Volume 60, Issue 6, Pages 941-952 (December 2015) Structural Basis of Neurohormone Perception by the Receptor Tyrosine Kinase Torso  Simon Jenni, Yogesh Goyal, Marcin von Grotthuss, Stanislav Y. Shvartsman, Daryl E. Klein  Molecular Cell  Volume 60, Issue 6, Pages 941-952 (December 2015) DOI: 10.1016/j.molcel.2015.10.026 Copyright © 2015 Elsevier Inc. Terms and Conditions

Molecular Cell 2015 60, 941-952DOI: (10.1016/j.molcel.2015.10.026) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 1 Structure of the mPTTH-sTorso Complex (A) Initial experimental electron density map after single anomalous diffraction (SAD) phasing and statistical density modification including 2-fold noncrystallographic symmetry (NCS) and multicrystal density averaging. The map was calculated to 4.5 Å resolution. Anomalous difference Fourier peaks are shown in green and orange for the tantalum and platinum 1 derivatives, respectively. The main chain of the final model is shown as black ribbon. (B) Final feature-enhanced electron density map at 2.7 Å resolution calculated with phenix.fem (Adams et al., 2010). (C) The heterotetramer complex. Molecule A of sTorso (right side) is colored blue (Domain I) and yellow (Domain II). The same domains of molecule B (left side) are colored light blue and khaki. Central dimer of mPTTH is colored green and lime. Modeled glycans are marked with asterisks. (D) sTorso is shown in surface representation, in two orthogonal views. The dimer of mPTTH from the complex is enlarged and boxed to the right. Strands of hairpins are labeled A–D for monomer A, and a–d for monomer B. Each cystine is labeled 1–7 to highlight disulfide pairing. See also Figure S1. Molecular Cell 2015 60, 941-952DOI: (10.1016/j.molcel.2015.10.026) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 2 sTorso’s Fold and Dimer Interactions (A) FN3 domains of sTorso are nearly orthogonal to each other. Cystines are numbered 1–8 in orange text. Disulfides are shown as sticks. Side chains involved in hydrophobic interactions between the N-terminal helix and the first domain are colored purple. The linker region between domains that could not be modeled (residues 156–170) is drawn as dashes. (B) N-terminal helix dimer interactions between receptor monomers. The view is from the perspective of the mPTTH dimer in Figure 1C. The Q43 label sits on the receptors’ 2-fold symmetry axis. Molecular Cell 2015 60, 941-952DOI: (10.1016/j.molcel.2015.10.026) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 3 mPTTH Binds Asymmetrically to the sTorso Dimer The 2-fold axes of the receptor and ligand dimers are shown in blue and green, respectively (center panel). Their point of intersection is shown as a black dot. The angle between these axes is ∼3.5°. A blue circle defines the plane of the membrane. Detailed interactions between the ligand and receptor are shown in four windows surrounding the center panel. Interactions observed in the complex with receptor A (right windows), but not maintained in the complex with receptor B (left windows) are labeled with red text. See also Figure S2. Molecular Cell 2015 60, 941-952DOI: (10.1016/j.molcel.2015.10.026) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 4 Analysis of Negative Cooperativity (A) SEC-MALS of mPTTH, sTorso, and different complex ratios. The molar mass determined by MALS is drawn as a heavy black line. Thin black dashed horizontal lines at 25, 35, 60, and 95 kDa identify the approximate expected masses for dimer mPTTH, monomer sTorso, heterotrimer, and heterotetramer, respectively. The vertical dashed lines mark the peak locations from the upper trace. (B) ITC of sTorso titrated into mPTTH. The isotherm is fit with an equation for two sequential binding events in the lower panel. (Inset) Thermodynamic parameters from the ITC experiment. First and second binding events are colored black and red, respectively. See also Figure S3. Molecular Cell 2015 60, 941-952DOI: (10.1016/j.molcel.2015.10.026) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 5 Structural Comparison to the IL-17R Complex (A) sTorso-mPTTH complex with sTorso molecule B removed for clarity. mPTTH is in surface representation with monomer B colored gray. Domain III of Torso, which is not included in our crystal construct, is shown as a red dashed oval. (B) The IL-17R complex (PDB 3JVF) aligned to strands of sTorso Domain II and colored as in (A) for clarity. (C) The mPTTH dimer shown as a cartoon and colored as in (A). (D) IL-17 dimer aligned manually to optimize cystine knot overlay with mPTTH. Molecular Cell 2015 60, 941-952DOI: (10.1016/j.molcel.2015.10.026) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 6 Torso’s Phylogenic Tree The Torso node of the protostomia RTKinome. Two main branches separate arthropod (lower) and nonarthropod (upper) sequences. The GenInfo Identifier (GI) number of each sequence is listed at the branch termini. Branches of related taxa are colored and labeled where appropriate. Complete sequences from known human parasites are labeled with an asterisk. See also Figures S4–S6. Molecular Cell 2015 60, 941-952DOI: (10.1016/j.molcel.2015.10.026) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 7 Model of Torso Activation (A) Schematic of the kinetic model for receptor-ligand interactions. L, R, C, and D represent ligand, receptor, complex, and dimeric receptor complex, respectively. VL and VR are the rates of ligand and receptor production, respectively; k1on, k2on, k1off, and k2off are binding rate constants; kRe is the receptor turnover rate; kCe and kDe are monomeric and dimeric endocytosis rates, respectively. (B) Kinetics of dimer concentrations for various combinations of receptor levels (R0/Kd1) labeled High, Medium, and Low and cooperativities (Kd2/Kd1). Kd1 is constant at 4 nM. (C) Heatmap showing the magnitude of dimensionless Mean Residence Time (MRT), defined as MRT∗kDe, for different receptor concentrations and cooperativities, as defined in (B). Receptor concentration as R0/Kd1 represents the extent and rate of the first binding step; values ≫1 represent a fast first binding step, and values higher than 1 can be used to study the second binding step. See also Table S2. Molecular Cell 2015 60, 941-952DOI: (10.1016/j.molcel.2015.10.026) Copyright © 2015 Elsevier Inc. Terms and Conditions