Review Binding in the Growth Hormone Receptor Complex James A

Home , Growth hormone receptor, Hormone receptor

Proc. Natl. Acad. Sci. USA Vol. 93, pp. 1-6, January 1996 Review Binding in the growth hormone receptor complex James A. Wells Department of Protein Engineering, Genentech, Inc., 460 Point San Bruno Boulevard, South San Francisco, CA 94080

ABSTRACT Binding reactions be- sons. First, the pharmacology of hGH is alents of hGHbp (28 kDa) ran as a single tween human growth hormone (hGH) and rather complex (3). For example it can peak (-75 kDa) with no evidence of ex- its receptor provide a detailed account of bind and activate at least two different cess free components. SDS/PAGE and how a polypeptide hormone activates its cloned receptors, the hGH (4) and pro- densitometry of the 75-kDa peak showed receptor and more generally how proteins lactin receptors (5). Moreover, hGH is a it had a composition ofone hGH molecule interact. Through high-resolution struc- member of the cytokine receptor super- per two hGHbp molecules. Furthermore, tural and functional studies it is seen that family (6), and thus an understanding of crystals of the complex were obtained and hGH uses two different sites (site 1 and how it binds its receptor may shed light on when dissociated gave a composition of site 2) to bind two identical receptor mol- the entire family. Finally, it is hoped that one hGH molecule per two hGHbp molecules. This sequential dimerization reac- through a detailed understanding of the ecules (10, 13). tion activates the receptor, presumably by structure and chemistry involved in the Two mutagenesis strategies, homolog- bringing the intracellular domains into binding reaction, one would be in a better scanning (11) and alanine-scanning (12), close proximity so they may activate cy- position to rationally design small mole- were employed to define binding determi- tosolic components. As a consequence of cules that could mimic the large interfaces nants in the hGH and the hGHbp (for this mechanism it is possible to build that are typical of protein-protein com- review see ref. 14). Initially, these studies antagonists to the receptor by introducing plexes. were performed without the aid of the mutations in hGH that block binding at structures of the complex or of the indi- site 2 and to build even more potent Basic Methods and Approaches vidual components. In homolog-scanning, antagonists by combining these with mu- segments (7-30 residues long) derived tants that enhance binding at site 1. Ala- Any detailed structural and functional from nonbinding homologs, such as pro- nine-scanning mutagenesis of all contact analysis is greatly facilitated by having an lactin or porcine growth hormone, were residues at the site 1 interface shows that abundant and recombinant source of the substituted into hGH. From the set of only a small and complementary set of component molecules. We were particu- segment-substituted molecules that dis- side chains clustered near the center of larly fortunate to have Escherichia coli rupted binding affinity, we could infer the interface affects binding. The most secretion systems for both hGH (7) and regions of the hormone that may contain important contacts are hydrophobic, and the hGHbp (8). These expression systems binding determinants. These regions were these are surrounded by polar and allowed rapid and high-level production of then subjected to alanine-scanning mu- charged interactions of lesser impor- these proteins in forms that interacted tagenesis in which each residue within the tance. Kinetic analysis shows for the most with virtually the same affinity as those disruptive segment was replaced by ala- part that the important side chains func- from natural or recombinant mammalian nine and the consequences for binding tion to maintain the complex, not to guide sources (Kd 0.3 nM). affinity were measured. In this way the the hormone to the receptor. Hormone- Determining the stoichiometry of the role of side-chain atoms beyond the /3-car- induced homodimerization or het- complex is fundamental to characterizing bon could be assessed. A panel of mono- erodimerization reactions are turning out any binding reaction. Initially, we pre- clonal antibodies that reacted with the to be pervasive mechanisms for signal sumed that growth hormone bound to native components, but not the unfolded transduction. Moreover, the molecular only one receptor in solution, because forms, were used to verify that these mu- recognition principles seen in the hGH- Scatchard analysis from an RIA showed a tations did not affect the overall fold ofthe receptor complex are likely to generalize 1:1 stoichiometry. As it turned out, how- protein. to other protein-protein complexes. ever, the receptor antibody (MAb5, ref. 9) Two different assays were used to de- used to precipitate the hGHhGHbp com- termine the effects of hGH mutants on How do hormones find their receptors? plex sterically excluded the second recep- binding. Using MAb5 to precipitate 1:1 Once there, what forces allow the hor- tor molecule from binding (10). This was complexes, we found a patch of mutations mone to bind? How does hormone bind- a blessing in disguise because more com- that disrupted binding to a region we ing lead to receptor activation? Most plex stoichiometries (such as the 1:2 stoi- called site 1. The second assay followed biological processes are regulated or chiometry that was later discovered) the hGH-induced dimerization of hGHbp structured by these reactions involving would have made interpretation more dif- molecules in solution by the quenching of noncovalent associations. Thus, an under- ficult than was the case for the single inter- a fluorescent tag placed near the C terminus standing of how hormones bind their re- face that was initially discovered (11, 12). of the hGHbp (10). The patch of alanine ceptors is broadly relevant to many other A variety of biophysical methods were mutations outside the site 1 patch that had molecular recognition events in biology. used to determine that the stoichiometry reduced affinity in this assaywe called site 2. This minireview will begin to address of the hGH-hGHbp complex in solution The solution of the x-ray structure of these questions from the perspective of a was 1:2 (10). One of the most powerful the 1:2 hGH-hGHbp complex (15) per- complex between human growth hormone was titration calorimetry, which showed (hGH) and the extracellular domain of its that the binding reaction was complete Abbreviations: hGH, human growth hormone; 1 was hGHbp, the extracellular domain of the hGH receptor, called the hGHbp (for general when equivalent of hormone added receptor; variant proteins are indicated by the reviews see refs. 1 and 2). This complex to 2 equivalents of the hGHbp. Gel filtra- single-letter code for the wild-type residue fol- has been intensively studied by mutational tion showed that mixtures containing 1 lowed by its position in the mature protein and structural methods for several rea- equivalent of hGH (22 kDa) plus 2 equiv- sequence and the substituted residue. 1 Downloaded by guest on September 26, 2021 2 Review: Wells ~~~~~~~~~~~~Proc.Natl. Acad. Sci. USA 93 (1996)

lized This causes a in A + B .A*B (Ka =kon/koff) [1] hGHbp. change data to be interpreted in structural con- kThfaff refractive index of the medium which is text for the first time. This was an enor- detected by a change in surface plasmon What role do contact side chains in mous advance. Not only did it reveal the play resonance of the gold film (called reso- on-rate or 1:2 natu're of the complex and the struc- determining (kon) off-rate nance units or RU). The stoichiometry of

tures of the bound components, it iden- (koff)? To address this question we focused binding can be calculated from the total tified all the contact residues at the two on the binding of hGH via site to the first change in RU, as this is related to the mass interfaces as well as hormone-receptor bound receptor. bound to the chip. On-rates are measured that the contact each showing receptors To study the kinetics of binding we used by the rate of change in RU as a function other (Fig. 1). a BlAcore (Pharmacia) (16). This device of hGH concentration. Off-rates are de-

has a flow cell with a film on Side Chains on the hGH Mostly Affect gold layered termined from rate of the decrease in RU Dissociation, Not Association the outside. Attached to the gold film is a upon release of hGH from a saturated layer of dextran fibers which extend into chip.

Binding affinity can be considered a sim- the flow cell to which we covalently at- To study the binding of hGH at site 1 ple balance of two reactions, association tached the hGHbp (17). As hGH flows without the complication of forming 1:2 and dissociation (Eq. 1). through the cell, it binds to the immobi- complexes, or binding through site 2, we

FIG. 1. Structural model of the 1:2 hGH-hGHbp complex taken from ref 15. hGH is shown in red, while the hGHbpl and hGHbp2 are shown in green and blue, respectively. The membrane bilayer is modeled below. Reprinted with permission from Science 255,257. Copyright 1992 American Association for the Advancement of Science. Downloaded by guest on September 26, 2021 Review: Wells Proc. Natl. Acad. Sci. USA 93 (1996) 3

prepared a receptor that could bind hGH tly influence the rate of association of the The hGH Receptor Is Activated by only at site 1 (17). This was done by hGH to the receptor, association is con- Sequential Dimerization introducing a mutation in the receptor trolled by diffusion, which is independent stem region (S201C) and fixing this by a of the side-chain composition of the in- Receptor oligomerization is a common, if disulfide linkage to the flow chip. Not only terface. Thus, the hormone finds its re- not pervasive, means by which extracellu- did this prevent receptor dimerization, it ceptor mostly by a random but rapid col- lar hormones transmit their signals to the also presented the hGHbp in a uniform lision process; the side chains function to inside of a cell without ever passing fashion to the hormone. Using this con- keep it bound once it has reached its through the membrane (for reviews see struction, we determined a stoichiometry receptor target. refs. 21-23). By a hormone simply bring- of binding of hGH-hGHbp of 0.84, a ko, of ing together two or more transmembrane 3 x 105 s-I.M-1, and a koff of 2.7 x 10-4 Binding Affinity Is Maintained by a membrane receptors, the intracellular do- M- 1. These values were virtually the same Small Cluster of Contact Residues mains of these can be juxtaposed so that for a variant of hGH (G120R) which is they may interact with other cellular com- blocked in binding to a second receptor by From the ratio of on- and off-rates, we ponents or catalyze a reaction (typically a mutation in site 2, indicating that the calculated the effects of the contact side protein phosphorylation). measured kinetic parameters reflected chains in site 1 on affinity. From this it The most striking aspect of the hGH- binding at site 1. Moreover, the affinity became clear that only a small set of the receptor complex is that one hormone constant was in close agreement with that buried side chains were necessary for tight binds and dimerizes two receptors. Muta- determined by RIA using MAbS. binding affinity. In fact, alanine substitu- tional and biophysical studies (10, 24) The side chains of 31 residues of hGH tions at only 8 of the 31 positions (K41, have shown that these sites do not react become buried to various degrees upon L45, P61, R64, K172, T175, F176, and randomly with the receptor but do so in a binding the first receptor (15). We con- R178) could account for -85% of the sequential fashion (Fig. 3). That is, hGH verted each of these residues to alanine binding energy. These formed two small reacts first with a receptor by using site 1 and measured their on-rates and off-rates patches near the center of the contact and then with a second receptor by using (17). The largest effects were for six mu- interface that we call the functional site 2. The basis for proposing this mech- tants (P61A, R64A, K172A, T175A, epitope (Fig. 2). anism was that mutations in site 2 do not F176A, and R178A) which individually A similar analysis (19, 20) was per- have an impact on the ability to form 1:1 caused a 5- to 30-fold increase in off-rate, formed on the receptor side, where 33 side complexes with hGH, whereas mutations but only a 1.1- to 2.5-fold decrease in chains become buried at the interface. in site 1 do. on-rate. The subtle effects on the on-rate Each of these residues (except G168 and The structure provided additional sup- were best correlated to changes in the the C108-C122 disulfide) were converted port for the sequential dimerization model electrostatics of the binding site. For ex- to alanine, and affinities of the mutated (15). For example, the contact epitope ample, the largest decreases in on-rate receptors to hGH were measured by RIA. between hGH and hGHbp1 buried about were for mutating positively charged res- Nine of these residues (R43, E44, 1103, 1300 A2, whereas that for hGH with idues, and the largest increases in on-rate W104, 1105, P106, 1164, D165, and W169) hGHbp2 buried only -850 A2. If, however, (up to 1.5-fold) were for mutating two could account for virtually all the binding hGHbp2 binds to the hGH hGHbpl com- negatively charged groups (E65A and affinity. These residues cluster at the cen- Rlex, then 1350 A2 becomes buried-850 E174A) at the binding site. ter of the contact interface (Fig. 2). A2 for the hGH-hGHbp2 interface plus Northrup and Erickson (18) have noted What structural features explain why 500 A2 for the hGHbp1lhGHbp2 interface. that protein-protein association generally some residues are important while others Thus, the binding of hGHbp2 requires occurs at rates that are 103 to 104 times faster are not? The functionally important resi- prior binding of hGHbpl because its bind- than would be expected from simple con- dues are those located near the center of ing site is composed of determinants from siderations of collision frequencies (-109 the contact epitope and contact those site 2 on hGH as well as the stem portion s-1.M-1) and strict orientation effects that found to be functionally important resi- of the bound hGHbpl. The reason that the assume productive binding occurs only dues on the other side. The most critical hormone does not form higher oligomers when the molecules collide within 2 A of interactions tend to be well-packed hydro- with the receptor is that the receptor uses their final binding site. These orientation phobic contacts. This is obvious on the virtually the same site to bind either site 1 effects are predicted to reduce the rate of receptor side, where W104 and W169 are or site 2 on hGH (15). Thus, one receptor productive collisions by _106 to produce by far the most important residues. The can bind only one hormone molecule at a on-rates of 103 s-' M-1. They argue that aliphatic portions of important charged time. long-lived collisions, or Brownian diffusion, and polar side chains from hGH, such as On the basis of this sequential dimer- would greatly accelerate the association. In D171, K172, and T175, pack against the ization mechanism and the structure of other words, when the proteins collide, they tryptophans on the receptor. No (or few) the complex it was possible to explain the do not diffuse away immediately but roll on buried waters are seen between the func- bell-shaped dose-response curve for acti- one another and thereby sample much more tional epitopes (20). vation of receptors by hGH (24-26). At surface area than would be the case for a The unimportant residues tend toward low concentrations, hGH can bind to re- single elastic collision. They suggest that the periphery of the contact or structural ceptors and easily find an empty receptor association rates between proteins are dom- epitope. These are often polar and incom- to form a dimer and effect a signal. How- inated by considerations of diffusion events pletely dehydrated. Thus, bridging water ever, at high concentrations receptors be- alone. molecules are seen between polar or come occupied to greater extents as 1:1 We can conclude that only a small set of charged contact residues in regions of the complexes, and thus fewer empty recep- side chains modulate these effects on the contact interface that are less important. tors are available to dimerize. affinity between hGH and the hGHbp and The extent of side-chain burial, or number This principle of sequential assembly that their major role is to slow the off-rate. of van der Waals contacts on their own, may be general to other hormone- This suggests there are many paths to are generally weak predictors of relative receptor complexes for the simple reason getting the hormone to the receptor. The effects on affinity when side chains are that it reduces the degrees of freedom for role of side chains is to keep the hormone converted to that of alanine (17, 20). association (for general review see ref. bound once it has reached the binding site, These correlations are much better, how- 27). In the hGH mechanism, the hormone not to get it there in the first place. Al- ever, when one considers only the burial of needs only to diffuse once in three dimen- though electrostatic interactions can sub- well-packed hydrophobic side chains. sions to reach the first receptor. The next Downloaded by guest on September 26, 2021 4 Review: Wells Proc. Natl. Acad. Sci. USA 93 (1996)

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FIG. 2. A series of space-filling models showing the complementary structural and functional epitopes on site 1 of hGH (dark gray, right) and the first bound receptor, hGHbpi (light gray, left). All contact residues are colored according to the effects that alanine substitutions have on binding affinity: light blue, <0.5 kcal/mol reduction in affinity (1 kcal = 4.18 U); dark blue, 0.5 to 1.5 kcal/mol reduction in affinity; and red, >1.5 kcal/mol reduction in affinity. Reprinted with permission from Science 267, 301 (see ref. 20). Copyright 1995 American Association for the Advancement of Science. association event occurs in the mem- membrane diffusion event would be sim- even soluble at the concentrations re- brane, which is two-dimensional. Fur- ilar except that a 1:1 hormone-receptor quired. Even for hGH, the half-maximal thermore, because the receptor is teth- complex would require finding another inhibitory concentration (IC5o) occurs at ered by its transmembrane domain this loaded receptor. For both steps, forming a -2 ,M, whereas the half-maximal excita- further limits two of the three degrees of 2:2 complex is far less efficient than form- tory concentration (EC5o) is 20 pM. This rotational freedom for the receptors to ing a 1:2 complex. 10,000-fold difference can be rationalized dimerize. A bell-shaped dose-response curve, if the step to dimerize and signal is much Contrast the sequential 1:2 dimeriza- which is consistent with formation of 1:2 more efficient than the initial binding tion model with one in which two separate complexes, may be difficult to see in other event. In other words, it is difficult to hormone molecules need to bind to dimer- hormone/receptor systems. To observe saturate the receptors as 1:1 complexes ize and signal (a 2:2 model, ref. 21). In this the inhibitory phase requires extremely before some have dimerized. Further- case, two three-dimensional diffusion high hormone concentrations. Some hor- more, the difference between EC50 and events would be required. The second mones may not be readily available or IC50 will be further expanded if only a Downloaded by guest on September 26, 2021 Review: Wells Proc. Natl. Acad. Sci. USA 93 (1996) 5

tures and likely share a common binding mechanism. Each hormone has at least hGH two sites for binding and oligomerizing its receptor(s). Such stoichiometry econo- mizes on the diffusion events-after diffusion to the first membrane-bound receptor the second reaction with a receptor is facilitated by diffusion in two dimen- sions instead of three. This mechanism should provide a common strategy for generating antagonists to these hormones. That is, one can produce hormone vari- ants that allow the first binding reaction but prevent downstream binding reactions. Such antagonists may be useful in disease states where excessive hormone levels are a problem, such as in the case of acromegaly produced by excessive levels of growth hormone. The growth hormone receptor interfaces are large, but only a few residues are critical in binding. The interface generally resembles the core of a folded protein: well-packed hydrophobic contacts are crucial and they are inside, while hydro- philic interactions appear less important and they are outside. Although the polar residues appear less important for affinity, they may be important for solubility of the hormone and for specificity of the binding interaction. We believe the implications of these findings are important for rational drug design. Perhaps small molecules can be built to bind to these large interfaces if Active (agonist) Inactive (antagonist) they are designed to bind the small functional epitopes. FIG. 3. Sequential dimerization model for activation of the hGH receptor taken from ref. 24. Copyright 1992 American Association for the Advancement of Science. hGH first binds via site Are these findings general to protein- 1 to receptor 1 and then through site 2 to the receptor 2. This dimerization event leads to signaling. protein interactions? Structures of antibody-antigen complexes show these inter- small number of receptors need to dimer- hancing) binding by site 1 while preventing faces to be large and it is likely that only a ize in order to signal (25). dimerization by site 2 (24). Indeed, potent small number of contact side chains are Mathematical models to describe the antagonists of hGH have been generated important in binding [see Davies and Co- oligomerization reactions on cell mem- by introducing a mutation (G120R) into hen review in this issue (38)]. Trapped branes with the appropriate dimensional site 2 that blocks dimer formation (24, 28). water molecules are also present at these constraints would be of use in trying to de- This same strategy has been used to create interfaces, whose functional importance convolute dose-response data in terms of antagonists to the prolactin receptor has yet to be determined. Although oligomerization parameters. Among other based on hGH or a homolog, human charged and polar side chains can be factors, the dimerization reaction will de- placental lactogen (29). critical in binding, it is noted that hydro- pend on receptor concentration or ten- As the literature accumulates on other phobic side chains in these and other dency for the receptor to preassociate, as helical cytokines it is becoming apparent protein-protein complexes [see Jones and well as on the association rate for the 1:1 that a number of their receptors such as Thornton review in this issue (39)] are hormone-receptor complex (25). These those for interleukin (IL)-2, IL-3, IL-4, very important. Whether these findings IL-5, IL-6, granulocyte colony-stimulating parameters will vary from one cell are general to most inter- surely factor (G-CSF), granulocyte-macrophage protein-protein type to another and from one hormone/ (GM)-CSF, and erythropoietin undergo faces remains an open question-one that receptor system to another. For these rea- will surely be tested as more complexes ligand-induced receptor homo- or hetero- sons and perhaps more, it may be difficult are to the same level of structural oligomerization (for reviews see refs. 23, brought to observe a bell-shaped dose-response 24, and 30). Moreover, mutational analy- and functional understanding. curve. Such behavior applies to receptor ses of the monomeric cytokines IL-2 (31), systems which form homodimers; het- IL-3 (32, 33), IL-4 (34), GM-CSF (35, 36), I thank my colleagues in the Protein Engi- erodimeric (or oligomeric) systems where neering Department at Genentech for their IL-5 (35), and IL-6 (37) suggest they may support and useful discussions, Kerri Andow the hormone has a single site for two have at least two receptor binding sites, a different receptors and binds them se- and Wayne Anstine for their graphics genius, circumstance that has allowed the gener- and Brian Cunningham, Bart de Vos, and Tony quentially may not be antagonized by high ation of hormone antagonists for IL-2, Kossiakoff for critical review of the manuscript. hormone concentrations because saturat- IL-3, IL-4, and GM-CSF. ing the first receptor would not reduce 1. Wells, J. A. & De Vos, A. M. (1993)Annu. binding of the second. Conclusions Rev. Biophys. Biomol. Struct. 22, 329-351. The sequential dimerization mecha- 2. Kossiakoff, A. A., Somers, W., Ultsch, M., nism predicted that antagonists of hGH The helical cytokines and their cognate Andow, K., Muller, Y. A. & De Vos, could be produced by allowing (or en- receptors form a family of related struc- A. M. (1994) Protein Sci. 3, 1697-1705. Downloaded by guest on September 26, 2021 6 Review: Wells Proc. Natl. Acad. Sci. USA 93 (1996)

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