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Topological Control of Cytokine Receptor Signaling Induces Differential Effects in Hematopoiesis Kritika Mohan, George Ueda, Ah Ram Kim, Kevin M

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Topological Control of Cytokine Receptor Signaling Induces Differential Effects in Hematopoiesis Kritika Mohan, George Ueda, Ah Ram Kim, Kevin M RESEARCH ◥ control the relative orientation of the ECDs in RESEARCH ARTICLE SUMMARY the dimeric complex. The “angle” series varied the scissor angle between the two ECDs, whereas the “distance” series varied their relative proxim- PROTEIN ENGINEERING ity. The designed DARPin ligands were validated by means of x-ray crystallography for representa- tive complexes. The systematic variation of angular Topological control of cytokine and distance parameters generated a range of full, biased, and partial agonism of EpoR signaling receptor signaling induces differential ◥ in the human erythroid cell ON OUR WEBSITE line UT7/EPO, as shown with flow-cytometry and effects in hematopoiesis Read the full article at http://dx.doi. immunoblotting for phos- Kritika Mohan*, George Ueda*, Ah Ram Kim†, Kevin M. Jude†, Jorge A. Fallas†, org/10.1126/ phorylated downstream science.aav7532 effectors. In general, in- Yu Guo†, Maximillian Hafer, Yi Miao, Robert A. Saxton, Jacob Piehler, .................................................. Vijay G. Sankaran, David Baker‡, K. Christopher Garcia‡ creasing the angle or dis- tance between the receptor ECDs resulted in a progressive partial agonism, as measured with INTRODUCTION: Receptor dimerization is a system, a well-characterized dimeric cytokine changes in maximum response achieved (Emax) fundamental mechanism by which most cyto- receptor system. and median effective concentration (EC50). Biased kines and growth factors activate Type-I trans- signal transducer and activator of transcription Downloaded from membrane receptors. Although previous studies RESULTS: We used the DARPin (designed (STAT) activation was elicited by some of the sur- have shown that ligand-induced topological ankyrin repeat protein) scaffold because of rogate DARPin ligands. We also evaluated the changes in the extracellular domains effects of these DARPin agonists on dif- (ECDs) of dimeric receptors can affect ferentiation and proliferation of hemato- signaling output, the physiological poietic stem and progenitor cells (HSPCs) relevance is not well understood. This that were maturing into the erythroid http://science.sciencemag.org/ is a difficult problem to study because lineage. The partial agonists displayed an engineered ligand system does not stage-selective effects on HSPCs, whereas exist that would enable a systematic the biased agonists more selectively pro- exploration of the relationship between moted signaling at either the early or late ligand-receptor dimer geometry and stages of differentiation. signaling. This question, as well as the capability of exploiting these structure- CONCLUSION: We have designed a activity relationships for drug discovery, series of surrogate ligands to system- are important because most cytokines atically alter receptor dimer topology and modulate signaling outputs. An exert pleiotropic effects that limit their on May 28, 2019 therapeutic utility. New approaches are important feature of these ligands is needed to modulate cytokine signaling that the dimerized modules are rigidly and identify clinically efficacious var- connected, thus allowing us to deter- iants. By contrast, small-molecule ligands mine structures of the ligand-receptor for G protein–coupled receptors have complexes, or model them accurately, been successfully discovered through and correlate precise geometries with medicinal chemistry and used to in- signaling output. Some variants induce duce conformational changes that lead stage-selective differential signaling in to physiologically relevant biased primary cells. This “topological tuning” signaling outputs. A comparable may be attributed to altered intracellular approach for dimeric receptors could Three different EpoR/DARPin complex topologies are shown orientations of Janus kinase 2 (JAK2) open a path to new pharmacological that elicit full, partial, and null agonism. Full agonism, red; relative to its substrates or mechanical parameters for cytokines and growth partial, green; and null, blue. The EpoR/DARPin structure shown distortion that leads to changes in com- factors. on the green cell is from a crystal structure, whereas the plex stability and receptor internalization. complexes shown on the red and blue cells are models. Although our experiments do not reveal a RATIONALE: In order to better un- predictive framework to relate topology to derstand how the extracellular structure its modular nature. We isolated a high-affinity signaling, this approach can be used to empirically of cytokine-receptor complexes affects down- DARPin to EpoR using yeast display and in vitro identify specific dimer topologies that correlate stream signaling events, we developed an evolution and determined the crystal structure with therapeutically desirable signaling outputs engineered ligand system to precisely con- of the DARPin/EpoR complex. We then con- for any dimeric receptor system.▪ trol the orientation and proximity of dimeric verted these monomeric DARPin binding mod- receptor complexes that would enable the ules into C2 symmetric homodimeric agonists The list of author affiliations is available in the full article online. measurement of structure-activity relation- by incorporating in silico designed dimerization *These authors contributed equally to this work. ships between receptor dimer geometry, interfaces. This rigidly connected dimeric DARPin †These authors contributed equally to this work. signaling, and function. We applied this scaffold then enabled us to design a series of ‡Corresponding author. Email: [email protected] (D.B.); [email protected] (K.C.G.) approach to design geometrically controlled extended ligands through sequential insertion Cite this article as K. Mohan et al., Science 364, eaav7532 “ ” ligands to the erythropoietin receptor (EpoR) of ankyrin repeat spacers to systematically (2019). DOI: 10.1126/science.aav7532 IMAGE: K. C. GARCIA AND E. SMITH Mohan et al., Science 364, 750 (2019) 24 May 2019 1of1 RESEARCH ◥ chemistry (21). Finding small-molecule drugs is RESEARCH ARTICLE not currently feasible for Type-I transmembrane receptors that bind to protein ligands, such as cytokines, through large extracellular domains PROTEIN ENGINEERING (ECDs). Nevertheless, ligand engineering strat- egies that take advantage of the potential for biased signaling behavior by dimeric cytokine Topological control of cytokine receptors could be used as probes to better un- derstand cytokine receptor signaling biology but also may have therapeutic potential. receptor signaling induces differential The overall geometry of the cytokine receptor dimer can influence the strength of the down- effects in hematopoiesis stream signal (6, 22–24); however, approaches do not exist to precisely control the orientation and Kritika Mohan1*, George Ueda2,3*, Ah Ram Kim4,5,6†, Kevin M. Jude7†, proximity of ligand-receptor complexes. The ability Jorge A. Fallas2,3†, Yu Guo8†, Maximillian Hafer9, Yi Miao1, Robert A. Saxton1,7, to topologically control receptor dimerization Jacob Piehler9,10, Vijay G. Sankaran4,5,6, David Baker2,3,11‡, K. Christopher Garcia1,7,12‡ could open access to a new pharmacologic metric for assessing structure-activity relationships Although tunable signaling by G protein–coupled receptors can be exploited through medicinal of cytokine and growth factor receptor signal- chemistry, a comparable pharmacological approach has been lacking for the modulation of ing. Previous studies have shown that intact signaling through dimeric receptors, such as those for cytokines. We present a strategy to antibodies (25, 26) and diabodies can act as sur- modulate cytokine receptor signaling output by use of a series of designed C2-symmetric rogate cytokine agonists capable of modulating Downloaded from cytokine mimetics, based on the designed ankyrin repeat protein (DARPin) scaffold, that can signaling outputs through receptor dimerization systematically control erythropoietin receptor (EpoR) dimerization orientation and distance (24, 27), but their segmental flexibility prevents between monomers. We sampled a range of EpoR geometries by varying intermonomer angle control of the precise geometry or distance be- and distance, corroborated by several ligand-EpoR complex crystal structures. Across the range, tween monomers. we observed full, partial, and biased agonism as well as stage-selective effects on We integrated a previous protein-binding hematopoiesis. This surrogate ligand strategy opens access to pharmacological modulation of scaffold technology, termed DARPin (designed http://science.sciencemag.org/ therapeutically important cytokine and growth factor receptor systems. ankyrin repeat protein) (28), with protein design and engineering approaches to create a self- assembling, rigidly connected dimeric scaffold ytokines are secreted proteins that medi- propagation of a phosphorylation cascade lead- that enables precise control of the relative orien- ateawiderangeoffunctions,principallyin ing to signal transducer and activator of tran- tations of the receptor ECDs in a dimeric complex the immune system, but they also exert scription (STAT) activation and induction of gene (Fig. 1A). We focused on the erythropoietin recep- C many other homeostatic functions that expression programs (3, 5). Structural and func- tor (EpoR), which plays a critical role in erythroid affect mammalian physiology (1–3). As one tional studies of cytokine-receptor complexes lineage commitment and differentiation to pro- example, hematopoiesis, the process by which together with similar studies
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