Structural basis of cytokine-mediated activation of ALK family receptors

Abstract

Anaplastic lymphoma kinase (ALK) 1 and the related leukocyte tyrosine kinase (LTK) 2 are recently deorphanized receptor tyrosine kinases (RTK) 3 . Together with their activating cytokines, ALKAL1 and ALKAL2 (also called FAM150A/FAM150B and AUGβ/AUGα) 4-6 , they are involved in neural development 7 , cancer [7][8][9] , and autoimmune diseases 10 . Furthermore, mammalian ALK recently emerged as a key regulator of energy expenditure and weight gain 11 , consistent with a metabolic role in Drosphila 12 . Despite such functional pleiotropy and growing therapeutic relevance 13,14 , structural insights into ALK and LTK and their complexes with cognate cytokines had remained elusive. Here, we show that the cytokine-binding segments of human ALK and LTK comprise an unprecedented architectural chimera of a permuted TNF-like module that braces a Glycine-rich subdomain featuring a hexagonal lattice of long polyglycine-II helices. The cognate cytokines ALKAL1 and ALKAL2 are monomeric three-helix bundles, yet their binding to ALK and LTK elicit similar dimeric assemblies with twofold symmetry, that tent a single cytokine molecule proximal to the cell membrane. We show that the membrane-proximal EGF-like domain dictates the apparent cytokine preference of ALK. Assisted by diverse structure-function findings, we propose a structural and mechanistic blueprint for complexes of ALK family receptors, thereby extending the repertoire of ligand-mediated dimerization mechanisms adopted by RTK. MainALK is an evolutionarily ancient RTK with the vertebrate orthologues uniquely endowed with an Nterminal heparin binding domain (HBD) (Fig. 1a and Extended Data Fig. 1a,b). Gene duplication in vertebrates spawned LTK as a second ALK-like receptor 16 , which evolved divergently with loss of the HBD and additionally the MAM-LDLa-MAM module in mammals (Extended Data Fig. 1c). The common architectural hallmark in the ectodomains of ALK and LTK comprises their cytokine-binding segment, which is unique among cytokine receptors and features an intriguing array of a TNF-like (TNFL) module, a glycine-rich (GR) region, and a membrane-proximal EGF-like module (EGFL) (Fig. 1a). Whereas ALKAL1 and ALKAL2 are both strong activators of LTK 4 , only ALKAL2 potently activates ALK 5,6 coupled to additional regulation via glycosaminoglycan binding to its HBD 15 .

A hypothalamic pathway for Augmentor α–controlled body weight regulation

“…Finally, we conclude that the Augα–Alk signaling cascade represents a neuronal signaling pathway controlling metabolic processes that was co-opted to induce a variety of human cancers by aberrant expression of activated Alk mutants. Moreover, the recent elucidation of the three-dimensional structure of free and Aug-occupied ligand-binding region of Alk provides important new insights about how Augα and Augβ bind to Alk and Ltk and stimulate receptor dimerization and activation ( 11 , 28 , 29 ). It is noteworthy that a molecular mechanism established for ligand-induced Alk activation ( 11 ) is consistent with the mode of action of Augα described in our manuscript.…”

Improving de novo Protein Binder Design with Deep Learning

“…The retrospective analysis in Fig 1 suggests incorporation of AF2 or RF2 into the design pipeline as a final evaluation filter could considerably increase the design success rate. To directly test this hypothesis, we carried out binder design campaigns on four new targets of considerable biological importance: ALK 6 , LTK 6 , IL10 receptor-ɑ 7 , and IL2 receptor-ɑ [8][9][10][11] (two sites). Using the protocol of Cao et al, we generated computational libraries of ~2 million designs for each target and filtered these down to ~20,000 designs to be experimentally tested for each target: ~15,000 designs using the physically-based filters of Cao et al and ~5,000 designs with AF2 pAE_interaction < 10 (these designs were also filtered by additional metrics as described in the Supplement).…”

The FUSION protein crystallization screen

“…Splitting the additive mixes into two different sets reduced the number of possible cross reactions, but some combinations of mixes were still prone to instability. The 'lanthanides' mix from MORPHEUS II, despite producing some exclusive crystal hits (De Munck et al, 2021;Modenutti et al, 2021), had to be excluded during preliminary tests because it led to precipitation or flocculation when combined with other mixes.…”