Crystal structure of human AKT1 with an allosteric inhibitor reveals a new mode of kinase inhibition

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.

Figure 1. Schematic structure of the AKT1/2 inhibitor.

Inhibitor VIII (EMD Chemicals) has IC₅₀'s of 58 nM, 210 nM, and 2119 nM against AKT1, -2, and -3, respectively .

Figure 2. Stereo view of the allosteric Inhibitor VIII binding site.

Inhibitor VIII is shown in green and Trp 80 is shown in orange.

Figure 3. Crystal structure of Inhibitor VIII bound to AKT1(1–443).

(A) Schematic representation showing the orientation of the PH domain (orange) relative to the N-lobe (pink) and C-lobe (yellow) of the kinase domain and Inhibitor VIII shown in green. (PDB code: 3O96). (B) In the same orientation as Panel A, the kinase domain is surface rendered. (C) Structure of AKT1(1–443):Inhibitor VIII rotated approximately 180° compared to Panel B.

Figure 4. Comparison of AKT1 allosteric and ATP competitive inhibitor binding sites.

(A) Superposition of AKT1 bound to Inhibitor VIII and activated AKT1 kinase domain bound to an ATP-competitive inhibitor (PDB code: 3CQW). In the allosteric inhibitor structure, the PH domain is shown as an orange surface, the kinase domain as a yellow cartoon, and Inhibitor VIII as green sticks. The activated kinase domain is colored blue and the ATP-competitive inhibitor is shown as magenta sticks. (B) Close-up view of the inhibitor binding positions. AKT1:Inhibitor VIII are represented as in Panel A. The ATP-competitive inhibitor and Phe 293 from PDB code 3CQW are shown in magenta. The DFG loop including Phe 293 from the Inhibitor VIII complex structure are colored green. Note the divergent positions of Phe 293 between the two structures and their superposition onto the converse inhibitor binding site. For clarity, the activated AKT1 protein backbone trace has been omitted from this panel.

Figure 5. AKT1 inter-domain and Inhibitor VIII interactions.

Close-up views of interaction regions for the PH domain (orange), kinase domain (yellow), and Inhibitor VIII (green). (A) Interactions between PH and kinase domains. The PH domain residues interacting with the kinase domain are shown in magenta and the reciprocally binding kinase domain residues are colored cyan. Shown in sticks are two ATP binding residues in the kinase domain interacting with residues from the PH domain. (B) AKT1 residues interacting with Inhibitor VIII. Interacting residues from the PH (magenta) and kinase (cyan) domains are labeled. Shown with a dotted line is the lone direct hydrogen bond between the inhibitor and protein.

Figure 6. Isozyme selectivity of allosteric inhibitors.

(A) Alignment of human AKT-1 -2 and -3. Residues are shaded to illustrate amino acid conservation. Above the sequence, denotes residues involved in inter-domain contacts, ║denotes residues contacting Inhibitor VIII, and marks residues involved in both inter-domain and Inhibitor VIII interactions. The arrow shows the position of the last residue in the crystallography construct, Thr 443. (B) Close-up view of non-identical regions in the Inhibitor VIII binding pocket with coloring as follows: PH domain (orange), kinase domain (yellow), and Inhibitor VIII (green). Residues with differences between AKT isozymes are shown in sticks and colored in cyan.

Figure 7. PH domain conformational differences.

(A) Schematic representation of the PH domain from the Inhibitor VIII bound structure (orange) and IP4 bound (PDB code 1UNQ) structure (magenta). The most dramatic differences between the PH domains of AKT1:Inhibitor VIII and the IP4 bound structure are in the VL3 loop containing Trp 80, which interacts with Inhibitor VIII, and loop 51–55 containing Asn 53, which interacts with IP4. The noted regions are colored yellow in the Inhibitor VIII bound structure and cyan in the IP4 bound structure. Trp 80 and Asn 53 are shown in stick representation. (B) Close-up view of the IP4 binding site. AKT1 PH domain (magenta) bound to IP4 (PDB code: IUNQ) is superposed on the AKT1:Inhibitor VIII structure. AKT1:Inhibitor VIII colored as follows: PH domain (orange), kinase domain (yellow), Inhibitor VIII (green sticks). Asn 53 in the multi-domain structure interacts with Asn 269 and is over 10 Å away from the IP4 binding site. In the Inhibitor VIII structure, Arg 86 points into the PH domain. A movement of 8 Å is required for Arg 86 to bind IP4.

Figure 8. Glu 17 interacts with Arg 273 in the kinase domain.

AKT1:Inhibitor VIII colored as follows: PH domain (orange), kinase domain (yellow), Inhibitor VIII (green sticks). A salt bridge between Glu 17 and Arg 273 is observed in the closed, ‘PH-in’ conformation.

Figure 9. Model of AKT activation and inhibition.

In the cytoplasm, the ‘PH-in’ and ‘PH-out’ conformations of AKT are in equilibrium. AKT is recruited to the plasma membrane via interactions with the products of PI3K and is subsequently phosphorylated on two sites, T308 and S473 in AKT1, which results in kinase activation. The allosteric inhibitor stabilizes the ‘PH-in’ form of the inactive enzyme (top left); whereas the ATP-competitive inhibitor binds to the activated form of the kinase (bottom left). Surface representations derived from the following structures: PDB code: 3CQW (active and ATP-competitive inhibitor bound kinase), PDB code: 1UNQ (membrane-bound PH domain), PDB code: 3O96 (cytoplasmic PH and kinase domains, and membrane-bound kinase domain). Coloring as follows: kinase domain (yellow), PH domain (orange), IP4 binding residues (cyan), phospho-T308 (red), allosteric inhibitor (green), ATP-competitive inhibitor (blue), PI3K products (violet). Phospho-S473 is not visible in these orientations of AKT.