Abstract
AKT1 (NP_005154.2) is a member of the serine/threonine AGC protein kinase family involved in cellular metabolism, growth, proliferation and survival. The three human AKT isozymes are highly homologous multi-domain proteins with both overlapping and distinct cellular functions. Dysregulation of the AKT pathway has been identified in multiple human cancers. Several clinical trials are in progress to test the efficacy of AKT pathway inhibitors in treating cancer. Recently, a series of AKT isozyme-selective allosteric inhibitors have been reported. They require the presence of both the pleckstrin-homology (PH) and kinase domains of AKT, but their binding mode has not yet been elucidated. We present here a 2.7 Å resolution co-crystal structure of human AKT1 containing both the PH and kinase domains with a selective allosteric inhibitor bound in the interface. The structure reveals the interactions between the PH and kinase domains, as well as the critical amino residues that mediate binding of the inhibitor to AKT1. Our work also reveals an intricate balance in the enzymatic regulation of AKT, where the PH domain appears to lock the kinase in an inactive conformation and the kinase domain disrupts the phospholipid binding site of the PH domain. This information advances our knowledge in AKT1 structure and regulation, thereby providing a structural foundation for interpreting the effects of different classes of AKT inhibitors and designing selective ones.
Introduction
Aberrant regulation of the PI3K/AKT pathway is implicated in the pathogenesis of several human cancers and inhibitors for multiple targets in this pathway are in clinical trials for the treatment of cancer [1]. There are three isozymes of human AKT (AKT1, 2, and 3, also known as PKB-α, -β and -γ), each containing an amino (N)-terminal PH domain, inter-domain linker, kinase domain and 21-residue carboxy (C)-terminal hydrophobic motif (HM) [2], [3]. The PH domain directs AKT translocation from the cytosol to the plasma membrane by binding to the membrane lipids phosphatidylinositide (PtdIns)(3,4)P2 and PtdIns(3,4,5)P3, which are products of phosphatidylinositide-3-kinase (PI3K). AKT is subsequently phosphorylated resulting in kinase activation [4]. Due to the tractability of kinases as pharmacological targets and the observed hyperactivation of AKT in many cancers, several small molecule inhibitors of AKT have been described (recently reviewed [5]). The majority of described AKT inhibitors are competitive with ATP, non-selective against AKT isozymes, and poorly selective against closely related kinases. Efforts to identify AKT specific and isozyme-selective inhibitors resulted in the discovery of novel selective, allosteric AKT inhibitors [6], [7]. As only a few kinases have been reported to be allosterically inhibited by small molecules [8], [9], [10], further investigation into the requirements for allosteric AKT inhibition was undertaken. Intriguingly, a new allosteric inhibition paradigm was revealed in which the presence of both the regulatory PH domain and catalytic kinase domain were required for allosteric inhibition. Subsequently the allosteric AKT inhibitors were optimized for clinical use and recently one, MK-2206, was reported to be well-tolerated in a Phase I clinical trial [11].
Comprehensive and elegant experimentation revealed substantial differences in the relative positions of the PH and kinase domains of inactive and membrane-associated AKT [12], [13], [14], [15], [16], resulting in the inactive form being termed the closed or ‘PH-in’ conformation; whereas the membrane-associated form is referred to as the open or ‘PH-out’ conformation. More in-depth characterization of Inhibitor VIII (Figure 1), a commercially available PH domain-dependent allosteric AKT1/2 inhibitor (Compound 16 h) [7], showed that Inhibitor VIII is dependent upon the presence of Trp 80 in the PH domain for its activity; and the inhibitor binds to a generally characterized ‘PH-in’ conformation of AKT1 [15], [17]. Therefore, in order to further our understanding of the regulation and inhibition of AKT and to aid in the design of selective AKT inhibitors, we extensively screened for crystals of AKT complexed to an allosteric inhibitor. Here we report the crystal structure of AKT1 complexed to Inhibitor VIII at 2.7 Å resolution (PDB code: 3O96).
Results
We expressed, purified, and set-up crystallization screens of full-length AKT1 and AKT2 pre-incubated with Inhibitor VIII. After 2.5 months, we observed a single small crystal in a full-length AKT1 crystal screen and determined from initial diffraction patterns that the crystal was proteinaceous. After the long time required for crystal growth, concern that the protein was proteolyzed in the crystallization drop arose, so the crystal was analyzed by sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis. We observed two major truncated forms of AKT1 protein from the crystal including one which was approximately 6,000 Daltons (Da) smaller than the starting material (Figure S1). We hypothesized that the most likely region for proteolysis resulting in a 50 kDa fragment would be in the disordered region encompassing the C-terminal HM segment. Therefore several carboxy-terminal truncated AKT1 (ΔHM-AKT1) constructs were expressed to identify a modestly truncated form suitable for crystallographic studies. ΔHM-AKT1(1-443) was successfully purified and its binding to Inhibitor VIII was analyzed using differential scanning fluorimetry [18] (Figure S2). We were able to co-crystallize ΔHM-AKT1(1–443) with Inhibitor VIII and to solve its structure to 2.7 Å resolution (Table 1 and Figure 2).
Table 1. Data collection and refinement statistics for AKT1:Inhibitor VIII.
Data collection | |
Space group | P21 |
Cell dimensions | |
a, b, c (Å) | 49.31, 69.94, 61.85 |
α, β, γ (°) | 90.0, 100.6, 90.0 |
Resolution (Å) | 25 – 2.7 (2.85 – 2.70) * |
Rmerge | 0.078 (0.382) |
I /σI | 8.1 (2.0) |
Completeness (%) | 99.9 (99.9) |
Redundancy | 3.5 (3.5) |
Refinement | |
Resolution (Å) | 25 – 2.7 (2.78–2.7) |
No. reflections | 11,464 (935) |
Rwork/Rfree | 0.245/0.307 (0.323/0.382) |
No. atoms | 3,095 |
Protein | 3,032 |
Ligand/ion | 42 |
Water | 21 |
Average B-factor | 52.4 |
R.m.s. deviations | |
Bond lengths (Å) | 0.005 |
Bond angles (°) | 0.88 |
The dataset was collected on one single crystal.
*Highest resolution shell is shown in parentheses.
Figure 3 shows the overall structure of the allosterically inhibited enzyme. The PH domain nestles between the N- and C-lobes of the kinase domain with Inhibitor VIII binding to all three regions. The conformation of the PH domain is similar to the previously determined apo structure [19] (RMSD = 1.14 Å for all Cα's) with significant conformational differences in the regions binding to the inhibitor and kinase domain described below. Both lobes of the kinase domain are in inactive conformations reminiscent of the unphosphorylated structure of AKT2 kinase domain [16]: residues 189–198 of the αC-helix and residues 299–312 of the activation loop are disordered, while the side chain of Phe 293 blocks the ATP binding site. In Figure 4 the complex structure of activated AKT1 kinase domain and an ATP-competitive inhibitor is superposed upon the co-crystal structure of inactive AKT1:Inhibitor VIII [20] illustrating not only the structural differences between the N-lobes of the kinase domain and Phe 293 positions, but also the >10 Å distance between inhibitor binding sites. The superimposed structures show the PH domain fills part of the space occupied by the αC-helix and phosphorylated activation loop in the activated AKT kinase domain structures [20], [21]; thereby sterically preventing the kinase domain from attaining an active conformation. Interactions between the PH domain and the kinase domain are concentrated in two regions of the kinase domain – in the N-lobe adjacent to the ATP-binding cleft and in the C-lobe (Figure 5A). The PH domain buries 1,526 Å2 of its surface in this complex and a combination of hydrogen bonds and nonpolar interactions are observed in both regions of this interface.
Inhibitor VIII binds to AKT1 in an allosteric binding site formed at the combined interface of the PH domain and the N- and C- lobes of the kinase domain. We were pleased to observe a ring-stacking interaction between Inhibitor VIII and Trp 80 (Figure 5B) as an alanine mutation of this residue in AKT1 has been shown to render Inhibitor VIII inactive [15], [17]. The position of Trp 80 in the inhibitor bound structure differs significantly from both the apo and Ins(1,3,4,5)P4 (IP4) bound PH domain structures, with an α-carbon displacement of 3.7 Å and 5.3 Å, respectively (Figure S3) indicating the variable loop 3 (VL3) loop shifts to accommodate various ligands. As shown in Figure 5B and Figure S4, Inhibitor VIII has several hydrophobic contacts with AKT1 that appear to drive compound binding while only a limited number of polar contacts are observed.
In the process of discovering Inhibitor VIII, modifications to the imidazoquinoxaline were found to impact AKT isozyme activity and selectivity [22]. Therefore, we mapped the amino acid differences between isozymes on the AKT1:Inhibitor VIII structure (Figure 6A) and identified only two regions of amino acid divergence. Both regions are located in the kinase domain and in the binding site for the tricyclic core. As shown in Figure 6B, Ser 205 has the only direct hydrogen bond to Inhibitor VIII. In AKT2 and AKT3, the corresponding residue is threonine. The second region consists of a three residue turn in AKT1 containing Glu 267, Lys 268, and Asn 269. This turn is not only one residue shorter in both AKT2 and AKT3 but the amino acids differ between isozymes. As illustrated in Figure 6B, this region is located on the opposite side of the tricyclic system from Trp 80. In addition to interacting with Inhibitor VIII, Lys 268 also has a polar interaction with the non-conserved binding site residue Ser 205. The IC50's for Inhibitor VIII are 58 nM, 210 nM, and 2119 nM for AKT1, AKT2, and AKT3, respectively [7]. As the majority of residues contacting Inhibitor VIII are conserved between isozymes, the approximately 35-fold difference in the inhibitor's activity between AKT1, and AKT3 are hypothesized to be due to the amino acid differences at the back of the pocket near the imidazole on the quinoxaline core. In AKT3, substitution of a threonine residue for serine at position 203 (equivalent to AKT1 Ser 205) may affect the ability of Inhibitor VIII to hydrogen bond to the protein and also alters the binding pocket by introducing an additional methyl group. Deletion of a turn residue in AKT3 located below Inhibitor VIII (corresponding to AKT1 Asn 269) is expected to change the position of the positively charged Lys residue so that it no longer interacts with Thr 203 (equivalent to AKT1 Ser 205) and to change the dimensions of the binding pocket. Therefore, the AKT1:Inhibitor VIII complex structure suggests further efforts to design AKT1 selective inhibitors should focus on optimizing specific interactions with Ser 205 and the Lys 268 loop.
An early regulatory step in the activation of AKT is its recruitment to the plasma membrane following interaction of the PH domain with the PI3K products, PtdIns-(3,4,5)P3 and PtdIns(3,4)P2. We noticed significant conformational and polar interactions differences in the PH domain when comparing the allosterically inhibited structure to the IP4 bound crystal structures (Figure 7). In the multi-domain AKT1 structure, the IP4 binding site is both rearranged and blocked by the C-lobe of the kinase domain. The most dramatic difference is observed for Asn 53 and the surrounding loop of residues 49-55 (Figure 7A). This major structural change appears to be the result of multiple interactions between the PH and kinase domains and may be partially stabilized by the presence of Inhibitor VIII. The network of inter-domain contacts and extensive rearrangement in and near the IP4 binding site illustrates why the allosterically inhibited ‘PH-in’ conformation does not bind PtdIns(3,4)P2 or PtdIns(3,4,5)P3 (Figure S5).
The transforming somatic mutation of Glu 17 to lysine (E17K) in AKT1 has been reported in several cancers including human breast, colorectal, ovarian and endometrial cancers [23]. AKT1(E17K) constitutively associates with the plasma membrane [23]. Lipid binding studies indicate the mutant's binding affinity for the constitutively present plasma membrane lipid, PtdIns(4,5)P2, is dramatically tighter than AKT1's (wt) affinity possibly due to favorable electrostatic interactions between Lys 17 and PtdIns(4,5)P2 [24]. Inhibitor VIII is reported to be 5-fold less potent on the E17K mutant of AKT1 than on AKT1(wt), indicating that Glu 17 either directly interacts with the inhibitor or affects the conformation required to bind inhibitor. We did not observe Glu 17 binding to the inhibitor in the co-crystal structure, however Glu 17 forms a salt bridge with the positively charged kinase residue Arg 273 (Figure 8) almost certainly providing additional stabilization to the closed ‘PH-in’ conformation. Conversely, in AKT1(E17K), the positively charged side chains of Lys 17 and Arg 273 will not form a stabilizing interaction between the PH and kinase domains thereby shifting the equilibrium from the closed, ‘PH-in’ conformation to the open, ‘PH-out’ conformation. Therefore the constitutive plasma membrane localization of AKT1(E17K) appears to be the result of both an equilibrium shift towards the open ‘PH-out’ form and a change in lipid selectivity.
Discussion
Both allosteric and ATP-competitive small molecule inhibitors of AKT are being investigated in preclinical and clinical testing. As shown in Figure 9, although these inhibitors target the same AKT kinase family, divergent profiles are reported relative to cellular localization and phosphorylation status. Remarkably, ATP-competitive inhibitors were shown to induce hyperphosphorylation via a membrane-dependent and kinase intrinsic mechanism [25]. Conversely, the allosteric AKT inhibitors prevent both membrane association and activation by phosphorylation [6], [15]. The conformation of the ATP binding site in the AKT1:Inhibitor VIII crystal structure clearly shows that the PH-in conformer is unable to bind ATP or ATP-competitive inhibitors. Not only does Phe 293 block the site, but critical ATP binding site residues interact with PH domain residues. Therefore, we hypothesize ATP-competitive AKT inhibitors are unable to bind the ‘PH-in’ conformer and only bind the membrane-associated and phosphorylated ‘PH-out’ form, which has a properly configured ATP binding site. When the competitive inhibitor occupies the ATP binding site, we propose that the PH domain cannot fully close onto the kinase domain and the phospholipid binding site remains exposed thus enhancing the propensity of AKT to localize to the membrane. In contrast, the AKT1:Inhibitor VIII co-crystal structure reveals the allosteric inhibitor locking AKT into a closed conformation with its phospholipid binding site blocked by the kinase domain. As a result, allosterically inhibited AKT remains cytosolic and is not activated via phosphorylation. From a clinical perspective, determining whether these two distinct mechanisms of directly inhibiting AKT will have different therapeutic outcomes has yet to be determined.
Materials and Methods
Expression and purification of AKT1(1–443) protein
AKT1(1–443) was expressed in Trichopulsia ni High Five (BTI-TN-5B1-4) cell line (Invitrogen, CA, USA) with a N-terminal hexa-histidine (His) tag that is followed by a thrombin cleavage sequence. The His-tagged AKT1(1–443) protein was enriched from the High Five cell lysate on Talon cobalt-affinity resins (Clontech, CA, USA) then eluted from the Talon beads in a buffer consisting of 25 mM Tris-Cl pH 8.0, 0.3 M NaCl, 0.05% (v/v) 2-mercaptoethanol, 100 mM imidazole, 10% (v/v) glycerol and Complete EDTA-free protease inhibitor (Roche, IN, USA). The N-terminal His tag was removed from the AKT1(1–443) protein by incubation with thrombin. The protein was further purified by Source Q15 anion-exchange chromatography (GE Health Biosciences, NJ, USA) with a linear NaCl salt gradient. Monomeric AKT1(1–443) protein was purified via Superdex 200 size-exclusion chromatography (GE Health Biosciences, NJ, USA) using the final storage buffer of 25 mM Tris-Cl pH 7.5, 100 mM NaCl, 10% glycerol and 5 mM DTT and concentrated to approximately 10 mg/ml. All purification steps were performed at 4°C and the final protein was stored at −80°C.
Crystallization
Crystals were grown by the vapor diffusion method. AKT1 (1–443) at 4.7 mg/mL was incubated with Inhibitor VIII at 0.25 mM. Hanging drops were set up in the presence of 50% (v/v) precipitant, consisting of 12.5 mM Na-acetate, 37.5 mM Na-citrate pH 5.2, 21% PEG MME 2000 at 20°C. Co-crystals usually appeared within 1–2 days.
X-ray data collection, data processing, structure solution, crystallographic structure refinement
AKT1-Inhibitor VIII co-crystals were harvested into a solution of 25 mM Na-acetate, 25 mM Na-citrate, 21% PEG MME 2000, pH 5.0 and were cryoprotected with 70% harvest solution + 30% ethylene glycol. Cryoprotected crystals were flash cooled in a stream of dry nitrogen vapor held at 100 K. X-ray diffraction data were collected on a Rigaku FR-E Superbright rotating anode X-ray generator, fitted with a Cu anode and an RAXIS IV++ image plate detector (Rigaku, TX, USA). The diffraction data were processed using Mosflm [26] and scaled using the program Scala [27].
The crystals belonged to space group P21 with unit cell dimensions of a = 49.31 Å, b = 69.94 Å, c = 61.85 Å, β = 100.6°.
The crystal structure was solved by molecular replacement, with all calculations performed using the program Molrep [27]. The molecular replacement calculations were performed in two steps: In the first step, a search model consisting of residues 147–440 of the inactive AKT2 kinase domain (PDB code: 1MRV) was used in a standard rotation function/translation function calculation, resulting in a single solution with an R-factor of 0.505 (similar searches with an active conformation of AKT1 kinase domain failed to find a reliable solution). The quality of this molecular replacement solution was improved slightly by brief crystallographic refinement to 2.8 Å resolution in Refmac5. In the second step, a search model consisting of residues 2–106 of the unliganded AKT1 PH domain (PDB code: 1UNP) was used in a rotation function/phased translation function search, using phase information calculated from the coordinates of the AKT2 kinase domain solution. A single PH domain plus kinase domain solution was found with an R-factor of 0.417. This combined solution was subjected to multiple cycles of refinement in Refmac5 to 2.7 Å resolution [28], followed by model rebuilding in the program O [29]. The final round of model rebuilding was guided by the use of simulated annealing composite omit maps followed by crystallographic refinement in CNX 2005 [30] to generate the final molecular model.
The final structure contains all residues of AKT1 from 2–429 except for 45–48, 114–144 (the linker region), 189–198 (the αB and αC helices) and 299–312 (the C-terminal end of the activation loop). The model also has 21 ordered water molecules and a single copy of Inhibitor VIII. The R-factor of the final model is 0.245 with an Rfree value of 0.307. 312 residues (98.7%) lie in the “most-favored” or “additional” regions of the Ramachandran plot, with 2 residues (0.6%) in the “generously allowed” region and 2 (0.6%) in the “disallowed” regions. All figures were generated using Pymol [31].
Supporting Information
Acknowledgments
We thank Tony Celeste, Katie Lundeen, and Joshua Ballard for their technical assistance. We thank our colleagues at Array BioPharma and Genentech for many stimulating discussions. In particular we thank Nicholas Skelton, James Graham, James Blake, Lesley Murray and the AKT Team.
Footnotes
Competing Interests: Array BioPharma Inc. and Genentech, Inc. funded these studies. Wen-I Wu, Walter Voegtli, Hillary Sturgis, Guy Vigers, and Barbara Brandhuber are employees and stockholders of Array BioPharma Inc. Faith Dizon is a former employee of Array BioPharma Inc. Genentech, Inc. has products in development in this pathway. The authors agree to adhere to all the PLoS ONE policies on sharing data and materials, as detailed online in the PLoS ONE guide for authors. Array BioPharma Inc. and Genentech, Inc. approved this publication and adherence to the material and data sharing policies of the publishing journal.
Funding: Array BioPharma Inc. and Genentech, Inc. funded and approved the publication of this work. The companies had a role in this study due to employment of one or more authors of this study.
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