The Ligand-Induced Dimer of the dEGFR Extracellular Region is Asymmetric
The key to understanding negatively cooperative growth factor binding to the Drosophila
EGF receptor lies in the asymmetry of the Spitz-induced s-dEGFR dimer shown in . We crystallized a form of s-dEGFR bound to the EGF-like domain of Spitz (SpitzEGF
, encompassing residues 48–105), and determined its structure to 3.2 Å resolution (Table S1
). The overall domain architecture of s-dEGFR is very similar to that in human EGFR (Burgess et al., 2003
), with which it shares 39% sequence identity over domains I to IV. The ‘solenoid’ domains I and III both contact the same bound growth factor molecule in ligand-stabilized dimers of Drosophila
() and human () sEGFR. Domains II and IV are cysteine-rich laminin-related domains, and domain II harbors the ‘dimerization arm’ that drives core receptor • receptor contacts in both dimers. The complete dEGFR extracellular region contains an additional cysteine-rich domain of ~150 amino acids that is absent in hEGFR (domain V, which resembles domains II and IV). Previous small-angle X-ray scattering (SAXS) studies of s-dEGFR showed that domain V projects as a linear extension from the domain IV C-terminus (Alvarado et al., 2009
). Removing domain V (to yield s-dEGFRΔV) was essential for crystallization of the ligand • receptor complexes reported here.
An Asymmetric Ligand-Induced s-dEGFRΔV Dimer
Whereas ligand-bound dimers of the human EGFR extracellular region () are symmetric, the (SpitzEGF
dimer shows clear asymmetry (). This asymmetry is most evident at the dimer interface, close to the domain II amino-terminus (marked with an asterisk in ). Indeed, significant differences in the relationships between domains I, II, and III are seen when the two subunits from the s-dEGFRΔV dimer are overlaid in (the left-hand molecule from is colored green, and the right-hand molecule is red) – yielding an overall rms deviation in Cα positions of 3.4 Å. further shows that the right-hand molecule of the (SpitzEGF
complex (red) closely resembles the unligated s-dEGFRΔV structure (cyan) that we recently described (Alvarado et al., 2009
), overlaying with Cα position rms deviation of just 1.3 Å. Ligand binding has therefore not altered the domain I/III relationship in the right-hand molecule of . Direct interactions between these two ligand binding domains that stabilize the unligated conformation are retained – but remodeled in detail (Figure S1
). By contrast, upon binding to the left-hand subunit (green in ), SpitzEGF
‘wedges’ itself between the two ligand-binding domains, and pushes them apart as indicated by the double-headed arrow in to break the direct domain I/III interactions seen in unligated s-dEGFRΔV (Figures S1A and S1C
). Moreover, separating domains I and III with this ‘ligand wedge’ distorts domain II (which connects them) and forces a substantial reorientation of the dimerization arm (). By distorting domain II in only one of the two s-dEGFRΔV molecules in the dimer (the left-hand one), ligand binding induces the marked asymmetry seen in , and allows formation of a more extensive (asymmetric) dimer interface than would otherwise be possible. Indeed, as described in detail later, the asymmetric dimer interface in buries a total surface area of 3,396 Å2
, some 33% greater than the 2,553 Å2
buried between the two nearly-identical subunits of the symmetric human s-EGFR dimer (which overlay with a Cα position rms deviation of just 0.8 Å as shown in ).
Inequivalence of the Two Ligand-Binding Sites in the dEGFR Dimer Suggests Cooperativity
One important consequence of asymmetry in the (SpitzEGF)2 • (s-dEGFRΔV)2 dimer is that the two ligand-binding sites differ significantly from one another (), whereas the two binding sites in the human s-EGFRΔIV dimer are almost identical (). Differences between the two ligand-binding sites in the (SpitzEGF)2 • (s-dEGFRΔV)2 dimer are most apparent where SpitzEGF contacts domain I. shows the two s-dEGFRΔV molecules from overlaid using the bound ligand as reference (the left-hand molecule from is colored green, and the right-hand molecule red). By ‘wedging’ domains I and III apart, SpitzEGF has shifted domain I of the green molecule towards the top left corner of the figure by 3–5 Å compared with its position in the red s-dEGFRΔV molecule, and has displaced the N-terminal αhelix of domain I by ~7 Å along its axis (see green arrows in ). Because of this shift in domain I position, its interactions with SpitzEGF are significantly altered in detail when the red and green sites are compared (upper inset in ), although they involve similar sets of dEGFR residues.
The Ligand-Binding Sites in the (SpitzEGF)2• (s-dEGFRΔV)2 Dimer are Inequivalent
The domain III/SpitzEGF interface is less altered between the two binding sites (lower inset of ). The change in domain III position with respect to bound ligand is small, and is mostly compensated for by small adjustments in rotamer positions of interfacial side-chains. In a few cases, dEGFR side-chains appear to replace one another functionally in the two domain III/SpitzEGF interfaces. For example, in the interface between SpitzEGF and the green binding site, the H433 side-chain (underlined in ) assumes the position occupied by E460 in the red binding site. Similarly, E400 substitutes spatially for S401 when the SpitzEGF-binding surfaces of the green and red binding sites are compared (, lower inset).
These differences in the way that SpitzEGF
interacts with the two binding sites in the asymmetric s-dEGFRΔV dimer can also be thought of as a displacement of ligand on the domain I and III surfaces, as illustrated in Figure S2
. In this view, it is clear that the SpitzEGF
A-, B-, and C-loops all make significantly different contacts with the receptor in the two binding sites. Only the location of the SpitzEGF
C-terminus on domain III is fixed between the two sites (Figure S2B
), consistent with previous studies that point to the C-termini of other EGF-like ligands as major determinants of binding affinity (Groenen et al., 1994
As mentioned above, the red binding site in reflects s-dEGFRΔV in a conformation that is unchanged from the unligated receptor (), whereas forming the green binding site requires the ligand to wedge apart domains I and III. When SpitzEGF binds to the green (wedged open) site, a total surface area of 4,030 Å2 is buried, compared with just 3,730 Å2 in the red (unaltered) site. Moreover, binding to the green site involves four additional predicted hydrogen bonds between ligand and receptor (an increase of 16% over the red site). Thus, the green s-dEGFRΔV molecule shown in and (the left-hand subunit in ) has characteristics expected for a higher affinity site. By contrast, the red s-dEGFRΔV molecule in and (the right-hand subunit in ) appears to be restrained in an unligated-like conformation, which may in turn compromise ligand binding so that this site has a lower binding affinity.
Negatively Cooperative Ligand Binding can be Recapitulated with s-dEGFR
The structural inequivalence of the two SpitzEGF binding sites in and prompted us to ask whether distinct classes of binding site (or negative cooperativity) can be detected in studies of SpitzEGF association with the isolated dEGFR extracellular region. We linked s-dEGFR molecules via their flexible C-termini to a solid support to approximate their arrangement at the cell surface while allowing dimerization. An AviTag sequence was introduced (via a unstructured linker) at the s-dEGFR C-terminus to allow enzymatic biotinylation of the protein and its capture on the surface of streptavidin-coated 96 well plates (see Experimental Procedures). SpitzEGF was labeled with Alexa Fluor-488 to monitor its binding to surface-bound s-dEGFR. The representative binding curve in cannot be fit satisfactorily with a simple hyperbola, but fits well to the Hill equation (red curve) with a low Hill coefficient (nH) of 0.31 that suggests negative cooperativity (the mean nH value for all experiments was 0.38±0.07, with a microscopic dissociation constant of 49.5 nM). Transformation of these data into a Scatchard plot () also reveals characteristic concave-up curvature of the sort seen for human EGF binding to its intact receptor at the cell surface. Parallel experiments using a non-dimerizing s-dEGFR mutant confirm that this behavior requires s-dEGFR dimerization, and showed simple hyperbolic binding curves () and linear Scatchard plots () with a best-fit nH value of 1.02 (0.97±0.1 over all experiments) and a KD value of 0.92 μM.
SpitzEGF Binding to s-dEGFR Yields Curved Scatchard Plots
Our studies of SpitzEGF
binding to dimerization-competent s-dEGFR that has been purified to homogeneity are consistent with the negative cooperativity seen for human EGF binding to its intact cell surface receptor (Macdonald and Pike, 2008
; Wofsy et al., 1992
). Importantly, whereas isolating the human EGFR extracellular region abolishes Scatchard plot curvature (Lax et al., 1991
; Lemmon et al., 1997
; Livneh et al., 1986
; Odaka et al., 1997
), our results show that this is not the case for Drosophila
EGFR. Concave-up Scatchard plots do not prove negative cooperativity, however. Indeed, the curves in can alternatively be fit by assuming the superposition of two hyperbolae that correspond to distinct (and independent) classes of binding site – as has traditionally been done for EGF binding to its cell-surface receptor. In such a fit for , a high-affinity site (KD
~ 4.7nM) could account for ~65% of the saturated SpitzEGF
binding signal, and an independent lower-affinity class of sites (KD
~ 1.3μM) could account for the rest. It seems unlikely that molecular heterogeneity is responsible for the Scatchard plot curvature seen for s-dEGFR. Indeed, these experiments were performed with highly purified protein. Moreover, data in for dimerization-defective s-dEGFRdim-arm
argue that dimerization of the dEGFR extracellular region is required for Scatchard plot curvature. Taken together, these findings support the hypothesis that (as for EGF binding to hEGFR) the binding curves in represent negatively cooperative binding of SpitzEGF
to s-dEGFR dimers, as suggested independently by the features of the asymmetric (SpitzEGF
dimer structure (and binding-site inequivalence) discussed above. It is also important to note that, both for s-dEGFR in our studies () and for intact EGFR in cells (Macdonald and Pike, 2008
), dimerization is required for the appearance of high-affinity ligand binding and for the manifestation of negatively cooperative ligand binding.
Half-of-the-Sites Reactivity in a Spitz • s-dEGFRΔV Crystal Structure
Intriguingly, we also obtained evidence for half-of-the-sites reactivity – the extreme of negative cooperativity – in crystallographic studies of s-dEGFRΔV bound to a variant of SpitzEGF
) with a C-terminal truncation that reduces its affinity for the receptor by ~12-fold (Figure S3
). Crystals that diffracted to 3.4 Å grew from a 1:1.2 mixture of s-dEGFRΔV and SpitzEGFΔC
, and molecular replacement (MR) identified excellent solutions for two s-dEGFRΔV molecules in the asymmetric unit. One was found using unligated s-dEGFRΔV (or the right-hand molecule in ) as the search model. The other could only be found in MR searches using the left-hand s-dEGFRΔV molecule from in which domains I and III are wedged apart. Unfortunately, a marked anisotropy in all datasets made full refinement of this structure impossible. We therefore used only rigid-body refinement, treating each domain of the two s-dEGFRΔV molecules as an independent body (see Experimental Procedures). For domains I and III, this seems well justified by the absence of ligand-induced structural changes in the individual domains of dEGFR or other ErbB receptors (Ferguson, 2008
). For domains II and IV, major structural changes may be missed with this approach – but we do not expect them.
The rigid-body refined structure of the s-dEGFRΔV/SpitzEGFΔC
complex () suggests a dimer with the same asymmetric arrangement of receptor molecules as seen in the (SpitzEGF
dimer discussed above, despite the fact that the crystal packing is quite different in the two cases. Most importantly, whereas one receptor molecule showed clear electron density for bound ligand in 2Fo
maps (), the other showed none () – even at very low contour levels. The absence of ligand from this second site cannot be explained by competing crystallization contacts. Thus, SpitzEGFΔC
appears to induce the formation of a singly-ligated asymmetric s-dEGFRΔV dimer in these crystals, depicted in . The left-hand (ligated) molecule in has the same conformation as the left-hand molecule in the (SpitzEGF
dimer in , with domains I and III wedged apart by the bound SpitzEGFΔC
. The right-hand molecule in has the same conformation as unligated s-dEGFRΔV, and its binding site is empty (or at least has very low occupancy). In parallel studies with Vein, a weak dEGFR activator (Schnepp et al., 1998
) with binding affinity even lower than that of SpitzEGFΔC
(data not shown), we determined a 3.4 Å rigid body-refined structure that also shows a singly-occupied asymmetric dimer. These apparently singly-ligated s-dEGFRΔV dimers suggest half-of-the-sites reactivity, where binding of SpitzEGFΔC
) to one site in the s-dEGFRΔV dimer prevents (or greatly impairs) ligand binding to the second site.
Half-of-the-Sites Reactivity in s-dEGFRΔV
Remodeling of the s-dEGFR Dimer Interface upon Ligand Binding
Unlike its human counterpart, the dEGFR extracellular region dimerizes even in the absence of ligand, albeit weakly (KD
~40 μM). Moreover, unligated s-dEGFRΔV crystallizes as a symmetric (crystallographic) dimer, illustrated in (Alvarado et al., 2009
). The interface of this symmetric dimer involves only the domain II dimerization arm, and the N-terminal parts of domain II are splayed apart (). The total surface area buried in the unligated dimer interface is just 2,262 Å2
Ligand Binding Promotes an Extensive Asymmetric Dimerization Interface
Binding of SpitzEGF
) enhances s-dEGFR dimerization by approximately 30-fold (Alvarado et al., 2009
), associated with an increase of more than 50% in the surface area buried at the dimer interface (to 3,396 Å2
). This large expansion of the interface arises primarily from direct contacts between the domain II amino-terminal regions that are seen only in the asymmetric ligand-induced dimer (). In binding to the left-hand molecule in , SpitzEGF
(magenta) has wedged itself between domain I (blue) and domain III (yellow), causing domain II (green) to become distorted or ‘bent’. The amino-terminal part of domain II in the left-hand molecule effectively ‘collapses’ onto its unaltered counterpart (grey) in the right-hand molecule, creating an additional set of intimate interfacial contacts (). The equivalent domain II regions are splayed apart in the symmetric unligated s-dEGFRΔV dimer (), presumably restrained by direct interactions between domains I and III of the receptor (Figure S1
) that we previously suggested may play an autoinhibitory role (Alvarado et al., 2009
). The additional >1,000 Å2
of surface area buried in the asymmetric dimer is likely to account for the 30-fold (~ 2 kcal/mole) increase in dimerization affinity observed upon ligand binding.
Formation of the more extensive asymmetric dimer interface seen in arises largely from a ligand-induced kink (of ~12 ) between modules m4 and m5 of domain II (marked with an arrow in ). Modules m2, m3 and m4 from domain II of the left-hand molecule (green) dock onto the domain II surface (grey) of the neighboring molecule in the dimer, without substantially altering the dimerization arm contacts (mediated by module m5). Side-chains from Q189 and R201 (in module m2), plus H205 (in module m3) of the ligand-bound receptor molecule make polar contacts across the dimer interface. Several additional side-chains, including those from P188 and P200 (from module m2) plus L206 and F207 (from module m3) also make van der Waal’s contacts with the opposing domain II in . Modules m2, m3, and m4 bury a combined surface of 1,160 Å2 in the asymmetric s-dEGFRΔV dimer interface (34% of the total interface), allowing an intimate set of receptor • receptor contacts to extend along much of the length of domain II in this dimer. Interestingly, Q189, P200, and H205 are conserved in hEGFR and human ErbB3. Q189 is also conserved in hErbB4, P200 in ErbB2, and H205 in hErbB2 and hErbB4. R201, L206 and F207 are not conserved in the human receptors.
Whereas the domain II amino-termini make intimate contacts across the interface of the (SpitzEGF
dimer, they contribute little to receptor • receptor contacts in the symmetric human sEGFR dimer (), burying just 476 Å2
(with no contribution from m4). Dimerization arm-mediated interactions are very similar in both Drosophila
and human sEGFR dimers (Garrett et al., 2002
; Ogiso et al., 2002
). However, additional differences between dEGFR and hEGFR are seen for interactions involving the carboxy-terminal part of domain II (modules m7 and m8). These modules make no direct contact across the interface in ligated human sEGFR dimers, whereas in s-dEGFRΔV they interact more extensively in the unligated dimer than in the ligand-induced dimer. As a result (and because of changes in the domain II/III relationship), ligand binding actually increases the distance separating the two copies of domain IV in the s-dEGFRΔV dimer by approximately 24 Å (); i.e.
ligand binding actually appears to drive apart domains IV of the two receptor molecules in the transition from a putative ‘pre-formed dimer’ () to a ligand-activated form ().
A Structural Model for Negative Cooperativity in an EGFR Extracellular Region
Levitzki et al. (1971)
pointed out four possible sources for half-of-the-sites reactivity in dimeric enzymes. In the first, the two ligand-binding sites are adjacent such that occupation of one site directly occludes the second. This cannot explain negative cooperativity in EGFR, where the two binding sites in the dimer are more than 50 Å apart. Two other models require non-identical binding sites in unligated dimers that arise either from asymmetric dimerization without ligand or from the existence of distinct subunit classes – neither of which are relevant for dEGFR. The fourth and final model involves a symmetric unligated dimer with two identical binding sites. Ligand binding to one of these sites induces conformational changes that promote asymmetry in the dimer, and restrain the vacant binding site so that its affinity for ligand is reduced. A model of this sort may explain the ligand-binding properties of dEGFR, as illustrated by the gallery of structures presented in .
Model for Negatively Cooperative Ligand Binding to s-dEGFR
Before interacting with SpitzEGF, the ligand-binding sites are identical in crystallographic pre-formed dimers, and presumably monomers, of s-dEGFR (). Ligand may bind to either species. In either case, the first (highest affinity) binding event yields the singly-ligated, asymmetric SpitzEGF • (s-dEGFRΔV)2 dimer shown in . High-affinity ligand binding appears to require receptor dimerization, since the s-dEGFRdim-arm mutant shows only low affinity SpitzEGF binding (). Indeed, formation of the asymmetric interface in will facilitate domain II bending in the left hand molecule, in turn promoting the wedging apart of domains I and III by the first ligand that binds – and enhancing its affinity.
In the asymmetric s-dEGFRΔV dimer formed after the first (high-affinity) binding event (), domain II in the unoccupied receptor is subjected to a new set of structural restraints. It can no longer ‘bend’ to allow SpitzEGF to wedge itself fully into the unoccupied ligand-binding site without disrupting the intimate asymmetric interface between amino-terminal parts of domain II shown in . This interface therefore restricts binding of ligand to the right-hand (unligated) receptor in . When a second ligand molecule does bind to the empty site in this dimer, two scenarios (at the extremes) are possible:
- The asymmetric dimerization interface seen in may be left intact. With the conformation of domain II fixed in the right-hand molecule so that it cannot bend, it will not be possible for a ligand to wedge domains I and III apart upon binding. This restriction will necessitate binding of ligand to an unaltered (suboptimal) site – as seen for SpitzEGF binding to the red binding site in . This would explain the reduced binding affinity of the second (right-hand) site in the s-dEGFR dimer.
- Alternatively, ligand binding to the second site in the dimer could wedge domains I and III apart exactly as in the first ligand binding event. The resulting domain II distortion would break the ‘extra’ contacts in the asymmetric domain II • domain II interface shown in , effectively ‘resymmetrizing’ the dimer. The work required to disrupt the asymmetric domain II • domain II interface will reduce the effective affinity of the second site for ligand.
The first of these possibilities is likely to explain why asymmetry is maintained in the (SpitzEGF)2 • (s-dEGFRΔV)2 dimer even after binding of the second ligand (). The energetic cost of disrupting the asymmetric dimer interface in presumably outweighs the gain in ligand • receptor interactions that can be achieved by wedging apart domains I and III to optimize the second binding site. The second SpitzEGF molecule therefore binds without altering the s-dEGFRΔV structure, and occupies a compromised binding site (red in ) with reduced contact area and fewer predicted hydrogen bonds (and therefore lower affinity). The ~12-fold reduced receptor-binding affinity of SpitzEGFΔC appears to prevent this ligand variant from occupying this compromised binding site altogether in our crystals, explaining the half-of-the-sites reactivity seen in the SpitzEGFΔC • (s-dEGFRΔV)2 dimer ().
The second of the possibilities outlined above is likely to explain the symmetry of the fully occupied human s-EGFRΔIV dimer illustrated in (Ogiso et al., 2002
). If domain II-mediated interactions in the asymmetric (singly-ligated) dimer are weaker in human EGFR than in Drosophila
, they will be disrupted more readily by binding of a second ligand molecule. There are several reasons to suspect that these interactions are indeed weaker in human EGFR than in its Drosophila
counterpart. Whereas the isolated extracellular region of dEGFR retains negatively cooperative ligand binding, contributions from the intracellular region are essential in the case of human EGFR (Livneh et al., 1986
; Macdonald-Obermann and Pike, 2009
). This argues that cytoplasmic domain interactions are required to stabilize the singly-ligated intact human EGFR dimers required for negatively cooperative EGF binding. Unlike its Drosophila
counterpart, isolated s-hEGFR does not form singly-ligated dimers in solution and does not exhibit negatively cooperative ligand binding (Lemmon et al., 1997
; Odaka et al., 1997
). However, negative cooperativity can be recapitulated in solution by fusing s-hEGFRΔIV to a dimeric IgG Fc domain (Adams et al., 2009
) – as also described for the insulin receptor (Bass et al., 1996
; Hoyne et al., 2000
). Studies of such artificial dimers may be needed in order to examine structural details of the singly-ligated hEGFR dimer inferred from cellular studies. It is also important to note that the intracellular regions of human EGFR and ErbB4 form asymmetric dimers (Jura et al., 2009
; Qiu et al., 2008
; Red Brewer et al., 2009
), which may contribute to the stabilization of asymmetric, singly-ligated dimers of the intact receptors. Residues in this intracellular dimer interface are not conserved in dEGFR, consistent with an increased reliance on extracellular interactions for negative cooperativity in Drosophila
Implications for the High- and Low-Affinity Binding Sites for Human EGF
Our studies suggest that the proposed ‘high-affinity’ and ‘low-affinity’ classes of EGF binding site at the cell surface do not reflect distinct EGFR populations. Rather, the characteristic curved Scatchard plots reflect negatively cooperative EGF binding to a single type of receptor species. In the binding scheme illustrated in , the first binding event – leading to the singly-ligated dimer in – could be considered as the ‘high-affinity’ site, and the second (leading to ) the ‘low-affinity’ site. Restraints imposed on the second binding site in an asymmetric, singly-ligated dimer can explain negatively cooperative ligand binding in a model that closely resembles mechanisms of negative cooperativity and half-of-the-sites reactivity reported for other multisubunit enzyme systems (Koshland, 1996
; Levitzki et al., 1971
We suggest that studies of the isolated human EGFR extracellular region have failed to recapitulate key receptor-receptor interactions required for its allosteric regulation. Intact human EGFR is reported to self-associate to some extent even in the absence of ligand (Chung et al., 2010
; Saffarian et al., 2007
), and is thought to form singly-ligated, asymmetric dimers required for negatively cooperative ligand binding at the cell surface (Macdonald and Pike, 2008
). Both of these properties are lost when the human EGFR extracellular region is studied in isolation (Lemmon et al., 1997
; Odaka et al., 1997
). A similar problem exists for the insulin receptor, where negative cooperativity in insulin binding is completely abolished when the extracellular region of the receptor is released from the membrane surface (De Meyts and Whittaker, 2002
). By contrast, the isolated extracellular region of the Drosophila
EGFR appears to maintain the self-association and allosteric properties of the intact receptor, allowing our studies of s-dEGFR to provide serendipitous insight into the mechanism of its allosteric regulation and structural basis for negative cooperativity.
Conclusions and Perspectives
The origin of concave-up Scatchard plots seen for EGF binding to its cell surface receptor over the past three decades has been contentious. The molecular nature of the ‘high-affinity’ and ‘low-affinity’ EGF-binding sites suggested by these curves has also been a subject of significant debate, although recent work suggests that they reflect negative cooperativity rather than distinct classes of site (Macdonald and Pike, 2008
). The studies described here provide a structural basis for understanding negative cooperativity in ligand binding to an EGF receptor. Our analysis suggests that high- and low-affinity binding sites for ligand do exist, but that they both occur in the same dimeric receptor complexes and arise from negative cooperativity rather than from distinct populations or ‘classes’ of receptor.
If the curved Scatchard plots seen in studies of cell surface EGF binding reflect negative cooperativity, how can apparent functional and structural differences between the presumed high-affinity and low-affinity classes of EGF receptors be explained? Early studies with antibodies reported to interfere only with high-affinity or low-affinity sites respectively concluded that the high-affinity subclass is necessary for early signaling responses to EGF (Bellot et al., 1990
; Defize et al., 1989
). Moreover, the route of EGFR internalization from the cell surface depends on the concentration of ligand used to activate the receptor, suggesting that the high-affinity and low-affinity classes of receptor may be subjected to different endocytic mechanisms (Sorkin and Goh, 2009
). At low EGF concentrations, EGFR internalization is primarily clathrin-mediated, whereas clathrin-independent mechanisms appear to dominate when very high EGF concentrations are applied. The negative cooperativity model of Macdonald and Pike (2008)
suggests that binding of the second ligand to an EGFR dimer reduces the affinity of the two receptors for one another by ~10-fold. Indeed, our s-dEGFR structures show how the second ligand-binding event must either compromise ligand/receptor or receptor/receptor contacts (). A weakened, doubly occupied, EGFR dimer could behave quite differently from one with only one site occupied – with altered dynamics and interaction (and dimer exchange) properties that might alter specificity, degree of autophosphorylation, mechanism of internalization, and other outcomes.
Differences in the signaling properties of singly- and doubly- occupied receptors may also explain the distinct biological outcomes when cells are treated with different agonists for the same ErbB receptor (Wilson et al., 2009
), or different concentrations of ligand. For example, EGFR agonists with low receptor-binding affinities (amphiregulin, epiregulin and epigen) might induce the formation of only (or primarily) singly-ligated dimers, whereas EGFR agonists with high affinity for the receptor (EGF, TGFα, betacellulin and HB-EGF) should be able to occupy both binding sites in the receptor dimer if present at sufficiently high concentrations. As a result, the receptor may be activated (and internalized – and ultimately deactivated) differently in response to the two ligand classes. A difference in signaling outcomes of this sort may be very important where EGFR ligands function as morphogens. Gradients of ligands for the Drosophila
EGFR (Spitz, Keren, Gurken, and Vein) function in tissue patterning in many developmental programs in D. melanogaster
). It is not clear how different concentrations of these ligands in morphogen gradients can induce different cell fates, which is crucial for ‘interpreting’ the gradients. Our work suggests one possibility. At the gradient peak, where ligand concentrations are high, dEGFR dimers will be fully occupied (with 2 ligands bound per dimer). By contrast, at the tail end of the morphogen gradient where ligand concentrations are low, only high-affinity sites will be occupied in the receptor dimers – yielding singly-ligated dEGFR dimers. If the doubly- and singly-ligated dimers have different signaling properties and internalization routes, distinct cell fates could be induced in the two regimes of receptor activation. This graded occupation of the two binding sites in the EGFR dimer would be substantially lessened in a non-cooperative system.