The T790M mutation in EGFR kinase causes drug resistance by increasing the affinity for ATP

Fig. 4. Representative electron density for the HKI-272/T790M mutant kinase. Note that the position and conformation of the inhibitor (yellow) is well defined and that there is clear, continuous density indicative of a covalent bond with the thiol of Cys-797 (Inset).

Fig. 5. T790M mutant EGFR kinase crystallizes with two molecules in the asymmetric unit, both of which make the activating "asymmetric dimer" interaction in the crystal lattice. The two molecules make essentially identical intermolecular contacts between their N-lobes and the C-lobe of an adjacent molecule in the crystal, but differ in the relative intramolecular orientations of their N-and C-lobes. In the asymmetric dimer, one molecule assumes the activating, cyclin-like role, by binding the N-lobe of the other and forcing an active, inward position of the C-helix (1). (a) Molecule A (red) assumes the activating role in one lattice contact. (b) Molecule B (green) assumes the activating role in the other. (c) Superposition of T790M asymmetric dimers shown in a and b with that of the WT EGFR kinase (yellow) (1, 2). The superposition is based on the C-lobe of the upper molecule. Note that the intermolecular register is identical in all three dimers, but that the relative orientation between the N-and C-lobes within each kinase varies.

1. Zhang X, Gureasko J, Shen K, Cole PA, Kuriyan J (2006) An allosteric mechanism for activation of the kinase domain of epidermal growth factor receptor. Cell 125:1137-1149.

2. Stamos J, Sliwkowski MX, Eigenbrot C (2002) Structure of the epidermal growth factor receptor kinase domain alone and in complex with a 4-anilinoquinazoline inhibitor. J Biol Chem 277:46265-46272.

Fig. 6. The gatekeeper mutation may promote a DFG-out conformation that unlocks the C-helix to facilitate conversion to the active state. Detailed views of the interaction of the C-helix with the DFG loop in three distinct states are presented. The DFG segment is shown as CPK spheres and colored green, hydrophobic residues at the base of the C-helix are shown as CPK spheres in yellow. The gatekeeper residue is shown in stick form, as are the side chains of Lys-745 and Glu-762, which form a key salt bridge in the active state. (a) In the fully inactive state, Phe-856 in the DFG loop interdigitates with Val-765, Met-766, and Val-769 in the C-helix, and it adopts a side-chain orientation that extends out of the plane of the page in the view presented. The interactions of the DFG loop in this state help to stabilize or "lock" the C-helix in the outward, inactive position [drawn from PDB ID code 2gs7 (1)]. (b) In molecule B of the T790M structure, we observe a DFG-out conformation in which Phe-856 interacts with the mutant gatekeeper methionine (Met-790, obscured by Phe-856 in this view). The C-helix is in the active position. We refer to this conformation as unlocked, as the DFG loop makes minimal contact with the C-helix. (c) In molecule A of the T790M structure, a fully active conformation is observed, with the DFG loop in the usual in position, where it again interacts extensively with the C-helix. As in the inactive state, Phe-856 interdigitates with Val-765, Met-766, and Val-769, but with a side-chain orientation that extends into the plane of the page in this view. In this state, the C-helix is locked in the active position.

1. Zhang X, Gureasko J, Shen K, Cole PA, Kuriyan J (2006) An allosteric mechanism for activation of the kinase domain of epidermal growth factor receptor. Cell 125:1137-1149.

Table 3. Crystallographic data and refinement statistics

R_merge= ∑|I_i - <I_i>|/∑I_i,,where I_i is the measured intensity of a single observation of the ith reflection.

R_cryst = ∑|F_o-F_c|/∑F_o, where F_o and F_c are observed and calculated structure factor amplitudes, respectively.

R_free is the R_cryst for reflections excluded from the refinement.

SI Text

Structural Insights into the Mechanism of Catalytic Activation by T790M. The crystal structure of the T790M mutant suggests a mechanism by which the substitution promotes catalytic activation of the enzyme. Briefly, we propose that the mutation lowers the energy required to transition between the inactive and active conformations by facilitating a DFG-out conformation of the kinase. The highly conserved Asp-Phe-Gly (DFG) sequence lies at the base of the kinase activation loop, where it intimately contacts the C-helix in its usual DFG-in orientation. An inactive, DFG-out conformation has been observed in a number of kinases, most notably Abl, where it is required for binding of the drug imatinib (1-3). However, no clear functional role for the DFG-out conformation has emerged. The crystals of the unliganded T790M mutant contain two independent copies of the molecule. Both molecules in the crystal make the "asymmetric dimer" interaction that is thought to reflect the cellular mode of dimerization of the cytoplasmic region of the EGFR (4). This interaction, between the N-lobe of one molecule and the C-lobe of the other, brings the C-helix of the first molecule into the active position. As noted above, both independent copies of the T790M protein make this interaction within the crystal lattice, and in both the C-helix is in the active, inward position (SI Fig. 5). However, only one of the two molecules is in the fully active conformation. In the other, the DFG loop is flipped into the out position, where it is stabilized by hydrophobic interaction between the mutant gatekeeper methionine and the phenylalanine residue of the DFG motif (Phe-856). In both the active and fully inactive states of the EGFR and other kinases, the DGF loop adopts the in position, where the phenylalanine residue interdigitates with hydrophobic residues on the C-helix (SI Fig. 6). In the EGFR, this interdigitation would appear to preclude the motions required for the C-helix to transit between its active and inactive states. Thus we hypothesize that the DFG loop functions as a lock on the C-helix and that concerted motions of the DFG loop and C-helix are required for transition of the C-helix between its active and inactive positions. The T790M structure suggests that the gatekeeper mutation may promote the unlocked DFG-out state, and therefore promote higher catalytic activity by lowering this putative kinetic barrier to activation. This hypothesis is consistent with studies that reveal correlations among the dynamics and accessibility of the DFG loop, the C-helix, the activation loop, and the gatekeeper residue in other kinases (5, 6). It is also consistent with a related hypothesis based on crystal structures and molecular dynamics simulations in Abl (3). We note that this mechanism of activation, effectively stabilization of an intermediate state, could also be expected to decrease the activity of a fully activated mutant. In this regard, we note that the K_cat of the L858R/T790M double mutant is decreased modestly relative to the highly active L858R enzyme (~3-fold; see Table 2). Another factor in the increased activity of the T790M mutant may be enhanced stability of the active conformation (relative to the inactive) in the T790M mutant. The mutant gatekeeper methionine makes favorable hydrophobic interactions with Met-766 and Leu-777 in the active state, and Met-766 in turn packs closely with the Phe-856 in the DFG loop. These residues are part of a "hydrophobic spine" that is characteristic of the active conformations of protein kinases (7). Stabilization of the hydrophobic spine is proposed to underlie activation of Src and Abl by their respective gatekeeper mutations (M. Azam, personal communication).

1. Nagar B, et al. (2002) Crystal structures of the kinase domain of c-Abl in complex with the small molecule inhibitors PD173955 and imatinib (STI-571). Cancer Res 62:4236-4243.

2. Schindler T, et al. (2000) Structural mechanism for STI-571 inhibition of abelson tyrosine kinase. Science 289:1938-1942.

3. Levinson NM, et al. (2006) A Src-like inactive conformation in the abl tyrosine kinase domain. PLoS Biol 4:e144.

4. Zhang X, Gureasko J, Shen K, Cole PA, Kuriyan J (2006) An allosteric mechanism for activation of the kinase domain of epidermal growth factor receptor. Cell 125:1137-1149.

5. Emrick MA, et al. (2006) The gatekeeper residue controls autoactivation of ERK2 via a pathway of intramolecular connectivity. Proc Natl Acad Sci USA 103:18101-18106.

6. Wiesner S, et al. (2006) A change in conformational dynamics underlies the activation of Eph receptor tyrosine kinases. EMBO J 25:4686-4696.

7. Kornev AP, Haste NM, Taylor SS, Eyck LF (2006) Surface comparison of active and inactive protein kinases identifies a conserved activation mechanism. Proc Natl Acad Sci USA 103:17783-17788.