1. Volume 11, Issue 2, Pages 507-517 (February 2003)
EGF Activates Its Receptor by Removing Interactions that Autoinhibit Ectodomain Dimerization 
Kathryn M Ferguson, Mitchell B Berger, Jeannine M Mendrola, Hyun-Soo Cho, Daniel J Leahy, Mark A Lemmon 
Molecular Cell 
Volume 11, Issue 2, Pages (February 2003)
DOI: /S (03)

2. Figure 1 Structure of the Autoinhibited sEGFR Monomer
(A) Ribbon representation of sEGFR, with domain I (L1) colored blue, domain II (S1) green, domain III (L2) yellow, and domain IV (S2) red. N and C termini are labeled. Disulfide bonds in domains II and IV that constitute C1 disulfide-bonded modules are black, while those in C2 modules are gray. Individual C1 and C2 modules are labeled in domain IV as described in the text. Disulfide bonds in domains I and III are colored yellow. The domain I-bound EGF molecule (see Figure 2) has been omitted for clarity.
(B) Detail of the region boxed in (A), showing autoinhibitory intramolecular interactions between the second C1 module of domain II (C1IIb) and the C1IVc and C1IVd modules of domain IV. Parts of domains II and IV shown have green and red surfaces, respectively, with backbones represented by green and red worms. Interdomain hydrogen bonds are drawn as white dashed lines.
Molecular Cell  , DOI: ( /S (03) )

3. Figure 2 Space-Filling Representation of sEGFR with Domain I-Bound EGF
sEGFR is shown in CPK representation, with subdomains colored as in Figure 1. Bound EGF is magenta. EGF is intimately associated with domain I (blue) but does not come closer than 4 Å away from domain III. The side chain of Y14 (projecting from the left-hand side of EGF) is too distant from domain III to participate in hydrogen bonding.
Molecular Cell  , DOI: ( /S (03) )

4. Figure 3 Binding of sEGFR to EGF Is Reduced at Low pH
Binding of sEGFR (at 150 nM) to immobilized EGF was measured at different pH values using BIAcore as described in the Experimental Procedures. The midpoint of this titration is at approximately pH 7.3.
Molecular Cell  , DOI: ( /S (03) )

5. Figure 4
Model for EGF-Promoted Domain Rearrangement in sEGFR, Leading to Dimerization
The domain rearrangement promoted by bivalent binding of EGF to both domains I and III was approximated as described in the text and is depicted with ribbon models and cartoon forms. Modeled structures (B–D) are shown on a gray background.
(A) sEGFR, in the same orientation and with the same coloring as in Figure 1A. Domain I-bound EGF is colored magenta. Key residues in EGF that must be docked onto domain III in the dimer (R41 and L47) are surrounded by an ellipse labeled “iii”. A hydrophobic pocket on domain III, into which L47 projects in the EGF•sEGFR dimer (Garrett et al., 2002; Ogiso et al., 2002), is circled and marked “i”. The side chain of D355 on domain III, which hydrogen bonds with R41 of EGF in the dimer, is also circled and marked “ii”. Domain I-bound EGF can be correctly docked on to domain III by 130° anticlockwise rotation of the domain I/II rigid body (plus EGF) about the axis marked by a filled black circle, plus a translation along this axis (into the page) of 20 Å.
(B) This domain rearrangement allows EGF binding to both domains I and III of an extended sEGFR molecule in which the domain II dimerization loop is exposed (and faces out of the page in this view). We have not attempted to model the polypeptide in the region of the rotation axis.
(C) The entire model for extended sEGFR has been rotated 90° about the vertical axis marked (from its orientation in [B]), to provide a view in which exposure of the domain II dimerization arm (C1IIb loop) and domain IV C1IVd loop (marked with an asterisk) can be appreciated.
(D) The extended sEGFR molecule, with EGF bound to domains I and III, dimerizes through domain II-mediated interactions, with possible additional contributions from domain IV (Berezov et al., 2002; Schlessinger, 2002). The dimer model shown uses the coordinates of the EGF•sEGFR dimer from Ogiso et al. (2002), to which we have added domain IV (by maintaining the domains III/IV relationship seen in [A]). While domains I, III, and IV are unaltered in structure and orientation in going from (C) to (D), the conformation of domain II is altered significantly (see text). For clarity, domain II on the right-hand sEGFR molecule in the dimer is colored black.
Molecular Cell  , DOI: ( /S (03) )

6. Figure 5
Mutations that Disrupt Intramolecular Domain II/IV Interactions Increase EGF Binding Affinity
EGF binding to wild-type and mutated sEGFR was measured using surface plasmon resonance (BIAcore). As reported previously (Ferguson et al., 2000), wild-type sEGFR (black curve) binds to EGF with a KD of 132 ± 9.9 nM. Mutation of K585 to alanine increased binding affinity to give a KD of 54.3 ± 8.3 nM (blue curve), mutation of both D563 and H566 to alanine gave a KD of 58.8 ± 5.5nM (green curve), and a triple D563A/H566A/K585A mutant binds with KD = 50.7 ± 6.3 nM (red curve). Mean results from at least three independent experiments are plotted, with errors shown as standard deviations.
Molecular Cell  , DOI: ( /S (03) )

7. Figure 6
Different Domain Orientations in Autoinhibited Structures of sEGFR and sErbB3
The structures of autoinhibited sEGFR and sErbB3 (Cho and Leahy, 2002) were overlaid by superimposition of domains III and IV.
(A) The sEGFR/sErbB3 overlay is shown in the orientation used for sEGFR in Figure 1A. sEGFR is gray; sErbB3 is magenta.
(B) The same sEGFR/sErbB3 overlay is shown in an orthogonal view to illustrate the different orientation of the domain I/II fragment in the two structures. The difference is approximated by a 60° rotation about the axis marked with a black circle.
Molecular Cell  , DOI: ( /S (03) )

8. Figure 7 Crystallographic Dimer of Inactive sEGFR
(A) Stereo view of the crystallographic dimer. One protomer (shaded gray, with domain IV black) is behind, while the other (with domains colored as in Figure 1A) is in front. EGF is colored magenta in both protomers. N and C termini and individual domains are all labeled. Three domain II side chains are shown: Y246, which is involved in the intramolecular domain II/IV interaction, plus N247 and D254, which make hydrogen bonds to domain IV of the adjacent molecule in this dimer (involving the carbonyl oxygen of N528 and amide nitrogen of V526, respectively, shown in ball and stick representation). If this dimer exists in vivo, the plane of the membrane would pass under the structure, perpendicular to the page.
(B) The dimer shown in (A) has been rotated by 90° about the horizontal axis shown to give a view facing up from the presumed membrane plane. The C termini project out of the page. Regions of intermolecular interaction between N247 and D256 on domain II and the backbone of domain IV are circled.
Molecular Cell  , DOI: ( /S (03) )