DE MUNCK STEVEN (BE)
UNIV GENT (BE)
WO2014159074A1 | 2014-10-02 |
US20200157236A1 | 2020-05-21 |
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Claims 1. Compositions in crystalline form selected from i) ALKTG (SEQ ID NO: 1) interacting with ALKAL2 (SEQ ID NO: 2) and ii) LTKTG (SEQ ID NO: 3) interacting with ALKAL1 (SEQ ID NO: 4) and Nb3.16 (SEQ ID NO: 5), characterized in that the crystals are: i) a crystal between ALKTG (SEQ ID NO: 1) interacting with ALKAL2 (SEQ ID NO: 2) in the space group P43212, with the following crystal lattice constants: a=97.57 Å ± 5%, b=97.57 Å ± 5%, c=355.35 Å ± 5%, α=β=γ=90°, and ii) a crystal between LTKTG (SEQ ID NO: 3) interacting with ALKAL1 (SEQ ID NO: 4) and Nb3.16 (SEQ ID NO: 5) in the space group P61, with the following crystal lattice constants: a=129.11 Å ± 5%, b=129.11 Å ± 5%, c=109.75 Å ± 5%, α=90°, β=90°, γ=120°. 2. The compositions of claim 1 which have a three-dimensional structure wherein the crystal i) comprises an atomic structure characterized by the coordinates depicted in the entry ID 7NWZ present in the on line RCSB protein database and wherein the crystal ii) comprises an atomic structure characterized by the coordinates depicted in the entry ID 7NX0 present in the on line RCSB protein database . 3. A computer-assisted method of identifying, designing or screening for a compound that can potentially interact with a crystal selected from a crystal i) or ii) as defined in claims 1 or 2, comprising performing structure-based identification, design or screening of a compound based on the compound’s interactions with a structure defined by the atomic coordinates as defined in claim 2. 4. A method for identifying a compound that can bind to the complex i) ALKTG - ALKAL2 or to the complex ii) LTKTG - ALKAL1 - Nb3.16, comprising dipping candidate small molecule compounds with the complex ALKTG - ALKAL2 or the complex LTKTG - ALKAL1 - Nb3.16L, and allowing co-crystallization, and screening candidate agonists or antagonists by using a method for measuring intermolecular interaction and comparing, designing and docking the 3D structures i) or ii) as defined in claim 2 and a candidate ligand by computer modeling. 5. A method of identifying, designing or screening for a compound that can interact with the complex i) ALKTG - ALKAL2 or to the complex ii) LTKTG - ALKAL1 - Nb3.16, including performing structure-based identification, design, or screening of a compound based on the compound’s interactions with the complex i) ALKTG - ALKAL2 or to the complex ii) LTKTG - ALKAL1 - Nb3.16. 6. A method for identifying an agonist or antagonist compound for the interaction with the complex i) ALKTG - ALKAL2 or with the complex ii) LTKTG - ALKAL1 - Nb3. comprising an entity selected from the group consisting of an antibody, a peptide, a non-peptide molecule and a chemical compound, wherein said compound is capable of enhancing or disrupting the interaction of the complex i) ALKTG - ALKAL2 or the interaction of the complex ii) LTKTG - ALKAL1 - Nb3.16 wherein said process includes: i) introducing into suitable computer program parameters defining an interacting surface based on the conformation of the complex i) ALKTG - ALKAL2 or the complex ii) LTKTG - ALKAL1 - Nb3.16 corresponding to the atomic coordinates 7NWZ or 7NX0 present in the on line RCSB protein database, wherein said program displays a three-dimensional model of the interacting surface, ii) creating a three-dimensional structure of a test compound in said computer program; iii) displaying a superimposing model of said test compound on the three-dimensional model of the interacting surface; iv) and assessing whether said test compound model fits spatially into an interaction site. |
Table 1: Crystallographic data and refinement statistics Table 2: Interaction interface analysis of ALK—ALKAL2 and LTK—ALKAL1 complexes List of contacting amino acids at the interfaces of the ALKAL2—ALKTG and ALKAL1—LTKTG complex. Hydrogen bonding residues were determined using the PISA server at EBI (https://www.ebi.ac.uk/msd- srv/prot_int/pistart.html) and confirmed by analysis in ChimeraX. van der Waals contacts were analyzed using the ‘find contacts’ function in ChimeraX. The components involved in the described interface are shown above each column. It is to be understood that although particular embodiments, specific configurations as well as materials and/or molecules, have been discussed herein for cells and methods according to the present invention, various changes or modifications in form and detail may be made without departing from the scope and spirit of this invention. The following examples are provided to better illustrate particular embodiments, and they should not be considered limiting the application. The application is limited only by the claims. Examples 1.Structure of ALK/LTK cytokine-binding domains ALK is an evolutionarily ancient RTK in C. elegans and D. melanogaster, where it is activated by HEN-1 18 and Jeb 19,20 , both featuring LDLa domains . The extracellular segment of invertebrate ALK resembles the one in vertebrates but lacks the N-terminal HBD. Whereas invertebrates encode a single ALK family receptor, gene duplication in vertebrates spawned LTK as a second ALK-like receptor 21 . During vertebrate evolution the ALK ectodomain remained constant and divergent evolution of LTK led to loss of the N- terminal HBD plus the MAM-LDLa-MAM cassette in mammals . Yet, the cytokine-binding segment in the ALK and LTK ectodomains bears no resemblance to any known protein-binding domain among cytokine receptors. To shed light onto the enigmatic structure-function landscape of ALK and LTK, we pursued crystal structures for human ALK and LTK ectodomains comprising their TNFL, GR, and EGFL membrane- proximal segments (Table 1). We produced glycan-shaved ALK TG-EGFL (residues 648-1030), its complex with a non-neutralizing Fab fragment 22 , and LTK TG (residues 63-380) in mammalian cells ( Fig.6a,b,c). We obtained crystal structures for ALK TG-EGFL and ALK TG-EGFL —Fab to 3 Å and 2.8 Å resolution, respectively, and for LTK TG to 1.3 Å resolution (Table 1). Unexpectedly, the TNFL and GR regions in ALK and LTK do not form separate domains, but are intimately interwoven into a large, continuous, and fully globular TG supradomain 23 (Fig. 1b,c, Fig. 6d,e,f). ALK TG and LTK TG display an unprecedented protein fold topology featuring a chimeric arrangement of subdomains with distinct secondary structure as a result of 6 crossover linkages (Fig.1b-d, Figure 6e,f). The TNFL subdomain is an anti-parallel β-sandwich while the GR subdomain folds into a honeycomb-like lattice of poly-glycine II-helices (Fig. 1e, Fig. 6g). TNFL and GR interface via an extensive hydrophobic groove lined by conserved residues (Fig.1f,g). The N-terminus of the TG supradomain maps to the first strand of its TNFL subdomain while its C-terminus is on the adjacent strand (Fig. 1d, Fig. 6d). In our structure of ALK TG-EGFL this connects to the membrane-proximal EGFL module (Cys987 to Pro1025) via a short N-glycosylated linker (Fig.1b, Fig.6e). Furthermore, the TG supradomain core is decorated by four peripheral α-helices with α1 tethered to the TNFL subdomain via a conserved disulfide, while the disulfide-linked α2 and α4 cluster at the tip of the GR subdomain together with α3 (Fig.6e,f). The GR subdomain displays 14 long and tightly packed pGII-helices arranged in a honeycomb-like lattice (Fig. 1e, Fig. 6g). Analogous pG-II helix networks, albeit much less extensive, have been observed in synthetic polyglycines 24 and four functionally diverse proteins spanning all domains of life 25,26,27,28 (Fig. 6h). Notably, the GR subdomain core has three pGII-helices (d, k, l) that exclusively contain glycine residues. Each of them is surrounded by six other pGII-helices, establishing an interaction network based on compact van der Waals contacts and hydrogen-bonding involving their main-chain amide and carbonyl atoms (Fig. 1e, Fig. 6g). Such tight packing appears to restrict the amino acid composition of these central pGII-helices, offering a rationale for the exquisite conservation of the participating poly- glycine sequences and for loss-of-function mutants in this region of Drosophila ALK 29 . 2.Evolution of ALK/LTK cytokine-binding domains A query in the DALI server 30 using our structural models for ALK TNFL and LTK TNFL retrieved TNF/C1q-class folds (e.g. r.m.s.d =2.8 Å against C1q and TNF, 72 Calpa atoms). However, the ALK TNFL /LTK TNFL chain topology is radically different and unprecedented (Fig.6i) 31 . Topology-independent searches 32 returned more extensive structural superpositions covering an additional ~20 residues in the canonical TNF fold, and structure-based sequence alignments clarified the sequence homology between the A, D and E β- strands in ALK/LTK TNFL and β-strands B, E and F in TNF or TRAIL. The shuffling of spatially equivalent beta- strands between ALK TNFL /LTK TNFL and TNF/C1q proteins goes far beyond a simple permutation (Fig.6i) 33 . The distinctly connected beta-strands in the ALK TNFL /LTK TNFL subdomain break up the alternating sheet- to-sheet register of the TNF/C1q beta-jellyroll, and instead permit the spatially contiguous sprouting of the three glycine-rich loop inserts (between beta-strands D and E, F and G, and H and H’) that go on to fold into the distinctive lattice of the ALK TNFL /LTK GR subdomain. There is no simple evolutionary path (by genetic reshuffling) that would lead to this unique, large-scale reconnection of the beta-jellyroll topology from the canonical TNF/C1q structure to the unique ALK TNFL /LTK TNFL fold––that sprouts its remarkable GR subdomain. Such spatial coalescence of three otherwise unremarkable (and conventionally unstructured) stretches of glycine-rich loop insertions to a topologically tortured TNFL domain argues for further study as a new and versatile scaffold for protein design 34,35 . 3.Assembly of ALK/LTK-cytokine complexes Since ALKAL1 has been reported as an LTK-specific cytokine and ALKAL2 activates both ALK and LTK 6–8 , we opted to pursue structures of ALK–ALKAL2 and LTK–ALKAL1 complexes. We could readily purify truncated versions of both ALKALs corresponding to the conserved C-terminal domains (termed ALKAL1 and ALKAL2) in HEK293T cells as well as full-length ALKAL1 (ALKAL1FL). All three purified ALKALs were monomeric (Fig. 7a) and could drive ALK-dependent Ba/F3 cell proliferation (Fig. 7b,c). Intriguingly, ALK/LTK-cytokine complexes displayed distinct receptor:cytokine stoichiometries. LTK TG-EGFL —ALKAL1 and LTK TG-EGFL —ALKAL2 complexes were measured as 2:1 stoichiometric assemblies (2 receptors + 1 cytokine) (Fig. 8a,b), whereas ALK TG-EGFL —ALKAL1 and ALK TG-EGFL —ALKAL2 complexes were only compatible with 1:1 stoichiometries (Fig. 8a,c). The same stoichiometric dissonance was observed for the complexes of ALK TG and LTK TG lacking the membrane-proximal EGFL domain (Fig. 8d,e). Such ALK/LTK–cytokine complexes appeared to deviate from the canonical 2:2 stoichiometries among RTK- cytokine complexes 36,37 , and were rather reminiscent of hematopoietic cytokine-receptor assemblies. After failing to obtain diffraction-quality crystals from purified LTK TG-EGFL —ALKAL1 and ALK TG-EGFL —ALKAL2 complexes, we leveraged ALK/LTK-cytokine complexes lacking the EGFL domain. In the case of LTK, we additionally employed a non-neutralizing single-domain VHH antibody (Nb3.16) fragment. Crystals of ALK TG —ALKAL2 and a tripartite complex comprising LTK TG —ALKAL1—Nb3.16 (Fig.8f,g) led to structures to 4.2 Å and 1.9 Å, respectively (Fig.2a,b, Table 1). Our structures revealed that the overarching assembly mode of ALK/LTK-cytokine complexes entails a 2:1 stoichiometric assembly where a single cytokine molecule is cradled proximal to the membrane by two copies of the receptor TG supradomains resulting in receptor dimerization (Fig.2a,b). The ALK/LTK- cytokine complexes feature three compact interaction interfaces. Sites 1 and 2 correspond to receptor— cytokine interactions and site 3 describes receptor—receptor contacts (Fig. 2c-f). The cytokine ligands are asymmetric three-helix bundles and use helices B and C to contact one copy of their receptors via site 1, and helix A to engage a second copy of the receptor via site 2 (Fig.2c,f). The dimerized ALK TG and LTK TG supradomains lean against each other at 45° to establish site 3 contacts contributed by the GR subdomains. This creates a surprising receptor assembly with C2-symmetry (Fig.2a,b,d,e) mediated by cytokines that lack twofold symmetry. Given how the EGFL domain connects to ALK TG (Fig. 1b), we envisage that ALKAL1/2 might be fully encapsulated by cognate full-length receptors over the cell surface. 4.Structure of ALKAL1/2 cytokines ALKAL1 and ALKAL2 adopt highly similar structures (r.m.s.d=0.54 Å, 56 Calfa-atoms) featuring a new type of disulfide-stabilized three-helix bundle, wherein αA connects via a conserved short loop to a helical hairpin constructed from αB and αC, which in turn are tethered by two conserved disulfides (Fig. 3a). The ALKAL1/2 fold is conspicuously open and lacks a classical hydrophobic core as observed in other helical cytokines 38,39 . Rather, αA only connects to the BC hairpin by inserting Ile77 into a hydrophobic pocket formed by Leu117, Tyr110 and the Cys104-Cys113 disulfide (Fig. 3a). Additional stabilization is provided by hydrogen-bonds between Lys73 and the main chain atoms of Cys104 and Tyr98 (Fig.3a). Despite their engulfment by the receptor dimers, both ALKAL1 and ALKAL2 display a solvent-exposed hydrophobic cavity defined by conserved residues: those lining the internal BC face (Leu117, Phe94, Tyr98) and the AB loop (Val84, Phe86) and those around the outside rim contributing hydroxyl groups (Fig.3b,). Such conservation of core residues and cysteine-disulfides suggest application of this fold to all vertebrate ALKALs. The few sequence differences map to two distinct patches contributed by the end of αC and the AB loop, and might provide insights into cytokine specificity. A conserved stretch of ~10 residues preceding αA was not ordered in the reported structures, suggesting they might help to stabilize the soluble forms of these cytokines rather than contribute to direct receptor engagement or possibly help reduce the entropic cost of binding. 5.ALK/LTK–cytokine interaction interfaces Despite being monomeric and lacking symmetry, ALKAL1 and ALKAL2 remarkably dimerize their cognate receptors into highly similar, twofold-symmetric assemblies reminiscent of receptor complexes mediated by erythropoietin and growth hormone 40,41 . However, these hematopoietic cytokines display a certain degree of pseudo-symmetry through their four-helix bundle. ALKAL1 and ALKAL2 contact the same binding sites on LTK and ALK but display different interaction modalities. In site 1, αB and αC use a hydrophobic epicenter surrounded by polar residues to contact the TNFL subdomain via a patch contributed by its D, E, H’’ and I strands and the H’’-I loop. In each complex, a trio of arginine residues in ALKAL1 (Arg102, Arg112, Arg119) and ALKAL2 (Arg123, Arg133, Arg140) engages two conserved glutamate residues at the periphery of site 1 (Fig.4a,b,Fig.9a, Table 2). These electrostatic interactions are supplemented by Arg115 of ALKAL1 (Arg136 in ALKAL2) running perpendicular to the crossover H’’-I loop of the TNFL subdomain and hydrogen-bonding to the main chain carbonyl group of a conserved valine (Fig. 4a,b). On the ligand side, site 1 encapsulates a hydrophobic core contributed by three conserved leucine residues in ALKAL1 and ALKAL2. Here, LTK offers a broader platform by presenting four conserved hydrophobic residues (Phe143, Leu361, Leu364, Val366) (Fig. 4b). Interestingly, the corresponding pocket on ALK substitutes for Phe143 and Leu361 via Ser758 and Thr967, respectively (Fig.4a, Fig.9b). In site 2, which is predominantly hydrophobic, the short ALKAL αA pairs with the tip of the BC hairpin to engage the second receptor molecule (Fig. 4c,d). In the LTK TG –ALKAL1 complex Phe80 protrudes from αA in ALKAL1 into a conserved hydrophobic pocket on LTK (Val366, Tyr124, Phe143). The neighboring Phe76 inserts between Leu361 and Leu364 at the edge of the site 2 interface. Surprisingly, the ALK TG – ALKAL2 complex is devoid of an equivalent residue for Phe143 (Fig.9c). Cytokine-mediated dimerization of the ALK TG and LTK TG supradomains induces receptor–receptor contacts (site 3) by locking α1 of the TNFL subdomain with α2 and α3 at the distal end of the GR subdomain (Fig.2d,e, Fig.9d). However, the two site 3 interfaces in LTK and ALK are distinct, with the latter being less extensive (Fig.4e,f, Fig.9d-f). In LTK, site 3 entails an interaction platform centered on His153 stacking against the peptide of Gly74, analogous to key interactions in IL-6/IL-12 family complexes 42 (Fig.4e). Additional interactions are mediated by Arg241 on an LTK-specific loop insert (Fig. 4e, Fig.9e). At the center of site 3, Asn369 hydrogen-bonds with its symmetry related counterpart (Fig. 9f). 6.Site 1 drives ALK/LTK–cytokine complexes To obtain insights into the contribution of the two distinct ALK/LTK–cytokine interfaces, we used mutants probing the polar interactions of site 1 and the central hydrophobic pocket in site 2. This is especially important to clarify for ALK since its extracellular domain, in contrast to LTK, does not readily proceed to 2:1 stoichiometric complexes with cytokines at the concentrations attainable in solution. Site 1 contacts were interrogated by introducing charge-reversal mutations of two conserved polar interactions resulting in ALKAL1 mutants ALKAL1 R102E , ALKAL1 R115E , and ALKAL1 R102E/R115E , and ALKAL2 mutants ALKAL2 R123E , ALKAL2 R136E and ALKAL2 R123E/R136E . We first established that ALKAL2 is the high- affinity cytokine for ALK and ALKAL1 for LTK and using these binding benchmarks concluded that all site 1 mutants for ALKAL1 and ALKAL2 drastically reduced their affinity to both receptors (Fig.10a,b). To probe site 2 we used mutants ALKAL1 F76E and ALKAL2 F97E aimed at their respective hydrophobic interaction patches, and a ALKAL2 H100A mutant. The interaction of ALKAL1 F76E , ALKAL2 F97E and ALKAL2 H100A with LTK resulted in a biphasic binding profile with one interaction obeying faster off-rates (Fig.10c,d). On the other hand, the binding profile of ALKAL2 F97E to ALK was similar to wildtype ALKAL2 (Fig.10e). We used purified ALKAL1/2 mutants to further investigate the role of sites 1 and 2 via our Ba/F3 cellular proliferation assay (Extended Data Fig.7f). Whereas wildtype ALKALs induced clear cell proliferation at 10 nM, site 1 double mutants did not (Fig. 4g,h, Fig. 10g). In contrast to our kinetic binding assays, ALKAL2 F97E appeared to abrogate the site 2 interaction showing cell proliferation similar to the functionally deleterious site 1 mutant ALKAL2 R123E/R136E (Fig.4g, Fig.10g). Moreover, we found that site 2 mutants retained their ability to form 1:1 stoichiometric complexes with both ALK and LTK, whereas site 1 mutants did not (Fig.10h). Finally, we interrogated the functional importance of site 3 in LTK and ALK. For LTK, mutants LTK R241A and LTK R241A/N369G had a decreased propensity to form the native 2:1 stoichiometric complexes with ALKAL1 (Fig. 10i). Given the resistance of ALK ectodomains to establish 2:1 stoichiometric complexes with cytokines at concentrations applicable in chromatographic methods, we established a Ba/F3 cell line expressing ALK M751T hypothesizing that this mutation would introduce an N-linked glycosylation site at Asn749. We found that ALK M751T was unable to drive cytokine-dependent cellular proliferation while it expresses very similarly to wildtype ALK (Fig.10j). The presented structural landscapes of ALK/LTK-cytokine complexes revealed key differences in site 2 and site 3, prompting the question whether evolutionarily conserved determinants might be at play. Indeed, we noted the absence of equivalents for Phe143 and His135 in ALK differentiates ALK from LTK across vertebrates. Together with an insert in the LTK e-f loop (Extended Data Fig.8a), these interactions create an LTK-specific zipper across the length of the site 3 interface acting as driving force for the apparent increased propensity to form a ternary complex (Fig.9e,f). Collectively, our structure-function data suggest that site 1 engagement is the driving force for establishing ALK/LTK-cytokine encounter complexes. Given the high sequence conservation of interfacing residues in both cytokines and receptors the observed ALKAL1/2 binding modes will likely apply to all vertebrate receptors consistent with the reciprocal species cross-reactivity of zebrafish ALKAL2a and human ALKALs 43 . Interestingly, surface amino acids on the opposite side of the ALK cytokine-binding interfaces are highly conserved, in contrast to LTK, suggesting that they might be relevant for interactions with the N-terminal domains, which are absent in LTK. 7.Mapping of somatic mutations in ALK To expand structure-function insights on ALK and LTK activation we structurally mapped mutations in ALK TG and LTK TG combining documented oncogenic potential and frequently occurring missense single- nucleotide polymorphisms 44,45 (Fig.11a,b). Mutations leading to constitutive receptor activation or that may enhance receptor–receptor contacts or stabilize active receptor states are widely studied to evaluate oncogenic potential. LTK variant R243Q and ALK variants G685R, G747R, and H694R locate at positions that would be compatible with such roles. Interestingly, H694R is a gain-of-function mutation in lung adenocarcinoma leading to constitutive activation of ALK 46 . On the other hand, mutations that may impact cytokine binding, e.g. by increasing affinity, are less common. Here, we identify two such candidate mutations: LTK variant P363L and ALK variant S737L mapping to interaction sites 2 and 1, respectively. ALK F856S , a gain-of-function mutation linked to acute myeloid leukemia 47 , and ALK R753Q identified in histiocytic neoplasms 48 , are roughly equidistant from the start of αC of bound ALKAL2 (Fig.11a). To gain insight into their possible mechanism, we established Ba/F3 cell lines expressing the two mutant ALK variants and found that while their expression levels were comparable to ALK WT , they both drastically increased cytokine-dependent cell proliferation (Fig.11c,d,e,f). Unlike previously reported 47 , ALK F856S was not constitutively active. In light of their structural context, these mutations might facilitate the reorganization of key regions of the cytokine-receptor interfaces. 8.Cytokine specificity in ALK by the EGFL domain Given the overall structural similarity of ALK TG /LTK TG –cytokine complexes and that EGFL modules can bind ligands 49 , we hypothesized that the membrane-proximal EGFL domain might underlie the apparent preference of ALK for ALKAL2 over ALKAL1. Benchmarking of the binding thermodynamics showed that even at micromolar concentrations, ALK TG-EGFL formed enthalpically-driven binary complexes with either cytokine characterized by a markedly higher affinity for ALKAL2 (K D = 40 nM) than ALKAL1 (K D = 600 nM) (Fig. 5a-c). In contrast, LTK TG-EGFL bound with high-affinity and 2:1 stoichiometry to both cytokines (K D, ALKAL1 = 10 nM, K D, ALKAL2 = 55 nM) (Fig.12a,b) in agreement with stoichiometries derived from small-angle X-ray scattering analyses (Fig.12c). As we could produce ALKAL1 carrying the N-terminal region absent in our structure, we titrated LTK TG-EGFL with ALKAL1FL and measured a modest increase in affinity compared to the truncated form (Fig.12d). Sequence differences in ALKAL1 and ALKAL2 map to distinct patches proximal to the EGFL domain (Fig. 12e). Remarkably, removal of the EGFL domain from ALK resulted in a drastic 30-fold decrease in affinity for ALKAL2 but only a 4-fold affinity reduction for ALKAL1 (Fig. 5d,e). In contrast, the LTK TG domain retained its nanomolar binding affinity to both ALKAL1 and ALKAL2 (Fig. 12f-h). Thus, while ALK TG is endowed with a baseline micromolar affinity for both cytokines, the receptor’s specificity for ALKAL2 is substantially enhanced by the EGFL domain. As ALK carries four additional N-terminal domains compared to LTK (Fig.1a), we measured the binding affinity of full-length ALK ectodomain (ALKFL) for ALKAL2. We obtained a similar affinity and 1:1 stoichiometry to the ALK TG-EGFL -ALKAL2 interaction indicating that the N-terminal domains of ALK play a negligible role in cytokine binding (Fig.12i). Nevertheless, in line with the reported interaction of canine ALK with heparin 9 , we found that heparin induced dimerization of a large fraction of human ALK FL in the presence or absence of bound cytokine (Fig. 12j). In hindsight, the inordinately high protein concentration in protein crystals compared to what can be attained in solution appears to have fortuitously compensated for the deficiency of ALK TG to undergo cytokine-induced dimerization in solution. 9.Mechanistic considerations Whereas cytokine-mediated dimerization of ALK and LTK leads to structurally similar ternary complexes, the mechanistic requirements for their assembly appear to be distinct. LTK-cytokine complexes are fully cytokine-driven whereas ALK-cytokine complexes might synergize with glycosaminoglycans (Fig.5f). Our reported ALK/LTK-cytokine complexes are likely representative in all vertebrates because of the strong sequence conservation in receptors and ligands, and their species cross-reactivity 6,43 . However, invertebrate ALK ligands are unable to activate human ALK and are structurally distinct from ALKAL1/2 50 . The currently known modes of ligand-induced activation of RTKs display a broad array of structural principles 36,37 , ranging from dimerized receptor assemblies exclusively driven by the activating ligands (e.g. Trka—NGF) 51 to complexes mediated fully via receptor contacts (e.g. EGFR—EGF) 52 . However, several cytokine-RTK assemblies feature an amalgamation of ligand- and receptor-mediated contributions (e.g. CSF-1/IL-34—CSF-1R) 53,54 including the involvement of accessory molecules (e.g. FGF—FGFR) 55 or co-receptors 56,57 , and the use of multiple cytokine copies to (e.g. EGFR and Insulin receptor) 52,58 . The utilization of a single copy of a monomeric cytokine by ALK family receptors to undergo dimerization with twofold symmetry introduces a novel cytokine-driven assembly mechanism among RTK (Fig.12k). It is now clear that key differences in the cytokine-binding regions of ALK and LTK dictate cytokine specificity and that receptor–receptor contacts are also important differentiating factors. The resistance of isolated ALK ectodomains towards cytokine-induced dimerization suggests that the reduced dimensionality of their membrane-proximal engagement and additional interactions of its N-terminal domains with glycosaminoglycans or proteoglycans 8 might be important for productive cytokine- receptor assemblies, much like bound heparin bridging receptors in FGF-FGFR complexes. In this context, the reported proteolytic shedding of the N-terminal segment of ALK’s ectodomain presents with a physiological conudrum 59 . Intriguingly, the EGFL module of ALK, but not LTK, appears to dictate cytokine specificity, such that the mode of its engagement in ALK-cytokine complexes may impact the orientation of the membrane-proximal domains (and their connected transmembrane helices) to fine tune signaling assemblies. We envisage application of our findings to further interrogate ALK/LTK signaling in physiology and disease, and in the therapeutic targeting of the ALK/LTK ectodomains 17 and their cognate cytokines 16 . Materials and methods No statistical methods were used to predetermine sample size. The experiments were not randomized and the investigators were not blinded to allocation during experiments and outcome assessment. 1.Plasmids, constructs, and cell lines for protein expression in mammalian cells Sequence optimized DNA for full length wild-type ALK (Uniprot ID Q9UM73), LTK (Uniprot ID P29376), ALKAL1 (Uniprot ID Q6UXT8) and ALKAL2 (Uniprot ID Q6UX46) were purchased from Genscript. DNA encoding for different human ALK constructs comprising either amino acids 19-1025 (ALKFL), 648-1025 (ALK TG-EGFL ) or 648-985 (ALK TG ) and human LTK constructs comprising amino acids 63-420 (LTK TG-EGFL ) or 63- 379 (LTK TG ) were cloned in the pHLsec vector 60 in frame with a N-terminal chicken RTPμ-like signal peptide sequence and a C-terminal caspase3 cleavable Fc-His x6 -tag at the C-terminus. Sequences encoding for ALKAL1 FL (residues 28-129), ALKAL1 (residues 57-129) and ALKAL2 (residues 78- 152) were cloned in the pHLsec vector in frame with a N-terminal chicken RTPμ-like signal peptide sequence followed by a caspase3 cleavable Sumostar-tag at the C-terminus. Sequence-optimized DNA encoding the light and heavy chains of Fab324 were purchased from IDT as GBlocks. The N-terminal signal peptide sequences were exchanged for a chicken RTPμ-like signal peptide sequence. The heavy chain was cloned in frame with a C-terminal caspase-3 site followed by an AVI-His 6x tag, while the light chain was cloned without a purification tag. 2.Protein expression in HEK293 and purification from conditioned media Production of all ALK TG-EGFL and ALK TG constructs was performed in adherently grown HEK293 MGAT1-/- cells 61 maintained in DMEM supplemented with 10% FCS. When cells reached 80% confluency they were transiently transfected using branched polyethylenimine 25kDa as transfection reagent in DMEM with 3.6 mM valproic acid and without FCS. Expression of the Fab fragment was achieved in adherent HEK293T cells using the same method. For the heterodimeric Fab fragment, plasmids encoding for each chain were co-transfected in a 1:1 ratio. Protein production for ALKFL, ALKAL1/2 and LTK constructs was performed in HEK293S cells grown in suspension and maintained in a mixture of 50% Freestyle (Thermofisher) / 50% Ex-Cell (Sigma-Aldrich) growth medium. Transient transfection was performed with linear polyethylenimine (Polysciences) 25kDa as transfection reagent. One day after transfection valproic acid was added until a final concentration of 1.5 mM 62 . For expression in suspension cells conditioned medium was harvested after five days and subsequently clarified by centrifugation for 12 minutes at 8000 xg while medium from adherently grown cells was harvested after 6 days and centrifuged for 15 minutes at 6000 xg. After centrifugation media were filtered through a 0.22 mm filter prior to chromatographic purification steps. ALK and LTK constructs were captured via their Fc-tag on a protein A column (HiTrap Protein A HP, Cytiva) and eluted via on-column digest with caspase 3 for 1 hour at 37°C followed by 2 hours at room temperature and eluted with HBS (HBS (20mM HEPES pH 7.4150mM NaCl). The eluted proteins were then concentrated and injected on a HiLoad 16/600 SD200 (Cytiva) size-exclusion chromatography column pre-equilibrated with HBS. Purified proteins were stored at -80°C until further use. ALKAL containing medium was fourfold diluted with 20mM HEPES pH 7.4 before loading on a cation exchange column packed with SP Sepharose Fast Flow resin (Cytiva) equilibrated in 20mM HEPES pH7.4 50mM NaCl. ALKALs were eluted using a NaCl gradient from 50mM-750mM for 20 minutes. Fractions containing ALKALs were immediately diluted with 20mM HEPES pH7.4 to a NaCl concentration of 200 mM and further supplemented with 0.1% (w/v) CHAPS. The C-terminal Sumo-tag was cleaved with caspase 3 overnight at 20°C. To remove undigested protein as well as the cleaved Sumo-tag, the digestion mixture was loaded on a MonoQ 5/50 GL column (Cytiva). Flowthrough containing the ALKALs was concentrated and injected on a Superdex 75 Increase 10/300 GL equilibrated in HBS supplemented with 0.1% CHAPS. Purified ALKALs were stored at -80°C at a concentration of 1mg ml -1 until further use. For ALKALs used in BaF/3 assays, endotoxin levels were measured using a Endosafe PTS limulus amebocyte lysate assay (Charles river) and were below 5 EU mg -1 . Fab fragments were captured using cOmplete His-tag purification resin (Roche) and eluted using HBS supplemented with 250 mM imidazole followed by buffer exchange to HBS on a HIPrep 26/10 desalting column. Caspase 3 was added to the purified Fab fragment in order to remove the AviHis tag of the heavy chain by overnight digestion at 20°C. The sample was loaded on an IMAC column in order to remove the enzyme and undigested protein. The flow-through containing tagless Fab fragments was concentrated and injected on a Superdex 200 Increase 10/300 GL (Cytiva) column pre-equilibrated in HBS. 3.Production of non-neutralizing single domain camelid VHH against LTK Single domain camelid VHHs (Nanobodies) against LTK were raised by immunizing llamas with LTK TG-EGFL and were selected for specific binding to LTK TG-EGFL via ELISA and BLI. Epitope binning via BLI led to the identification of candidate Nanobodies with non-neutralizing behavior with respect to cytokine binding. The sequences of such non-neutralizing Nanobodies were cloned in a MoClo derived yeast expression vector in frame with a N-terminal preproMF secretory leader sequence followed by the N-terminal His x6 tag and a caspase cleavage site. Komagataella phaffii cells were transformed by electroporation and grown on YPDS agar containing 500 µg/ml zeocin. One colony was picked to inoculate 500 ml of BMGY supplemented with 100 µg/ml zeocin and grown at 28°C for 24 hours. Next, cells were pelleted by centrifugation at 500xg for 7 minutes and resuspended in 500ml BMMY medium without zeocin and incubated O/N at 28°C. Expression was further induced by adding 2.5 ml of 50% methanol, the same volume of methanol was again added after 8h and 24h. After which cells were incubated for another 8h at 28°C. Finally, conditioned medium was harvested by centrifugation for 10 minutes at 6000xg. His-tagged camelid single domain VHHs were captured by addition of 2ml cOmplete resin (Roche) to 500 ml conditioned medium followed by overnight incubation at 4°C while shaking. Nanobodies were eluted in HBS supplemented with 250mM imidazole and buffer exchanged to HBS on a HiPrep 26/10 desalting column (Cytiva). The N-terminal Hisx6 tag was removed by an overnight caspase3 digest at 20°C. Undigested protein and the enzyme were removed via IMAC. The flowthrough containing tagless nanobody was concentrated and injected on an SD75 Increase 10/300 GL column (Cytiva) pre- equilibrated in HBS. Purified nanobody was concentrated to a concentration of 4 mg ml −1 and flash- frozen and stored at − 80 °C. 4.Crystallization and crystal structure determination. For ALK TG-EGFL —Fab324 crystals, a 1.5 molar excess of Fab324 was added to ALK TG-EGFL and further subjected to an over-night enzymatic digestion of N-linked glycans with EndoH. The complex was polished by SEC on a Superdex 200 Increase 10/300 GL (Cytiva) and concentrated to 13.5 mg ml -1 . Commercial sparse matrix crystallization screens were set up using a Mosquito liquid handling robot (TTP Labtech) in sitting drop format using 100 nL protein mixed with 100 nL mother liquor in SwissSci 96-well triple drop plates incubated at 287 K. A first hit in the Morpheus II screen 63 was further optimized to a condition consisting of (40 mM Polyamines, 0.1 M Gly-Gly/AMPD pH 8.5, 11% w/v PEG4000 , 19 % w/v 1,2,6-Hexanetriol) in sitting drop format with a 100 nl protein mixed with 200 nL mother liquor geometry. Crystals were cryoprotected in mother liquor containing 25% w/v 1,2,6-Hexanetriol prior to being cryocooled in liquid nitrogen. Diffraction data was collected at 100 K at the ID23-2 beam line at ESRF, Grenoble. The datasets were processed using XDS 64 . Initial phases were calculated by molecular replacement with PHASER 65 using the coordinates of a Fab fragment exhibiting the highest sequence identity (PDB: 5nuz, chain A ) followed by rigid body refinement in Buster 66 . A partial polyalanine model was built into the visible electron density in Coot 67 followed by density modification via Resolve 68 . The density modified map showed density for several aromatic sidechains allowing for assignment of the correct register and tracing of the ALK sequence. Additional refinement steps were carried out in PHENIX 69 using individual B-factor refinement in combination with TLS, XYZ refinement, optimizing the X-ray/geometry weights as well as local torsion angle non-crystallographic symmetry (NCS) restraints. For crystals of ALK TG-EGF , glycans were trimmed by over-night enzymatic digestion with EndoH in HBS. The complex was polished via SEC and concentrated to 10.5 mg ml -1 . Commercial sparse matrix sitting drop crystallization screens were set up as described. One hit was obtained in the Morpheus II screen and optimized to 0.5mM Manganese(II) chloride tetrahydrate, 0.5mM Cobalt(II) chloride hexahydrate, 0.5mM Nickel(II) chloride hexahydrate, 0.5mM Zinc acetate dihydrate, 13% w/v PEG 3000, 28% v/v 1,2,4- Butanetriol, 1% w/v NDSB 256 in hanging drop format. Crystals were cryoprotected in mother liquor containing 25% 1,2,4-Butanetriol prior to being flash frozen in liquid nitrogen. Diffraction data was collected at 100 K at the P14 microfocus beam line at PETRA III, Hamburg and integrated using XDS with standard parameters except for the ‘‘BEAM_DIVERGENCE’’ parameter which was doubled. Initial phases were obtained using maximum likelihood molecular replacement in Phaser using the structure of the ALK TG domain. The structure was refined using Phenix.refine followed by manual building in COOT. The EGF-like domain was manually built into the electron density. Refinement in phenix followed the same protocol as for the ALK TG-EGFL —Fab324 structure except for the absence of NCS restraints and implementation of additional reference restraints based on the structure of ALK TG in complex with Fab324. For crystals of LTK TG , purified LTK TG was concentrated to 10 mg ml -1 and used to set up sparse matrix screens as previously described. Crystals appeared in a condition of the Morpheus II screen with the final optimized condition containing (MOPSO/Bis-Tris pH 6.3, 12% PEG 20000, 26% Trimethyl propane 1% w/v NDSB 195 5mM Yttrium (III) chloride hexahydrate, 5mM Erbium (III) chloride hexahydrate, 5mM Terbium(III) chloride hexahydrate, 5mM Ytterbium (III) chloride hexahydrate) set-up in sitting drop format with a 100 nL protein mixed with 200 nL mother liquor geometry was cryoprotected in mother liquor and cryo-cooled in liquid nitrogen. Diffraction data was collected at 100K at the P13 microfocus beam line at PETRAIII, Hamburg and processed using XDS as previously described. Phases were obtained by single wavelength anomalous dispersion making use of the anomalous signal from lanthanide atoms. Determination of the lanthanide substructure for four sites was performed by the hybrid substructure search as implemented in Phenix. Phases were calculated using Phaser-EP. The density was readily interpretable, and a model was manually built in Coot and further refined in Phenix implementing an anisotropic individual B-factor model. For LTK TG —ALKAL1—Nb3.16 crystals a 3-fold molar excess of Nb3.16 was added to the LTK TG —ALKAL1 complex, concentrated and injected into a Superdex 200 Increase 10/300 GL (Cytiva) equilibrated in HBS. Eluted fractions were concentrated to 13.5 mg ml -1 and used to set up sitting drop crystallization screens as described. Crystals were obtained in a condition consisting of MOPSO/BIS-TRIS pH 6.313% PEG 8000, 22% w/v 1,5-pentanediol, 5mM sodium chromate tetrahydrate, 5mM sodium molybdate tetrahydrate, 5mM sodium tungstate tetrahydrate, 5mM sodium orthovanadate tetrahydrate. Crystals were cryoprotected in mother liquor containing 25% 1,2,4-Butanetriol prior to being flash frozen in liquid nitrogen. Diffraction experiments were performed at 100 K Proxima 2 microfocus beam line at the Soleil synchrotron. Initial phases were obtained by maximum likelihood molecular replacement using Phaser with the previously obtained LTK TG structure. A model for Nb3.16 was automatically built using ArpWarp 70,71 followed by manual building of ALKAL1 in Coot. Refinement was performed in Phenix with individual anisotropic ADP parameters with a TLS model. For ALK TG —ALKAL2 crystals a 3-fold molar excess of ALKAL2 was added to ALK TG and subjected to an overnight EndoH digest, concentrated and injected into a Superdex 200 Increase 10/300 GL (Cytiva) equilibrated in HBS. Eluted fractions were concentrated to 8 mg ml -1 and used to set up sitting drop crystallization screens as described. Initial hits were obtained in a condition consisting of 0.1M MES pH 6.515% w/v PEG 60005% w/v MPD. A single crystal was used to prepare a seed stock 72 using the PTFE seed bead (Hampton Research). The best diffracting crystals were obtained by seeding with a 1:1000 dilution of the seed stock into the optimization screen. Crystals were cryoprotected in 78% (v/v) mother- liquor supplemented with 22% (v/v) ethylene glycol before flash freezing in liquid nitrogen. Diffraction data were collected at 100 K at the Proxima 2 microfocus beam line at the Soleil synchrotron. Data as processed as described above with the difference that anisotropy correction was implemented by the UCLA diffraction anisotropy server 73 . Initial phases were obtained by molecular replacement in Phaser using the previously determined ALK TG and ALKAL1 structures. First refinement cycles were performed in Buster followed by iterative refinement using Coot and Phenix. B-factors were refined using two isotropic atomic displacement parameters complemented by TLS. During refinement structures of ALK TG and ALKAL1 provided reference model restraints. For Fab324 crystals, tag-free Fab324 was concentrated to 18.5 mg ml -1 and a pH versus (NH 4 ) 2 SO 4 concentration screen was set up in sitting drop format resulting in crystals in a condition consisting of (1.5M (NH4)2SO440 mM Glycine pH 9.5). The crystal was cryoprotected using a saturated (NH 4 ) 2 SO 4 solution. Diffraction experiments were performed at the P13 beamline PetraIII, Hamburg and data were processed using XDS. Phases were obtained by molecular replacement using our structure of Fab324 obtained from the ALK TG-EGFL —Fab324 complex. The structure was refined using Coot and Phenix. All display items containing structures were generated using the PyMOL Molecular Graphics System, version 2.0.5 (Schrödinger). 5.Isothermal Titration Calorimetry (ITC) Experiments were performed using a MicroCal PEAQ-ITC instrument at 310K. Proteins used in ITC experiments were expressed in HEK293S cells grown in suspension. As a final purification step all proteins were buffer exchanged to the same HBS buffer via size-exclusion chromatography. Titrations were preceded by an initial injection of 0.5 µL. The injection spacing was optimized per experiment to allow for the signal to get back to a stable baseline. Throughout the titration the sample was stirred at a speed of 750 r.p.m. Data were analyzed using the PEAQ-ITC analysis software (version 1.1.0.1262, Malvern) and fit using a “one set of sites” model. 6.Multi-angle Laser Light Scattering (MALLS) Protein samples of 100 µl at approximately 1.0 mg ml -1 were injected onto a Superdex 200 increase 10/300 GL column (Cytiva) connected in line to a UV-detector (Shimadzu), a miniDawnTReos (Wyatt) multi-angle laser light scattering detector and an optilab T-Rex (Wyatt) refractometer. The refractive increment value (dn/dc) was 0.185 mL g -1 . Band broadening was corrected for using reference measurements of BSA (Pierce). Data analysis was carried out using the Astra 6.1.6 software and standard deviations were calculated using Prism8 (Graphpad Software). 7.Biolayer interferometry (BLI) Screening of mutant ALKALs was performed by immobilizing wild-type and mutant ALKAL1/2 variants. To this end, residues 57-129 of wild-type ALKAL1 and interface mutants (R102E, R115E, R102E/R115E, F76E and F80E) as well as residues 78-152 of wild-type ALKAL2 and interface mutants (R123E, R136E, R123E/R136E, F97E and H100A) were cloned in the pHLsec vector in frame with a C-terminal Avi tag. All constructs were transiently cotransfected in suspension grown HEK293S cells together with a BirA expression plasmid (pDisplayBirA-ER 74 ) as previously described and supplemented with 100 μM biotin upon transfection. After 4 days of expression, excess biotin was removed by desalting the conditioned media to HBS on a HIPrep 26/10 desalting column (Cytiva). All measurements of binding kinetics and dissociation constants were performed using an Octet Red 96 (Forté Bio) in assay buffer (20 mM HEPES pH 7.4, 150 mM NaCl, 0.02% (w/v) BSA, 0.002% (v/v) Tween 20) at 298K. ALKALs were immobilized to a level of 0.5 nm on streptavidin-coated biosensors (Forté Bio). To verify that no aspecific binding was present during the assay, non-functionalized biosensors were used as a control by measuring in parallel all ligand concentrations as well as running buffer. For all mutants a two-fold dilution series from 6.4 μM-400 nM was employed. Data analysis was performed using the Data Analysis software 9.0.0.14 (Forté Bio) and binding curves were exported to Prism8 (Graphpad Software) for plotting of curves. 8.Small angle X-ray scattering (SAXS) SEC-SAXS data were collected at the SWING beamline at SOLEIL (France) using an integrated online HPLC set-up. Purified samples of ALK TG-EGFL —ALKAL2 (18.5 mg ml -1 ) and LTK TG-EGFL —ALKAL1 (19 mg ml -1 ) expressed in HEK293SMGAT -/- cells were injected on a Biosec-3 column (Agilent) with HBS as a running buffer. The scattering data were collected in continuous flow mode with a flow speed of 0.3ml/min and a 1 s exposure time per frame. Buffer and sample frames were selected and subtracted using the program RAW 75 . Theoretical scattering curves and fitting to experimental scattering data was performed with AllosMod-FoXS. Briefly, a model for the C-terminal EGFL domain of LTK was generated by homology modeling starting from the crystal structure of the ALK EGF-like domain using the SWISS-MODEL server 76 . This model was manually placed and connected to the C-terminus of the LTK TG domain in Pymol based on the ALK TG-EGFL structure. Missing regions in the ALK and LTK structures were added using MODELER 77 . The resulting models were subsequently energy minimized using Rosetta-Relax, and used as an input for AllosMod to add N-linked glycans on positions Asn709, Asn808, Asn886 and Asn986 for ALK and Asn380 and Asn412 for LTK, and calculated resulting model energy landscapes. The output of AllosMod was then used in AllosMOD-FoXS to calculate fits with theoretical scattering curves during fast AllosMod simulations at 300 K. 9.Cell culture and retroviral transduction Ba/F3 cells (murine pro-B cell line) cells were cultured in RPMI/10% FCS supplemented with mouse interleukin-3 (IL-3, 1 ng ml -1 ). Ba/F3 cell line was not listed in the database of commonly misidentified cell lines maintained by ICLAC and NCBI Biosample. Ba/F3 cells were transduced with viral supernatant MSCV-ALK WT /ALK R753Q /ALK F856S /EV-IRES-GFP (EV; empty vector) for 2 days in RPMI/10% FCS supplemented with mouse + IL-3 as previously described 78 . GFP-sorted cells were used for the cell growth assays and western blot. After removal of IL-3 from the media, the cells were stringently washed with PBS for three times. The cell growth curves and heatmaps were made using GraphPad Prism 9 software as mean values, with error bars representing standard deviation. 10.Reagents For Western blotting, the following antibodies were used: ALK (Purchased from Cell Signaling Technologies; catalog no.: 3633; dilution: 1:1,000), Phospho-ALK (Tyr1278) (Cell Signaling Technologies; 6941; 1:250), Phospho-ALK (Tyr1604) (Cell Signaling Technologies; 3341; 1:250), Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (Cell Signaling Technologies; 4370; 1:2,000), p44/42 MAPK(Erk1/2) (Cell Signaling Technologies; 9102; 1:1,000), β-actin (Sigma-Aldrich; A-5441; 1:2,000), GAPDH (GENETEX; GTX100118; 1:2000). For the cell growth assay, Crizotinib (Sigma-Aldrich; PZ0191-5MG), DMSO (Signa- Aldrich; D8418-100ML), and IL-3 (Peprotech; AF-213-13) were used. 11.Quantification and statistical analysis Statistics and reproducibility Ba/F3 assay Statistical significance was determined by two-way ANOVA followed by Tukey’s multiple comparison test where multiple comparisons should be adjusted. Data were plotted using GraphPad Prism 9 software as mean values, with error bars representing standard deviation. Heatmaps were also made using GraphPad Prism 9 software based on mean values. *P < 0.05, ** P < 0.01 and *** P < 0.001, respectivNb3.16ely, unless otherwise specified.
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