Crystal structure of the ALK (anaplastic lymphoma kinase) catalytic domain

Abstract

ALK (anaplastic lymphoma kinase) is an RTK (receptor tyrosine kinase) of the IRK (insulin receptor kinase) superfamily, which share an YXXXYY autophosphorylation motif within their A-loops (activation loops). A common activation and regulatory mechanism is believed to exist for members of this superfamily typified by IRK and IGF1RK (insulin-like growth factor receptor kinase-1). Chromosomal translocations involving ALK were first identified in anaplastic large-cell lymphoma, a subtype of non-Hodgkin's lymphoma, where aberrant fusion of the ALK kinase domain with the NPM (nucleophosmin) dimerization domain results in autophosphosphorylation and ligand-independent activation. Activating mutations within the full-length ALK kinase domain, most commonly R1275Q and F1174L, which play a major role in neuroblastoma, were recently identified. To provide a structural framework for understanding these mutations and to guide structure-assisted drug discovery efforts, the X-ray crystal structure of the unphosphorylated ALK catalytic domain was determined in the apo, ADP- and staurosporine-bound forms. The structures reveal a partially inactive protein kinase conformation distinct from, and lacking, many of the negative regulatory features observed in inactive IGF1RK/IRK structures in their unphosphorylated forms. The A-loop adopts an inhibitory pose where a short proximal A-loop helix (alphaAL) packs against the alphaC helix and a novel N-terminal beta-turn motif, whereas the distal portion obstructs part of the predicted peptide-binding region. The structure helps explain the reported unique peptide substrate specificity and the importance of phosphorylation of the first A-loop Tyr1278 for kinase activity and NPM-ALK transforming potential. A single amino acid difference in the ALK substrate peptide binding P-1 site (where the P-site is the phosphoacceptor site) was identified that, in conjunction with A-loop sequence variation including the RAS (Arg-Ala-Ser)-motif, rationalizes the difference in the A-loop tyrosine autophosphorylation preference between ALK and IGF1RK/IRK. Enzymatic analysis of recombinant R1275Q and F1174L ALK mutant catalytic domains confirms the enhanced activity and transforming potential of these mutants. The transforming ability of the full-length ALK mutants in soft agar colony growth assays corroborates these findings. The availability of a three-dimensional structure for ALK will facilitate future structure-function and rational drug design efforts targeting this receptor tyrosine kinase.

The R1275Q Neuroblastoma Mutant and Certain ATP-competitive Inhibitors Stabilize Alternative Activation Loop Conformations of Anaplastic Lymphoma Kinase

“…3 Although the F1174L muta- Other aspects of the ALK F1174L kinase domain structure also resembled features observed in the wild-type protein, notably the conformation of the DFG motif at the beginning of the A-loop, the position of the ␣C-helix, and the relative degree of closure between the N-and C-terminal lobes of the kinase. These features, together with the positioning of the C-terminal portion of the A-loop that sterically blocks the substrate binding site, have all been discussed previously as contributing to an overall inactive kinase conformation (38,39). In contrast to the F1174L ALK crystal structure, the structure of the R1275Q ALK kinase domain showed a dramatic difference in the activation loop conformation compared with the wild-type protein (r.m.s.…”

“…Several structural features of the published, unphosphorylated ALK kinase domain differ from the structural template provided by the IRK ternary structure and interestingly, ALK also differs from the unphosphorylated, inactive form of IRK kinase domain (43). These differences have been described elsewhere (38,39).…”

“…A structural understanding of inhibitor binding to ALK was recently enabled by the publication of crystal structures of the ALK kinase domain both alone and in complex with ATP-competitive inhibitors (38,39). The structures revealed that the ALK kinase domain adopts the canonical kinase-fold, but that it also contains two notable features.…”

“…All of the structures described in this work use ALK(1084 -1410) C1097S. By way of comparison, ALK kinase domain structures have also been determined using constructs with the boundaries 1072-1410, 1094 -1407, 1093-1411, and 1069 -1411 (19,38,39,52).…”

“…All of the mutations cluster around the active site of ALK. One of the most commonly mutated residues, Phe 1174 , sits in a hydrophobic pocket at the base of the ␣C-helix, where it is involved in hydrophobic stacking interactions with other phenylalanine residues from the N-terminal ␤-turn region (Phe 1098 ), the activation loop (Phe 1271 ), and C-terminal kinase domain (Phe 1245 ) as described by others (35,38,39). The second commonly mutated residue, Arg 1275 , is found in the activation loop where it is part of the ␣-helix formed in the unphosphorylated ALK kinase domain structures.…”