and PKB are the only phosphatidylinositol-3,4,5-trisphosphate (PIP3, PtdIns(3,4,5)- members that possess PtdIns(3,4,5)P3-binding PH domains. P3) second messenger signal produced by the phosphoinositide 3 kinase (PI3-kinase) in response to insulin and growth Received: April 1, 2022 factors.2 Signaling pathways regulated by the phosphorylation Accepted: June 23, 2022 of protein kinases are essential for many biological processes.3 Published: July 15, 2022 Uncontrolled PDK1 downstream signaling has been related with many different diseases.4 Hence, PDK1 is overexpressed in a variety of human cancers, contributing to the proliferation, © 2022 The Authors. Published by American Chemical Society https://doi.org/10.1021/acsomega.2c02020 25186 ACS Omega 2022, 7, 25186−25199 ACS Omega http://pubs.acs.org/journal/acsodf Article Numerous experiments in vitro and in cells suggested that the explanation is that residue Lys465 forms key hydrogen bonds activation of PKB by PDK1 was precisely meant to rely on the with the D3 and D4 phosphates of PIP3, which are lost in the mutual binding of the PDK1 and PKB PH domains to K465E mutant.22 Using a time-resolved fluorescence resonance PtdIns(3,4,5)P3; this interaction would mediate the trans- energy transfer assay, it was also detected that PDK1(mutated location and colocalization of these two kinases at the plasma K467A) interacted with all D5-phosphorylated inositides with membrane, allowing a tight interaction and the phosphor- a lower affinity. Additionally, the overlay of the uncomplexed ylation of PKB by PDK1 at the Thr308 activating residue.15,16 PDK1 PH domain structure with the structures of the PDK1 PKB binding to PIP3 does not really activate the PKB kinase PH−PIP3 and PH−PIP4 complexes indicate that the PH but rather triggers a conformational change that facilitates the domain does not undergo a conformational change when it phosphorylation by PDK1. In live cells, this induced binds to phosphoinositides, as suggested previously. conformational change alters the interaction between the PH The role of the PDK1 PH domain in the activation of PKB and KD domains, thus changing from an inactive PKB-in was then analyzed by knock-in mutation. First, knock-in conformer in which the PH and KD domains establish close PDK1(mutated LLL) embryonic stem cells were generated by interactions that hide the activation sites to a PKB-out mutating Arg472, Arg473, and Arg474 to three Leu groups.23 structure suitable for phosphorylation and activation.17 It was observed that the lack of interaction of PDK1 with the Interestingly, several studies have suggested a direct role for membrane resulted in reduced PKB activation, but PDK1 the binding of the PH domain of PDK1 to 3-phosphoinositides expression levels were also reduced. However, further research in activating the protein, possibly by inducing a conformational could not be done because the PDK1(mutated LLL) knock-in change like that of PKB.10 In particular, the autophosphor- mice died at embryonic age. The 1W1G structure of the PDK1 ylation of Thr513 in combination with the binding of the PH PH−PIP3 complex revealed that the Arg473L mutation could to PIP3 could trigger the structural modification that leads to cause PH domain instability, leading to hypomorphic the PDK1 active form. However, the regulation of PDK1 expression levels of the mutant protein that could in turn activity in live cells seems to depend on a homodimerization cause the lethal phenotypes. process rather than an intramolecular conformational To validate the mechanism for the activation of PKB by change.14,18 A computational model of the PDK1 full-length PDK1 and at the same time generate new tools to manipulate homodimer with an autoinhibitory PH−PH configuration was PKB activation, a rationally designed structural-based point built using a docking procedure.19 The authors proposed that mutation was engineered to replace Lys465 within the PDK1 the dimer would be disrupted upon translocation and PH domain with Glu. While the overall folding of the PDK1− autophosphorylation, leading to an active PDK1 monomer. PH(mutant K465E) crystal (PDB ID 2VKI) was preserved, The first crystal structure of the human PDK1 kinase thereby accounting for the stability of the mutated PH domain, domain cocrystallized with an ATP molecule was released in the positions of the charged side chains of close residues lined 2003 (PDB ID 1H1W). The structure defined the presence of at the PH−membrane interface were different and, con- a hydrophobic pocket, denominated the PIF-pocket, within the sequently, the positive charge of the PtdIns(3,4,5)P3 binding small lobe of the PDK1 catalytic domain, which was meant to site was severely reduced.24 be required for the binding of PDK1 to the hydrophobic motif Functional analysis of the PDK1 K465E mutation was next of its substrates; an adjacent phosphate-binding site was also addressed in vivo by generating knock-in mice physiologically revealed.20 The most recent human PDK1 KD crystallographic expressed from the endogenous locus of the mutant rather structure in complex with a peptide bound to the PIF-pocket than the wild-type version of PDK1. Since the anticipated lack was launched in 2016.21 In that study, the authors described of activation of PKB could have led to embryonic lethality, the the allosteric connection between the ATP-binding site and the expression of the PDK1 K465E protein was circumscribed to PIF-pocket, which was modulated by small compounds bound muscle tissues using Cre-Lox methodologies.5,25 In contrast to to the active site. Additionally, molecular dynamics simulations PDK1(mutated LLL), the PDK1 K465E mutation did not of the KD showed that there was a dynamic equilibrium compromise PDK1 expression levels. Puzzlingly, it was between the open and closed conformations of the KD domain previously found that the activation of PKB by insulin and validated the key roles of Lys144 and Lys199 in stabilizing administration was selectively, albeit only modestly, diminished the closed-active and open-inactive forms of the kinase, but not abolished in muscle protein extracts of the mutant respectively.21 Crystallographic structures of the uncomplexed mice when compared to the wild-type controls (Bayascas and PDK1 PH domain (PDB ID 1W1H), the PDK1 PH domain Alessi, unpublished data). To rule out whether the observed complexed with PIP3 (PDB ID 1W1G), and the PDK1 PH PKB activation reflected the partial penetration of the mutation domain complexed with inositol (1,3,4,5)-tetrakisphosphate into the skeletal muscle, full-body knock-in mice were then (Ins(1,3,4,5)P4, PIP4) (PDB ID 1W1D) were launched in generated, which were viable, smaller in size, and exhibited no 2004, which revealed significant differences compared to overt phenotypes. In tissues derived from these mice, a partial standard PH folds.22 Thus, the PDK1 PH domain shows a N- rather than total ablation of PKB activation was confirmed, terminal extension of two extra antiparallel β-sheets and an although the PDK1 K465E mutant protein was incapable of extra α-helix, which create a new hydrophobic core. Moreover, PtdIns(3,4,5)P3 binding in vivo.24 the PDK1 PH domain possesses a much more spacious ligand- The finding that PDK1(mutated K465E) was still able to binding site. As in other PH domains, the PDK1 PH domain is activate PKB suggested that there was another mechanism for bound to the phosphate groups of phosphoinositides at the the phosphorylation of PKB that was independent of plasma membrane by several positive residues (Arg472, PtdIns(3,4,5)P3 binding. Although the insulin-like growth Arg474, Arg521, Lys465, and Lys467, among others) lined factor 1 (IGF1) induced recruitment of the isolated PDK1 PH along the VL1−3 loops. Using a qualitative protein− domain to the plasma membrane was avoided in cells with the phospholipid overlay assay, it was observed that the mutant K465E mutation, the dynamic translocation of the isolated PH PDK1(mutated K465E) failed to bind phosphoinositides. The domains was found to function differently from that of full- 25187 https://doi.org/10.1021/acsomega.2c02020 ACS Omega 2022, 7, 25186−25199 ACS Omega http://pubs.acs.org/journal/acsodf Article length PDK1; indeed, PDK1 was mainly cytosolic, with only a resulting structures were used to build the model of the KD small fraction present at the membrane, and did not markedly domain with residue Ser241 of the activation loop phosphory- translocate to the plasma membrane following the stimulation lated. In the 5LVP structure, PDK1(50−359) was cocrystal- of cells with IGF1.22 A pool of full-length PDK1 was previously lized in the presence of the phosphorylated peptide KGAG- noticed to be located at the plasma membrane in resting cells, GGGFPQFS(P)YSA (underlined residues were conserved in which in contrast to PKB was not further enriched by growth the hydrophobic motif (HM) of Akts or PKBs, SGKs, S6Ks, factors.11 This PtdIns(3,4,5)P3-independent plasma membrane and PKCs). Thus, in our second KD model, we have included localization most likely relies on binding to other anionic the HM-phosphorylated peptide sequence FPQFS(P)YS. phospholipids such as phosphatidylserine.26,27 Additionally, Hydrogen atoms were added with the module HBUILD the kinase domain of PDK1 has been shown to play a from the program CHARMM,38,39 and the protonation states prominent role in PDK1 membrane localization.28 Once at the of the titrable residues were determined with the PROPKA membrane, the prior phosphorylation of the PKB hydrophobic algorithm.40 In accordance with the high pKa (8.5) calculated motif at Ser473 by mTORC2 may drive the docking of the by PROPKA, the residue Glu188 was protonated in the 5LVP PDK1 K465E protein to the PIF-pocket, allowing the model. The modeled fragment was first minimized by 100 phosphorylation and activation of PKB by PDK1, albeit to steps with the adopted basis Newton−Raphson (ABNR) lower levels.29,30 method of the program CHARMM,38 then 100 steps of ABNR Although subtle, this signaling lesion resulted in knock-in minimization were performed on the side chains of all the male and female mice smaller than those that presented insulin residues in the KD domain. These minimizations were done in resistance24 and neuronal dysfunctions.29,31,32 More recently, the presence of the crystallographic waters. behavioral analysis of the PDK1(mutated K465E) knock-in The same protocol was used to obtain a model of the PH mice has enhanced the relevance of this mutation, which fine- domain that included polar hydrogens and the side chains of all tunes the regulatory mechanism of PKB activation and leads to the residues in optimized positions. several physiological responses in vivo that are sex-, age-, and 2.2. Models of the KD−PH Complex. 2.2.1. Complex genetic load-dependent.33,34 KD(1H1W)−PH(Wild-Type)-In. The KD model built from the To gain insights into the mechanism of regulation of PDK1, 1H1W structure (as described above) was docked with the PH we aimed to understand how the PDK1 K465E mutant kinase model (described above) to generate representative structures could still activate PKB in the absence of phosphoinositide of the KD−KD complex. The docking was performed in the binding. To that end, docking and molecular dynamics (MD) absence of water molecules with the program ZDOCK.41 calculations were performed for the first time to model and ZDOCK performs global searches were one of the molecules is simulate the wild-type KD−PH and the KD−PH(mutant fixed (the KD domain in our case) and the other is docked in K465E) PDK1 complexes. The same analyses were performed different positions (PH domain). The results are filtered such with PH-in and a PH-out conformations of wild-type PDK1 that only those with specified residues (defined by the user) in and a PH-in model of KD−PH(mutant K465E) PDK1. In the binding site are returned. From the 2000 structures addition, three KD−PH complexes were simulated, including a generated, only 13 structures accomplished the restriction that substrate analogue bound to the PIF-pocket. By comparing the was used in this work to define the PH-in conformation, that is, different in silico KD−PH complexes, we found that only the that all the PIF-pocket residues of the KD and the Lys465 of PH-out conformation was active for the wild-type PDK1, the PH domain were in the binding region simultaneously. whereas the active conformation of the PDK1 K465E mutant These 13 structures were re-ranked with the ZRANK was PH-in, since the ATP remained stable at the active site program42 (after hydrogen atoms were added to the while the PIF-pocket was more accessible to the peptide ZDOCK generated files), and the lowest-energy structure substrate. We corroborate that both the docking-site binding was selected as the input of the ROSIE server.43 The and the catalytic activity were in fact enhanced in the PDK1 RosettaDock Server performs a local docking search. That is, K465E protein compared to the wild-type, even in the algorithm will search a set of conformations near the given unstimulated cells. Introducing the K465E amino acid starting conformation for the optimal fit between the two substitution in the K465E PH-in conformer might have partners. We used the Docking 2 protocol. From the structures evoked the Thr513 phosphorylation-mediated PDK1 activa- created in the docking run, we selected the one with lowest tion mechanism;18 indeed, both glutamate substitution and interface energy. This final structure was used to perform 780 phosphorylation resulted in the incorporation of additional ns MD simulations with the program AMBER,44,45 (see section negative charges into the VL-loop region, improving the PH-in 3). In these simulations, the ATP molecule was added to the conformation and leading to the enhanced activation of the KD domain using the original crystallographic coordinates. enzyme. This model is a representation of the wild-type KD−PH interaction in the PH-in configuration. 2. SIMULATED MODELS 2.2.2. Complex KD(1H1W)−PH(Mutant K465E)-In. We 2.1. Models of the PH and KD Domains. In this work, started from the complex KD(1H1W)−PH(wild-type)-in by we built several models of the KD−PH complex. In all cases, mutating the residue Lys465 to a Glu residue with the VMD the initial coordinates for the PH domain were taken from the program.46 We then added the original ATP coordinates. After crystallographic structure with PDB ID 1W1H.22 For the KD a first period of minimization and equilibration, we performed domain, we used two different sets of coordinates correspond- 780 ns MD simulations with the program AMBER (see section ing to PDB IDs 1H1W and 5LVP.20,21 In both cases, there are 3). This model is a representation of the KD−PH(mutant missing residues that belong to the activation loop (residues K465E) interaction in the PH-in configuration. 233−236 in 1H1W and residues 231−239 in 5LVP). These 2.2.3. Complex KD(1H1W)−PH(Wild-Type)-Out. To gen- were modeled with the program MODELER35,36 using the erate a representative structure of the wild-type KD−PH 3HRF structure as a template for the missing segment.37 The interaction in the PH-out configuration, a docking procedure 25188 https://doi.org/10.1021/acsomega.2c02020 ACS Omega 2022, 7, 25186−25199 ACS Omega http://pubs.acs.org/journal/acsodf Article was followed with the ZDOCK program without using any 1. Two hundred energy minimization steps, with restraints restrictions or blocking residues. The ten lowest-energy applied on the Cartesian coordinates of the protein structures generated by the program were re-ranked. domains and ligands (ATP and HM-peptide). Representative configurations of the PH-out state were 2. Two hundred energy minimization steps for the entire selected using the following criteria: (1) a distance larger molecular system. than 25 Å between the residue Leu155 of the PIF-pocket and the residue Lys465 of the VL1-loop and (2) the VL-loops 3. Periodic boundary MD simulations at constant volume should be rotated out of the protein surface region and back to were performed for 10 ps of heating up to 300 K, the PIF-pocket. Under these criteria, only 2 of the 10 lowest- followed by 110 ps of equilibration through periodic energy structures mentioned above presented a PH-out boundary MD simulations at constant temperature and configuration, and the one with the lowest energy was chosen pressure. The weak-coupling algorithm and the as the input of the RosettaDock server to follow the same Berendsen barostat50 were employed for temperature docking protocol and MD procedure mentioned in section and pressure regulation, respectively. The SHAKE 2.2.1 (with the ATP molecule included in the MD algorithm51 was applied to constrain bond distances simulations). involving H atoms. 2.2.4. Models from 5LVP the Crystallographic Structure: 4. P a r t i c l e m e s h E w a l d m o l e c u l a r d y n a m i c s KD−PH(Wild-Type)-In, KD−PH(Mutant)-In, and KD−PH- (PMEMD)52,53 simulations to reach 780 ns. Along the (Wild-Type)-Out Models with the HM-Phosphorylated production period under periodic boundary conditions, Peptide Sequence in the PIF Pocket. With the most recent the temperature was controlled by Langevin dynamics54 X-ray crystallographic structure, we followed a slightly different and the pressure was regulated by the Berendsen strategy to obtain the models of the KD−PH wild-type or barostat. No restraints were applied besides those mutant complexes in the two different configurations that imposed by the SHAKE algorithm. included the HM peptide. The KD model built from the 5LVP A time-step of 2 fs was used for the MD trajectories. structure (as described above) was docked, without using any The minimization, heating, and equilibration steps (1−3 restrictions or blocking residues, with the PH model above) were performed with the SANDER module of (described above) to generate representative structures of the AMBER 14 program.44 The production stage was the KD−PH complex. carried out with the AMBER 16 GPU (CUDA) version Among the ten most-stable structures (re-ranked by the of the PMEMD package.55,56 ZRANK program), we selected one that was more We analyzed the trajectories generated for the six models of representative of the PH-in configuration (that is, with the the KD−PH complex in different configurations using the VL-loops rotated toward the internal zone of the complex, program CPPTRAJ (C++ Process Trajectory). In all cases, we facing the PIF pocked and the helix-αC) and another used the rmsd command to perform RMSD calculations and representative of the PH-out configuration (under the criteria the hbond and distance commands to analyze inter atomic described above). Both structures were refined with the interactions between parts of the system. During the RosettaDock Server. The lowest-interface-energy structures simulation, the trajectory was saved every 5000 steps, that is, were selected for both the in and out configurations and every 10 ps, for analysis. completed with the ATP molecule in its X-ray position in the KD domain before a long MD simulation was initialized to 4. RESULTS AND DISCUSSION study the stability and behavior of the ternary complexes in the We first sought to biochemically determine the subcellular two different PH configurations (in and out). localization of PDK1 to clarify whether agonist stimulation, In addition, the mutant KD−PH-in model was built from PtdIns(3,4,5)P3 production, or phosphoinositide binding the initial wild-type KD−PH-in model complex by mutating dynamically relocated PDK1 pools. To that end, cytosolic the residue Lys465 to a Glu residue with the VMD program.46 and membrane protein fractions obtained from muscle tissue Next, a MD simulation was carried out for this model, similar extracts derived from PDK1 K465E mutant and PDK1 wild- to that for the mutant complex built from the 1H1W structure. type control littermate mice, who were or were not injected with insulin, were analyzed by Western blot. As depicted in 3. SIMULATION PROTOCOL Figure 1, PDK1 is mainly cytosolic in the unstimulated control tissues, and only a small proportion of the total protein was Each model was solvated with a nearly cubic pre-equilibrated detected in the membrane fractions in both the wild-type and box of TIP3P47 water molecules (of dimensions around 194 Å mutant samples. In contrast to the PKB kinase, which is × 185 Å × 180 Å). The final solvated models had around excluded from the membrane in resting conditions and 100 000 atoms each. A buffering distance of 12 Å was enriched in these fractions by insulin in both control and established between the atoms of the enzyme and the edge of mutant tissues, membrane-associated levels of PDK1 are the box. Water molecules closer than 1.0 Å to any atom of the neither further increased by insulin in wild-type or mutant ligands or the enzyme were removed. The total charge of the samples not excluded from the membrane in the mutant system was neutralized by including one Cl− ion or one Na+ samples by the PDK1 K465E mutation. Therefore, a pool of ion depending on the model. The AMBER ff14SB48 force field PDK1 protein constitutively associated with membranes in an was employed to define the protein residues, and the agonist-independent manner is definitively demonstrated; this phossaa1049 force field was used to define the molecular is preserved in the PDK1 K465E samples, thereby evidencing mechanics (MM) parameters of the ATP phosphate groups. the participation of other mechanisms, such as the suggested The following simulation protocol was used for all the phosphatidylserine binding site or the kinase domain, in models: anchoring PDK1 to the plasma membrane.27,28 25189 https://doi.org/10.1021/acsomega.2c02020 ACS Omega 2022, 7, 25186−25199 ACS Omega http://pubs.acs.org/journal/acsodf Article constructed, the RMSD calculation (Figure S1) shows that the complex system has stable dynamical behavior. The PH domain is more mobile than the KD domain in all cases. Visual inspection of the trajectories indicates that the two domains, KD and PH, interact all along the simulations and that the complex rotates as a whole. In the analysis of the trajectories generated for each of the models, we focused our attention on interactions or conforma- tional changes related to key residues of the enzymatic system whose roles in catalysis or kinase activation have been Figure 1. Subcellular localization of endogenous PDK1. Mice were proposed in the literature. In particular, fasted overnight and intravenously injected with either saline (CONTROL) or 0.5 mU/g insulin (INSULIN). Protein extracts 1. The dynamical behavior of ATP in the active site and its were obtained from knock-in mutant (PDK1 K465E) and control interactions with the conserved residue Lys111 or wild-type (PDK1 wild-type) muscle tissues, which were fractionated others. into cytosolic (CYT) and membrane (MEM) fractions. Samples were 2. The salt bridge between Lys111 and Glu130, a highly subsequently analyzed by Western blot with PKB, PDK1, the conserved salt bridge between a Lys that positions the cytosolic marker GAPDH, and the plasma membrane marker Na-K- phosphate of ATP at the active site and a Glu from helix- ATPase antibodies. The results are representative of three experi- αC (Lys111 and Glu130 in PDK1), because it is widely ments, where each lane represents a sample derived from a different mouse. considered a hallmark of active structures of protein kinases.20,57 This PDK1 K465E enzyme anchored to the membrane in a 3. The salt bridge formed by Lys144 and Asp138, which PtdIns(3,4,5)P3-independent manner has been widely shown was experimentally observed only in the closed form of to be unproficient at phosphorylating PKB.23,29 The surfing of the complex and not in the open form, where it was membrane-bound wild-type PDK1 to PtdIns(3,4,5)P3-en- replaced by the Lys199-Asp138 interaction. riched rafts, where PKB will be expected to be located upon 4. The distance between the conserved PH residue K465 agonist stimulation, could account for normal PKB activation, (or, when mutated in the mutant models, E465) and the which will be prevented in the mutant samples. Yet, in the PIF-pocket (residues Lys115, Ile118, and Ile119 in the PDK1 K465E mice, the considerable activation of PKB was αB-helix; residues Val124 and Val127 in the αC-helix; still observed. We hypothesize that changing Lys at the critical and residue Leu155 in β-sheet5). position 465 into Glu may have structural and functional Figure 2a shows the relative positions of the above- consequences for PDK1 regulation beyond preventing PtdIns- mentioned residues in the initial structure of the complex (3,4,5)P3 binding, which could explain the remaining PKB KD(1H1W)−PH(wild-type)-in. Lys111 is located between the activation in the mutant tissues. To explore that possibility, PIF-pocket (around 8−10 Å from it) and the ATP molecule. It docking and molecular dynamics calculations of the wild-type interacts with the ATP phosphate arm and the residue Glu130 KD−PH and the mutant KD−PH(K465E) PDK1 complexes (represented in Figure 2a but not in Figure 2b) simultaneously were modeled as described in section 3. In all the six models through salt-bridge interactions. The Asp138 residue is located Figure 2. (a) Initial KD(1H1W)−PH(wild-type)-in model. The side chains of relevant kinase residues are represented in sticks and colored by atom type. The residues shown as green and red sticks correspond to the phosphate pocket and the PIF-pocket, respectively. (b) Final structure from the MD simulation of the KD(1H1W)−PH(wild-type)-in complex. See panel a for the meanings of the colors used. 25190 https://doi.org/10.1021/acsomega.2c02020 ACS Omega 2022, 7, 25186−25199 ACS Omega http://pubs.acs.org/journal/acsodf Article between the two lysine residues, namely, Lys144 and Lys199, phosphate group of ATP (with only 0.09% of the residence in an intermediate position (neither the closed nor open time), whereas at the beginning of the simulation it competes configuration). The VL-loops are formed by positively charge with Glu130 and Asp223 in their interactions with Lys111. residues (Lys465, Arg472, Lys512, etc.) that interact with Finally, the interaction of the O1A atom of ATP with the NZ negatively charged residues of the small lobe of the KD domain atom of Lys111 is lost, and ATP moves away to find other (for example, Asp132 and Glu153). interactions, as shown in the final structures of the simulation. Figure 3a shows the movement of ATP from its initial Regarding the dynamics of the protein, at the start of the interaction with Lys111. The ATP molecule goes far from the simulation, the Asp138 residue interacts with Lys144. However, after approximately 80 ns, it rotates to interact with Lys199, thus going from the closed form to the open form (see Figure S3a). During the simulation, the distance between the geometric center of the PIF-pocket residues and the geometric center of a group of four VL-1 residues around Lys465 (residues 463−466) increases from 12−14 Å to 16−18 Å (Figure S4a). When the simulation is carried out with the mutant model complex (complex KD(1H1W)−PH(mutant K465E)-in), the ATP molecule remains at the same position in the active site for the first quarter of the simulation, as can be seen in Figure 4a. After that, it moves to interact with positively charged Figure 3. The KD(1H1W)−PH(wild-type)-in complex. (a) Distances between Lys111 or Lys86 and ATP. (b) The salt bridge between Lys111 and Glu130. active site to interact with Lys86, whose position in the protein complex can be seen in Figure 2b. Figure 3b indicates that the salt-bridge interactions between the conserved residues Lys111 and Glu130 do not significantly change despite the ATP movement. Figure S2a compares the RMSDs of ATP, which were calculated with the first structure or the average structure as references, respectively, revealing that the ATP movement occurs at the very beginning of the simulation. The analysis of the structures along the trajectory shows that at the beginning of the simulation Glu130 simultaneously interacts with the adenine ring of the ATP molecules (N6 atom), the NH3+ side- chain of Lys111, and the −NH main-chain groups of residues Figure 4. The KD(1H1W)−PH(mutant K465E)-in complex. (a) Gly225 and Phe224 located in a loop nearby the activation Distances between Lys111 and ATP. (b) The salt bridge between Lys111 and Glu130. loop. Another negative side-chain residue in the same loop, Asp223, approaches Lys111 (the distance between them is reduced by 3 Å along the first quarter of the trajectory) and residues of the activation loop (Arg238 and Lys235) and a anchors it near Glu130. The stability of the Asp223−Lys111 positively charged residue in the VL loop (Lys512). The and Glu130−Lys111 interactions is supported by residence movement of ATP far from Lys111 correlates with a times of 28.92% and 37.07%, respectively, as indicated in Table strengthening of the Lys111−Glu130 interaction (as seen in S1 of the Supporting Information file. This seems to be the Figure 4b). It is interesting to observe that there is a clearer main reason why Lys111 loses its initial interaction with the α- oscillation of the enzymatic system between the open and 25191 https://doi.org/10.1021/acsomega.2c02020 ACS Omega 2022, 7, 25186−25199 ACS Omega http://pubs.acs.org/journal/acsodf Article closed forms during the time ATP and Lys111 maintain their Figure 6b shows the relative positions of the key residues at interaction, as shown in Figure S3b. After that, the system the end of the simulation, showing that they do not dynamics stabilize the open-form configuration. significantly change from the beginning of the simulation and For the mutant model, the distance between the PIF thus supporting the stabilization of the system in the PH-out residues and the VL-1 residues is slightly shorter (11−16 Å) configuration with the ATP molecule within the active site. than the corresponding one in the wild-type model (Figure Interestingly, Asp138, which also changes its interaction with S4b). Lys144 by interacting with Lys199 as in the PH-in Figure 5 shows the relative positions of the key residues configuration, is more mobile in this PH-out model, as seen mentioned above at the end of the complex KD(1H1W)− when comparing Figure S3a and c in the Supporting PH(mutant K465E)-in simulation. Information file. The KD(1H1W)−PH(mutant K465E)-out complex was not modeled because the PH-out conformation with the PH domain well-oriented to interact with the cellular membrane could never be stabilized in the mutant, as unfavorable electrostatic interactions with PtdIns(3,4,5)P3 prevented binding. The results obtained for the models built from the 5LVP X- ray structure of the KD domain shed light on the effect of the substrate in the active site. Figure 8a shows the initial disposition of the key residues of the system at the beginning of the simulation for the model KD(5LVP)−PH(wild-type)-in. The position of the HM-peptide substrate (in gray) that interacts with the PIF-pocket residues is shown. As occurred for the model built from the 1H1W structure, in the case of the PH-in configuration of the wild-type complex, the ATP molecule migrates to interact to other positively charged residues other than Lys111. However, in this case, instead of forming interactions with KD residues, it is the VL- loop residue Lys465 that mainly stabilizes the final position of the ATP molecule (see Figure 8b). A general difference between the PH-in structures generated from 5LVP and those generated from 1H1W is the larger distance in the former (23−30 Å in comparison to 12−18 Å) between the PIF-pocket and the VL-loops (see Figure S4d), what may explain the different behavior of ATP. In addition, that larger difference in the VL-loops−PIF Figure 5. Final structure of the KD(1H1W)−PH(mutant K465E)-in complex from the MD simulation. See Figure 2 for the meanings of distance, the presence of the substrate, and perhaps other the colors used. causes that we have not been able to detect result in the stabilization of the ATP molecule in the active site of the mutant model complex KD(5LVP)−PH(K465E)-in. Figure 9a The results obtained for the third model built from the shows that ATP interacts with Lys111 all along the simulation. 1H1W structure, namely, complex KD(1H1W)−PH(wild- That interaction, with a residence time of 95.55%, is correlated type)-out where the interaction between the KD and the PH with a weaker interaction of the salt bridge Lys111−Glu130 domain is not though the VL-loops but instead between the (Figure 9b) and a clearer oscillation of the system between the large lobe of the KD domain and the helices of the PH domain open and closed forms (Asp138 alternatively interacts with (see Figure 6a), indicate that the ATP molecule maintains its Lys144 and Lys199 along the simulation, Figure 9c). It is position in the active site for the entire simulation (Figure 7a). worth noting that, as seen in the previous models, Glu130 this might be correlated with the fact that the distance between establishes hydrogen-bond interactions with the main-chain Glu130 and Lys111 is larger than those in the previous models −NH groups of residues 223−225 (see the larger residence (see Figure 7b), as the interaction between Lys111 and ATP time interaction values of 61.63% and 25.36% for this model in weakens the Lys−Glu salt-bridge. The analysis of the Table S1) and is closer to the αC-helix. Therefore, the interactions established by Glu130 along the MD trajectory mutation in the VL-loops in the presence of the substrate suggests that this residue, together with the loop formed by seems to widen the space between the 130−223−225-αC-helix residues Gly225 and Phe224, which are hydrogen-bonded to region and the Lys−ATP dyad, thus contributing to reinforcing Glu130 (as seen in the wild-type and mutant PH-in model the interactions between the latter two residues. The larger complexes), has enough space to move away from the separation between Glu130 and the Lys111−ATP dyad Lys111−ATP dyad. Finally, Glu130 interacts with another explains the stability of ATP in the active site and seems to positive residue, Arg131, as seen from the snapshot at around be correlated with the oscillation between the open and closed 650 ns. The residence time of this interaction is 38.15% (see forms of the KD domain, which is associated with the active Table S1). This new interaction is established as an alternative state of the enzyme, as mentioned in the literature. This active to the salt bridge with Lys111, which now anchors the ATP state is reached when ATP and then the substrate have entered molecule in the active site with a residence time of 70.08%. in the active site but not in the absence of the substrate, as 25192 https://doi.org/10.1021/acsomega.2c02020 ACS Omega 2022, 7, 25186−25199 ACS Omega http://pubs.acs.org/journal/acsodf Article Figure 6. (a) Initial structure of the KD(1H1W)−PH(wild-type)-out complex from the MD simulation. See Figure 2 for the meanings of the colors used. (b) Final structure of the KD(1H1W)−PH(wild-type)-out complex from the MD simulation. See Figure 2 for the meanings of the colors used. suggested by the negative (no oscillation) result obtained for interacting with Lys144 and Lys199 along the simulation. our theoretical models without the substrate. These changes may have promoted a partial transition of the Figure 10 shows the final positions of the key residues in the mutant PDK1 enzyme to the out conformation, analogous to active site at the end of the simulation. The data collected in that induced by the binding of the wild-type PH domain to Table S1 of the Supporting Information file reinforce the idea PIP3 but less efficient. This conformation may have enabled that the mutant model KD(5LVP)−PH(K465E)-in is closer to PDK1 K465E to dock to the phosphorylated hydrophobic the out conformation of the KD(5LVP)−PH(wild-type)-out motif of membrane-located PKB,58 allowing Thr308 phos- complex (see below) than to the in conformation of the phorylation. To test these predictions, a peptide encompassing KD(5VLP)−PH(wild-type)-in complex. the hydrophobic motif sequence of the PKC-related protein Finally, the results obtained for the model built from the kinase 2 (PRK2), termed PIF-tide (from PDK1 interacting 5LVP structure representing the PH-out configuration, namely fragment peptide) was employed to perform the affinity KD(5LVP)−PH(wild-type)-out, indicate that the PH-out purification of PDK1 from protein extracts derived from PDK1 configuration of the system also maintains the ATP ligand in wild-type and PDK1 mutant mouse tissues. As shown in Figure the active site in the presence of the HM-peptide substrate. 15, the interaction of PDK1 with the docking-site-emulating This is shown by the analysis of the trajectory (see Figure 11), peptide was enhanced by the PDK1 K465E mutation in most which goes from the initial configuration represented in Figure tissues analyzed. By contrast, PDK1 was purified with PIF- 12a to the final snapshot represented in Figure 12b. sepharose from liver protein extracts derived from PDK1 wild- Interestingly, the structures generated with this model in the type and PDK1 K465E knock-in mice with the same efficiency, PH-out configuration, the substrate HM-peptide, and the ATP an unexplained tissue singularity that was observed pre- ligands in the active site clearly oscillate between the open and viously.24 closed forms of the KD domain (see Figure 13), and a looser Moreover, the catalytic activity of PDK1 immunoprecipi- salt-bridge interaction between Lys111 and Glu130 is present tated from wild-type and mutant mouse muscle samples was (see Figure 14). That result supports an equilibrium between assayed in vitro against two different peptides, the T308tide the open and closed conformations of PDK1, as proposed corresponding to the T-loop of PKB, and the PDKtide, which previously.21 consisted of both the sequence of the T308tide and the In the PDK1 K465E protein, molecular modeling data sequence of the PIFtide. As shown in Figure 16, the catalytic suggested that replacing lysine at position 465 with glutamate activity of PDK1 against the T308tide is similar in the wild- resulted in the stabilization of the ATP molecule at the active type and the mutant samples and is not enhanced by insulin site. This was accompanied by a clear oscillation between open administration, thereby supporting the notion of PDK1 as a and closed forms, as revealed by Asp138 residue alternatively constitutively active enzyme. As reported previously, the 25193 https://doi.org/10.1021/acsomega.2c02020 ACS Omega 2022, 7, 25186−25199 ACS Omega http://pubs.acs.org/journal/acsodf Article increased by insulin, twofold in the wild-type samples. Of note, in the PDK1 K465E samples, the PDK1 activity against PDK1tide was found to be induced threefold compared to that in the T308 assay, thereby demonstrated that the improved binding of the PDK1 K465E mutant protein to the substrate- docking site might have been sufficient to enable the phosphorylation of PKB in the mutant mouse samples even in the absence of PtdIns(3,4,5)P3 binding. 5. CONCLUSIONS In summary, the results presented here for the PDK1 wild-type enzyme in the PH-in configuration are coherent with the generally accepted idea that one of the factors regulating PDK1 activity is its change to a configuration where the VL-loops of the PH domain do not face the PIF-pocket but are oriented in an opposite direction toward the external face of the KD−PH complex (PH-out configuration), which is triggered by the interaction with the PtdIns(3,4,5)P3 lipid at the cellular membrane. This is supported by the fact that in our PH-out models (KD(1H1W)−PH(wild-type)-out and KD(5LVP)− PH(wild-type)-out) the ATP molecule is stable in the active site independent of the presence or absence of the HM of the substrate in the active site, whereas the PH-in modeled configurations (KD(1H1W)−PH(wild-type)-in and KD- (5LVP)−PH(wild-type)-in) are not able to maintain the reactive disposition of the ATP ligand. Importantly, the results obtained for the simulated K465E mutation (complexes KD(1H1W)−PH (mutant K465E)-in and KD(5LVP)−PH- (K465E)-in) suggest that the PH-in configuration of the mutant might be partially active, as ATP is maintained in the active site, especially in the presence of the HM of the Figure 7. The KD(1H1W)−PH(wild-type)-out complex. (a) substrate. The presence of the negatively charge amino acid Distances between Lys111 and ATP. (b) Thee salt bridge between Glu rather than the positively charge Lys at position 465 could Lys111 and Glu130. have promoted the repulsion of the VL-loops of the PH domain away from the substrate-docking site. Similarly, the presence of a docking site sequence next to the T-loop in incorporation of negative charges via the phosphorylation of PDKtide enhances the PDK1 activity, which is not further the Thr at position 513 has been shown to be a driving force in Figure 8. (a) Initial structure of the KD(5VLP)−PH(wild-type)-in complex from the MD simulation. See Figure 2 for the meanings of the colors used. (b) Final structure of the KD(5VLP)−PH(wild-type)-in complex from the MD simulation. See Figure 2 for the meanings of the colors used. 25194 https://doi.org/10.1021/acsomega.2c02020 ACS Omega 2022, 7, 25186−25199 ACS Omega http://pubs.acs.org/journal/acsodf Article Figure 10. Final structure of the KD(5VLP)−PH(mutant K465E)-in complex from the MD simulation. See Figure 2 for the meanings of the colors used. Figure 11. The KD(5VLP)−PH(wild-type)-out complex. Distances between Lys111 and ATP. Figure 9. The KD(5LVP)−PH(K465E)-in complex. (a) Distances between Lys111 and ATP. (b) Salt bridge between Lys111 and Glu130. (c) Distances between Asp138 and Lys144 or Lys199. 6. EXPERIMENTAL METHODS 6.1. Mice. The generation and genotyping of PDK1 K465E knock-in mice expressing the single-amino-acid substitution from Lys465 to Glu in the PDK1 PH domain were described PDK1 activation. As predicted by the model, this enhanced previously.32 Mutant mice and matched wild-type littermates both docking site binding and catalytic activity in vivo. Thus, were used in all the experiments. Mice were maintained on a the theoretical results presented here for the mutant models 12 h light−dark cycle (lights on at 07:00) with ad libitum are coherent with those obtained experimentally. In addition, access to food and water. All animal procedures were performed in accordance with 2010/63/UE regarding the our simulations shed light on phenomena that seem to care and use of animals for experimental procedures. Protocols characterize the active configurations (the ones represented by were approved by the Ethical Committee for Human and the PH-out wild-type and PH-in mutant models). Specifically, Animal Experimentation, Universitat Autònoma de Barcelona, the more dynamical behavior of the enzyme with enhanced and performed under a Generalitat de Catalunya Project oscillation between the open and closed forms, which License. The study complies with the ARRIVE guidelines characterizes the respiration of the KD domain, as suggested developed by the NC3Rs.59,60 by Schulze et al.,21 and the conservation of a looser salt-bridge 6.2. Protein Extracts. Tissues were rapidly extracted, freeze clamped in liquid nitrogen, and homogenized on ice interaction (in comparison to the corresponding stronger using Kinematica Polytron (Brinkmann, CT, USA) in a 10-fold interaction in the PH-in wild-type models) between Lys111 mass excess of ice-cold lysis buffer (50 mM Tris-HCl pH 7.5, 1 and Glu130, which is flexible enough to allow the ligand to mM EGTA, 1% (by mass) Triton-X 100, 1 mM sodium move during catalysis. orthovanadate, 50 mM sodium fluoride, 5 mM sodium 25195 https://doi.org/10.1021/acsomega.2c02020 ACS Omega 2022, 7, 25186−25199 ACS Omega http://pubs.acs.org/journal/acsodf Article Figure 12. (a) Initial structure of the KD(5VLP)−PH(wild-type)-out complex from the MD simulation. See Figure 2 for the meanings of the colors used. (b) Final structure of the KD(5VLP)−PH(wild-type)-out complex from the MD simulation. See Figure 2 for the meanings of the colors used. Figure 13. The KD(5LVP)−PH(wild-type)-out complex. Distances Figure 14. The KD(5LVP)−PH(wild-type)-out complex. The salt between Asp138 and Lys144 or Lys 199. bridge between Lys111 and Glu130. pyrophosphate, 0.27 M sucrose, 0.1% (by volume) 2- resulting supernatant was centrifuged at 200 000 × g for 60 mercaptoethanol, and the complete proteinase inhibitor min at 4 °C to obtain the cytosolic fraction (supernatant) and cocktail (one tablet per 50 mL)). Lysates were then the membrane fraction (pellet), which was homogenized in centrifuged at 4 °C for 10 min at 13 000 × g to remove buffer A containing 1% (w/v) Triton X-100. insoluble material. The supernatant was aliquoted, snap frozen 6.4. Affinity Purification of PDK1. PDK1 was affinity in liquid nitrogen, and stored at − 80 °C. purified on PIF-Sepharose from tissue lysates as described 6.3. Subcellular Fractionation. Tissues were rapidly previously.23 Briefly, 10 μL of streptavidin-Sepharose was extracted, freeze clamped in liquid nitrogen, and homogenized conjugated to 0.5 μg of biotinylated PIF peptide (Biotin-C6 on ice using a Dounce homogenizer (50 strokes) in a 10-fold spacer-REPRILSE-EEQE-MFRD-FAYI-ADWC) and incu- mass excess of ice-cold lysis buffer A (10 mM HEPES, 10 mM bated with 0.5 mg of precleared tissue lysates at 4 °C for 1 KCl, 1.5 mM MgCl2, and the complete protease inhibitor h on a shaking platform. The pull-downs were washed twice cocktail). The homogenates were centrifuged twice at 3000 × g with 1 mL of lysis buffer, resuspended in SDS sample buffer, for 10 min at 4 °C to remove unbroken cells and nuclei. The electrophoresed and immunoblotted for PDK1. 25196 https://doi.org/10.1021/acsomega.2c02020 ACS Omega 2022, 7, 25186−25199 ACS Omega http://pubs.acs.org/journal/acsodf Article and ab7671, respectively), while the p44/42 MAPK ERK1/2 antibody was from Cell Signaling (catalogue no. 9102). 6.6. Protein Kinase Assay. The activation state of PDK1 was assessed following the immunoprecipitation of PDK1 from cell extracts. The activity was measured against the T308tide peptide (KTFC-GTPE-YLAP-EVRR, which corresponded to the T-loop of PKB, and the PDKtide, which consisted of the sequence of both T308tide and PIFtide (KTFC-GTPE-YLAP- EVRR-EPRI-LSEE-EQEM-FRDF-AYIA-DWC), as described previously.61 ■ * ASSOCIATED CONTENT sı Supporting Information The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.2c02020. Additional figures illustrating the evolution of RMSDs Figure 15. Protein extracts derived from the indicated tissues were with time, some selected distances between relevant obtained from PDK1 knock-in (K465E) and control wild-type (wild- residues for the different KD−PH models, and a table type) mice. PDK1 was purified by affinity on PIF-Sepharose (right with the calculated residence times for selected hydro- panel); as controls, PDK1 protein expression levels were measured on gen-bond interactions (PDF) total tissue extracts (left panels). Representative Western blots are shown, where each lane corresponds to a sample derived from a different mouse. ■ AUTHOR INFORMATION Corresponding Authors À ngels González-Lafont − Departament de Química, Universitat Autònoma de Barcelona, Bellaterra, Barcelona 08193, Spain; Institut de Biotecnologia i de Biomedicina (IBB), Universitat Autònoma de Barcelona, Bellaterra, Barcelona 08193, Spain; orcid.org/0000-0003-0729- 2483; Email: angels.gonzalez@uab.cat Jose Ramón Bayascas − Institut de Neurociències, Universitat Autònoma de Barcelona, Bellaterra, Barcelona 08193, Spain; Department of Biochemistry and Molecular Biology, Biochemistry Unit of the School of Medicine, Universitat Autònoma de Barcelona, Bellaterra, Barcelona 08193, Spain; Email: JoseRamon.Bayascas@uab.cat Authors Mireia Garcia-Viloca − Departament de Química, Universitat Autònoma de Barcelona, Bellaterra, Barcelona 08193, Spain José M. Lluch − Departament de Química, Universitat Autònoma de Barcelona, Bellaterra, Barcelona 08193, Spain; Institut de Biotecnologia i de Biomedicina (IBB), Universitat Autònoma de Barcelona, Bellaterra, Barcelona 08193, Figure 16. PDK1 was immunoprecipitated from muscle extracts of Spain; orcid.org/0000-0002-7536-1869 the indicated genotypes and its activity was assayed using either the T308tide or PDKtide peptides as the substrates. Each point represents Complete contact information is available at: the mean activity ± the standard error of the mean for three different https://pubs.acs.org/10.1021/acsomega.2c02020 samples, each of which was assayed in triplicate. **P < 0.005 and *P < 0.05 compared to controls. Notes The authors declare no competing financial interest. 6.5. Western Blot. The protein concentration was determined by the Bradford method using bovine serum ■ ACKNOWLEDGMENTS The authors acknowledge the Spanish “Ministerio de Ciencia e albumin as the standard. Samples were electrophoresed, Innovación” for Grants PID2020-113764GB-I00 and transferred onto nitrocellulose membranes on a Bio-Rad RTI2018-101249-B-I00 Mini-PROTEAN system, and immunoblotted with the indicated antibodies. The PDK1 antibody was raised in sheep against the sequence RKIQ-EVWR-QQYQ-SNPD- AAVQ, corresponding to residues 540−559 of mouse PDK1. ■ REFERENCES (1) Mora, A.; Komander, D.; van Aalten, D. M. F.; Alessi, D. R. PDK1, the Master Regulator of AGC Kinase Signal Transduction. The PKB total antibody was raised in sheep against the Seminars in Cell and Developmental Biology 2004, 15 (2), 161−170. sequence RPHF-PQFS-YSAS-GTA, corresponding to residues (2) Pearce, L. R.; Komander, D.; Alessi, D. R. The Nuts and Bolts of 466−480 of rat PKBα. Anti-GAPDH and anti-Na-K ATPase AGC Protein Kinases. 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