Hormone Receptor Binding Energetics

The energetics of the high-affinity site 1 hGH-hGH-R1 binding that forms the intermediate 1:1 complex has led to a

Table I Receptor-Receptor H-Bonding Interactions (<3.2 Â) for PRL-R and hGH-R Ternary Complexes

rPRL-R1

rPRL-R2

hGH-R1

hGH-R2

I188O

Q196Ne2

S145OY

D152OS2

Y200N

Q194Oe1

L146N

S201OY

D201O81

K198N

T147OY

D152OS1

E187Oe2

K198N

H150Ne2

N143OS1

D152O82

Y200OV

S201OY

Y200OV

number of important insights into the relationships between binding and specificity determinants and protein-protein interactions, in general [28]. In particular, the seminal work of Wells and colleagues [28-31] demonstrated that the hGH-hGH-R ECD1 protein-protein interactions are characterized by binding "hot-spots" that focus binding energies within a cluster of relatively few residues. Thirty residues of hGH make contact in the site 1 interface with hGH-R1; however, it was shown that about 85% of the binding energy was developed through only eight of the residues, and there were no apparent distinguishing characteristics between the interactions that were energetically important and those of the other side chains that were energetically null [31]. This very efficient use of the binding interface characterized by the concentration of the binding determinants within a relatively small area of the contact surface allows for separate adjacent areas to be used for specificity determinants without compromising the binding.

It has been determined that a similar type of hot-spot also exists for the hGH-hPRL-R1 site 1 interaction and that the interface encompasses the same binding footprint as the hGH-hGH-R1 counterpart [9,31]. The two systems differ, however, in how the energy is distributed within the footprint. For instance, residues such as E174 and R167 make little energetic contribution to binding in the hGH-hGH-R case but play extremely important roles in the hGH-hPRL-R contact surface («800-fold difference in binding between the two systems when alanine is substituted at each of these sites). Interestingly, R167 makes a salt bridge to an acidic side chain in both receptors (to E127 in hGH-R and D124 in hPRL-R), yet Ala-scan mutagenesis indicates that only in the case of hGH-hPRL-R does this salt bridge have a positive effect on binding [31]. This is an example of how the energetics of interactions is very context dependent on protein-protein interfaces [3].

The binding properties of ECD2 are intrinsically different than those for ECD1, because they involve the combined effects of two spatially distinct binding surfaces: binding site 2 on the hormone and a contact with the C-terminal domain of the bound ECD1 (Fig. 1). Neither of these surfaces alone supports binding without the interaction of the other. In glaring contrast to the extensive characterization of the energetics of site 1 binding, there is considerably less biochemical and biophysical information for site 2 in GH/PRL systems. A study by Cunningham et al. [32] showed that site 2 did not contain a well-defined hot-spot, as was the case for site 1. However, there is no information about whether the spatially independent hormone-receptor and receptor-receptor interfaces act in additive or cooperative fashion. This deficiency has greatly limited our understanding of the overall energetics driving the homodimerization process. In this regard, recent data show that the receptor-receptor interface contains a hot-spot in the hGH-hGH-R system and probably plays a more important role than the hormone site 2-receptor contact in stabilizing tertiary forms of the complex (Bernat and Kossiakoff, unpublished data).

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