A two-site flexible clamp mechanism for RET-GDNF-GFRα1 assembly reveals both conformational adaptation and strict geometric spacing.

RET receptor tyrosine kinase plays vital developmental and neuroprotective roles in metazoans. GDNF family ligands (GFLs) when bound to cognate GFRα co-receptors recognize and activate RET stimulating its cytoplasmic kinase function. The principles for RET ligand-co-receptor recognition are incompletely understood. Here, we report a crystal structure of the cadherin-like module (CLD1-4) from zebrafish RET revealing interdomain flexibility between CLD2 and CLD3. Comparison with a cryo-electron microscopy structure of a ligand-engaged zebrafish RETECD-GDNF-GFRα1a complex indicates conformational changes within a clade-specific CLD3 loop adjacent to the co-receptor. Our observations indicate that RET is a molecular clamp with a flexible calcium-dependent arm that adapts to different GFRα co-receptors, while its rigid arm recognizes a GFL dimer to align both membrane-proximal cysteine-rich domains. We also visualize linear arrays of RETECD-GDNF-GFRα1a suggesting that a conserved contact stabilizes higher-order species. Our study reveals that ligand-co-receptor recognition by RET involves both receptor plasticity and strict spacing of receptor dimers by GFL ligands.

A secondary RET mutation in the activation loop conferring resistance to vandetanib.

Resistance to vandetanib, a type I RET kinase inhibitor, developed in a patient with metastatic lung adenocarcinoma harboring a CCDC6-RET fusion that initially exhibited a response to treatment. The resistant tumor acquired a secondary mutation resulting in a serine-to-phenylalanine substitution at codon 904 in the activation loop of the RET kinase domain. The S904F mutation confers resistance to vandetanib by increasing the ATP affinity and autophosphorylation activity of RET kinase. A reduced interaction with the drug is also observed in vitro for the S904F mutant by thermal shift assay. A crystal structure of the S904F mutant reveals a small hydrophobic core around F904 likely to enhance basal kinase activity by stabilizing an active conformer. Our findings indicate that missense mutations in the activation loop of the kinase domain are able to increase kinase activity and confer drug resistance through allosteric effects.

Drugging the catalytically inactive state of RET kinase in RET-rearranged tumors.

Oncogenic fusion events have been identified in a broad range of tumors. Among them, RET rearrangements represent distinct and potentially druggable targets that are recurrently found in lung adenocarcinomas. We provide further evidence that current anti-RET drugs may not be potent enough to induce durable responses in such tumors. We report that potent inhibitors, such as AD80 or ponatinib, that stably bind in the DFG-out conformation of RET may overcome these limitations and selectively kill RET-rearranged tumors. Using chemical genomics in conjunction with phosphoproteomic analyses in RET-rearranged cells, we identify the CCDC6-RETI788N mutation and drug-induced mitogen-activated protein kinase pathway reactivation as possible mechanisms by which tumors may escape the activity of RET inhibitors. Our data provide mechanistic insight into the druggability of RET kinase fusions that may be of help for the development of effective therapies targeting such tumors.

RET recognition of GDNF-GFRα1 ligand by a composite binding site promotes membrane-proximal self-association.

The RET receptor tyrosine kinase is essential to vertebrate development and implicated in multiple human diseases. RET binds a cell surface bipartite ligand comprising a GDNF family ligand and a GFRα coreceptor, resulting in RET transmembrane signaling. We present a hybrid structural model, derived from electron microscopy (EM) and low-angle X-ray scattering (SAXS) data, of the RET extracellular domain (RET(ECD)), GDNF, and GFRα1 ternary complex, defining the basis for ligand recognition. RET(ECD) envelopes the dimeric ligand complex through a composite binding site comprising four discrete contact sites. The GFRα1-mediated contacts are crucial, particularly close to the invariant RET calcium-binding site, whereas few direct contacts are made by GDNF, explaining how distinct ligand/coreceptor pairs are accommodated. The RET(ECD) cysteine-rich domain (CRD) contacts both ligand components and makes homotypic membrane-proximal interactions occluding three different antibody epitopes. Coupling of these CRD-mediated interactions suggests models for ligand-induced RET activation and ligand-independent oncogenic deregulation.

Adenosine-binding motif mimicry and cellular effects of a thieno[2,3-d]pyrimidine-based chemical inhibitor of atypical protein kinase C isoenzymes.

The aPKC [atypical PKC (protein kinase C)] isoforms ι and ζ play crucial roles in the formation and maintenance of cell polarity and represent attractive anti-oncogenic drug targets in Ras-dependent tumours. To date, few isoform-specific chemical biology tools are available to inhibit aPKC catalytic activity. In the present paper, we describe the identification and functional characterization of potent and selective thieno[2,3-d]pyrimidine-based chemical inhibitors of aPKCs. A crystal structure of human PKCι kinase domain bound to a representative compound, CRT0066854, reveals the basis for potent and selective chemical inhibition. Furthermore, CRT0066854 displaces a crucial Asn-Phe-Asp motif that is part of the adenosine-binding pocket and engages an acidic patch used by arginine-rich PKC substrates. We show that CRT0066854 inhibits the LLGL2 (lethal giant larvae 2) phosphorylation in cell lines and exhibits phenotypic effects in a range of cell-based assays. We conclude that this compound can be used as a chemical tool to modulate aPKC activity in vitro and in vivo and may guide the search for further aPKC-selective inhibitors.

Molecular recognition of the Tes LIM2-3 domains by the actin-related protein Arp7A.

Actin-related proteins (Arps) are a highly conserved family of proteins that have extensive sequence and structural similarity to actin. All characterized Arps are components of large multimeric complexes associated with chromatin or the cytoskeleton. In addition, the human genome encodes five conserved but largely uncharacterized "orphan" Arps, which appear to be mostly testis-specific. Here we show that Arp7A, which has 43% sequence identity with ß-actin, forms a complex with the cytoskeletal proteins Tes and Mena in the subacrosomal layer of round spermatids. The N-terminal 65-residue extension to the actin-like fold of Arp7A interacts directly with Tes. The crystal structure of the 1-65(Arp7A)·LIM2-3(Tes)·EVH1(Mena) complex reveals that residues 28-49 of Arp7A contact the LIM2-3 domains of Tes. Two alanine residues from Arp7A that occupy equivalent apolar pockets in both LIM domains as well as an intervening GPAK linker that binds the LIM2-3 junction are critical for the Arp7A-Tes interaction. Equivalent occupied apolar pockets are also seen in the tandem LIM domain structures of LMO4 and Lhx3 bound to unrelated ligands. Our results indicate that apolar pocket interactions are a common feature of tandem LIM domain interactions, but ligand specificity is principally determined by the linker sequence.

Synthesis, structure-activity relationship and crystallographic studies of 3-substituted indolin-2-one RET inhibitors.

The synthesis, structure-activity relationships (SAR) and structural data of a series of indolin-2-one inhibitors of RET tyrosine kinase are described. These compounds were designed to explore the available space around the indolinone scaffold within RET active site. Several substitutions at different positions were tested and biochemical data were used to draw a molecular model of steric and electrostatic interactions, which can be applied to design more potent and selective RET inhibitors. The crystal structures of RET kinase domain in complex with three inhibitors were solved. All three compounds bound in the ATP pocket and formed two hydrogen bonds with the kinase hinge region. Crystallographic analysis confirmed predictions from molecular modelling and helped refine SAR results. These data provide important information for the development of indolinone inhibitors for the treatment of RET-driven cancers.

Structure of a conserved dimerization domain within the F-box protein Fbxo7 and the PI31 proteasome inhibitor.

F-box proteins are the substrate-recognition components of the Skp1-Cul1-F box protein (SCF) E3 ubiquitin ligases. Here we report a structural relationship between Fbxo7, a component of the SCF(Fbxo7) E3 ligase, and the proteasome inhibitor PI31. SCF(Fbxo7) is known to catalyze the ubiquitination of hepatoma-up-regulated protein (HURP) and the inhibitor of apoptosis (IAP) protein but also functions as an activator of cyclin D-Cdk6 complexes. We identify PI31 as an Fbxo7.Skp1 binding partner and show that this interaction requires an N-terminal domain present in both proteins that we term the FP (Fbxo7/PI31) domain. The crystal structure of the PI31 FP domain reveals a novel alpha/beta-fold. Biophysical and mutational analyses are used to map regions of the PI31 FP domain mediating homodimerization and required for heterodimerization with Fbxo7.Skp1. Equivalent mutations in Fbxo7 ablate interaction with PI31 and also block Fbxo7 homodimerization. Knockdown of Fbxo7 does not affect PI31 levels arguing against PI31 being a substrate for SCF(Fbxo7). We present a model for FP domain-mediated dimerization of SCF(Fbxo7) and PI31.

Crystallization and preliminary X-ray diffraction analysis of vaccinia virus H1L phosphatase.

The cysteine-based protein phosphatase H1L was the first reported dual-specificity protein phosphatase. H1L is encapsidated within the vaccinia virus and is required for successful host infection and for the production of viable vaccinia progeny. H1L has therefore been proposed as a target candidate for antiviral compounds. Recombinant H1L has been expressed in a catalytically inactive form using an Escherichia coli host, leading to purification and crystallization by the microbatch method. The crystals diffract to 2.1 A resolution using synchrotron radiation. These crystals belong to space group P422, with unit-cell parameters a = b = 98.31, c = 169.15 A, and are likely to contain four molecules in the asymmetric unit. A sulfur SAD data set was collected to 2.8 A resolution on beamline BM14 at the ESRF to facilitate structure determination. Attempts to derivatize these crystals with xenon gas changed the space group to I422, with unit-cell parameters a = b = 63.28, c = 169.68 A and a single molecule in the asymmetric unit. The relationship between these two crystal forms is discussed.

Structure and chemical inhibition of the RET tyrosine kinase domain.

The RET proto-oncogene encodes a receptor tyrosine kinase for the glial cell line-derived neurotrophic factor family of ligands. Loss-of-function mutations in RET are implicated in Hirschsprung disease, whereas activating mutations in RET are found in human cancers, including familial medullar thyroid carcinoma and multiple endocrine neoplasias 2A and 2B. We report here the biochemical characterization of the human RET tyrosine kinase domain and the structure determination of the non-phosphorylated and phosphorylated forms. Both structures adopt the same active kinase conformation competent to bind ATP and substrate and have a pre-organized activation loop conformation that is independent of phosphorylation status. In agreement with the structural data, enzyme kinetic data show that autophosphorylation produces only a modest increase in activity. Longer forms of RET containing the juxtamembrane domain and C-terminal tail exhibited similar kinetic behavior, implying that there is no cis-inhibitory mechanism within the RET intracellular domain. Our results suggest the existence of alternative inhibitory mechanisms, possibly in trans, for the autoregulation of RET kinase activity. We also present the structures of the RET tyrosine kinase domain bound to two inhibitors, the pyrazolopyrimidine PP1 and the clinically relevant 4-anilinoquinazoline ZD6474. These structures explain why certain multiple endocrine neoplasia 2-associated RET mutants found in patients are resistant to inhibition and form the basis for design of more effective inhibitors.