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 mol- ecules. 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.