Differential effects of zinc on functionally distinct human growth hormone mutations

K.M. Duda1 and C.L. Brooks1,2,3

1Ohio State Biochemistry Program and 2Departments of Veterinary Biosciences and Biochemistry, The Ohio State University, 1925 Coffey Road, Columbus, OH 43210, USA

3 To whom correspondence should be addressed. e-mail: brooks.8{at}osu.edu


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Human growth hormone (hGH) binds and activates lactogenic receptors by a sequential receptor dimerization mechanism. The affinity for the first lactogenic receptor is increased due to one zinc molecule linking hGH residues H18 and E174, located in helices 1 and 4, respectively, with two adjacent residues in the lactogenic receptor (D187 and H188). Two functionally unique groups of mutant hGHs have been identified. Addition of 25 µM zinc to lactogenic bioassays differentially affects mutant activities based on which group they belong to. One mutation (G120R) is located within the binding surface of hGH that interacts with the second lactogenic receptor. In the presence of endogenous zinc, G120R reduces the maximal activity of hGH without altering either the agonist or antagonist phases of the bell-shaped dose–response curve. Addition of zinc to this assay further reduces the activity of this protein. In contrast, mutations within a hydrophobic motif in hGH that functionally couples the two lactogenic receptor binding sites decrease the sensitivity (right-shift) of the agonist phase of the dose–response curve without similarly affecting the antagonist phase. The addition of zinc to these lactogenic assays increases the sensitivity (left-shifts) of the dose–response curves, largely negating the effect of these mutations. The effects of zinc differentiate between mutations within these two distinct functional motifs by limiting the pool of potential conformations that are available for binding within either of the receptor binding sites of this ligand.

Keywords: cytokine hormone–receptor complex/human growth hormone/protein–protein interaction/zinc


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Proteins exist as an ensemble of conformers that are determined by the amino acid sequence, chemical and physical environment and folding conditions. Hydrogen exchange (HX) NMR, studies pioneered by Englander (Englander, 2000Go) and colleagues, show that proteins are dynamic, existing in a large number of states where various local sections of the molecule transiently unfold. The impact of ligand binding on the conformer ensemble has been evaluated both by calculation and experimental studies (Freire, 1997Go, 1999, 2000; Luque and Freire, 2000Go; Pan et al., 2000Go) and demonstrates that ligand binding changes the probabilities of members of the conformer ensemble to exist. These studies also suggest that only a limited number of residues are required to transmit the influence of a binding event to distal locations in the protein that determine changes in the conformer population.

Human growth hormone (hGH) binds either lactogenic or somatotrophic receptors to create heterotrimeric complexes (Wells et al., 1993Go). Lactogenic receptors bind hGH in a sequential manner, with the first receptor binding to a structural topology composed of residues in helices 1 and 4 and the loop connecting helices 1 and 2 (site 1) (Somers et al., 1994Go). Subsequently, the second receptor binds in the surfaces between helices 1 and 3 (site 2) (Goffin and Kelly, 1997Go). We have shown that mutations of residues that form a motif of contiguous hydrophobic residues connecting sites 1 and 2 diminish the activity of site 2 in biological assays (Duda and Brooks, 2003Go). These residues articulate a site 1 binding-induced conformation change resulting in the selection of conformers that strongly promote site 2 binding. This interpretation of our data is enforced by structural data that show that the regions containing these motif residues undergo a substantial rearrangement with site 1 binding (PDB nos 1HGU and 1BP3) (Somers et al., 1994Go; Chantalat et al., 1995Go).

Zinc influences the activity of hGH in lactogenic, but not somatotrophic, binding and biological assays. In binding assays, the addition of zinc increased the affinity by 8000-fold when compared with the assays containing EDTA (Cunningham et al., 1990Go). Similarly, zinc increases the activity of hGH biological assays (Dattani et al., 1993Go; Fuh et al., 1993Go). Zinc binds hGH on residues H18 and E174 of hGH and residues D187 and H188 of the extracellular domain of the human prolactin receptor (hPRLbp) docked at site 1 (Somers et al., 1994Go). Zinc binding serves two functions. First, it participates in the binding of hGH and receptor at site 1, linking the two proteins and maturing their affinity. Secondly, zinc binding stabilizes hGH structure. Zinc binding to sequentially distant residues (residues 18 and 174) of hGH confines the articulation of helices 1 and 4 in which the zinc binding residues are located, influencing the population of conformers. In contrast, zinc binding to the lactogenic receptor will only affect the orientation of the two adjacent binding residues (D187 and H188): zinc is less likely to stabilize global hPRLbp structure because the two residues involved in metal binding do not link sequentially distant structural elements.

The ultimate effects of ligand binding to hGH can be judged by their biological actions in lactogen-dependent cell lines (Cunningham et al., 1990Go; Dattani et al., 1993Go; Fuh et al., 1993Go). Dose–response studies using extended concentration ranges of lactogens describe agonist and antagonist phases (Fuh et al., 1993Go). The biological effects of structural changes within the receptor-binding surfaces of lactogenic hormones have been described. Changes include coordinated shifts in agonist and antagonist phases of the dose–response curve when structural changes are introduced within site 1 and diminution of the maximal response when structural changes are introduced within site 2 (Ilondo et al., 1994Go; Goffin et al., 1996Go). We have recently reported that structural changes outside the receptor-binding surfaces, but within a motif that functionally couples sites 1 and 2, produce a third distinct pattern in cellular bioassays (Duda and Brooks, 1999Go); the agonist phase of the dose–response curve shifts, while the antagonist phase does not change. In these studies, the coupling of sites 1 and 2 is influenced, reducing the ability of site 1 binding to influence the function of site 2. In these instances, site 1 function is unchanged, as evidenced by no changes in the site 1-dependent antagonist phase of the dose–response curve; the uncoupling is observed as a decrease in sensitivity of the agonist phase of the dose–response curve, which is influenced by the function of both sites 1 and 2.

In previous mutagenic studies (Duda and Brooks, 1999Go, 2003) interpreted with the aid of available structural data (Somers et al., 1994Go; Chantalat et al., 1995Go), we suggested that site 1 binding of hPRLbp to hGH constrained helix 3 relative to helix 1 to populate conformers able to bind hPRLbp at site 2. We provided functional evidence for the mechanism by which this conformational motif influences the conformer ensemble of hGH that facilitates site 2 binding (Duda and Brooks, 2003Go). In the present study, we compare the effects of the presence or absence of exogenous zinc on the effects of mutations located within site 2 (G120R hGH) or in the conformational motif that transmits site 1 binding to site 2 (five mutations). We find that the influence of zinc on these mutant hGHs has distinct effects that can be understood by evaluating the structural consequences of zinc binding to hGH.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Plasmids and bacterial strains

An f1 origin of replication was inserted into the pT7-7 plasmid (kindly provided by S.Tabor, Harvard Medical School, Boston, MA, USA) generating the pT7-7 f(–) phagmid. The positive strand was used for cloning, ssDNA production and expression of hGH. Escherichia coli strains DH5{alpha}, RZ1032 and BL21 (DE3) were used for cloning, production of ssDNA and protein expression, respectively. This system was previously described (Peterson et al., 1999Go).

Expression, purification and characterization of recombinant hGHs

Proteins were expressed in BL21 (DE3) cells and purified by anion exchange chromatography as previously described (Peterson et al., 1999Go). Proteins were evaluated for size and purity by 15% SDS–PAGE under reducing and non-reducing conditions. Proper protein folding was confirmed from absorption, fluorescence and circular dichroism spectra that were collected at 20°C in 10 mM Tris pH 8.2, 150 mM NaCl. Mutants constructed from wild-type methionyl hGH included F44E, L93E, G120R, Y160E, L163F and Y164E.

FDC-P1 lactogenic assays

FDC-P1 cells expressing the human prolactin receptor were a gift from Genentech Inc. (San Francisco, CA, USA). hGH dose–response curves were obtained as previously described (Peterson et al., 1999Go). Wild-type and all mutant hGHs were tested in a single assay that was independently performed at least three times. Variation within an assay is normally smaller than that between assays. But the relative differences observed between the ED50 values of hGHs were present and consistent in each assay. Protein concentrations were measured by the bicinchroninnic acid (BCA) protein assay (Smith et al., 1985Go).


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Protein characterization

Yields of purified proteins were >20 mg/l of bacterial fermentation and purity >95% as judged by SDS–PAGE. The characteristics of absorption, fluorescence or circular dichroism spectra indicated that the proteins were properly folded and were consistent between preparations (data not shown).

Lactogenic bioassays: coupling motif

When lactogenic bioassays are performed in endogenous zinc (total zinc was estimated to be ~3 µM), mutations within the coupling motif of hGH increased their ED50 between 3- and 279-fold (Table I and Figure 1). Dose–response curves showed that, with endogenous zinc, each mutation within the coupling motif shifted the agonist phase of the dose–response curve to the right, but failed to affect the antagonist phase of the curve. These data are similar to those previously observed (Duda and Brooks, 1999Go, 2003) and are explained by a loss in the efficiency by which site 1 binding selects conformers with high affinities at site 2. The greatest losses of activity are at residues closest to site 1 (F44E; 279-fold), while the smallest losses of activity are distal to site 1 (L93E; 4.2-fold).


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Table I. Effects of zinc and mutations on the activities of hGH in the FDC-P1 lactogenic bioassay
 


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Fig. 1. FDC-P1 cells transfected with the hPRL receptor stimulated with hGHs supplemented with 25 µM zinc (bottom) or with endogenous zinc levels (top). The y-axis represents the normalized scale indicating the reduction of Alamar Blue.

 
When 25 µM zinc sulfate was included in the assay, the lactogenic activities of wild-type and each of the mutants in the coupling motif were significantly increased (Table I and Figure 1). The ED50 of wild-type hGH was reduced from 3.25 to 0.42 nM (an ~8-fold increase in activity). When zinc was added to the FDC-P1 lactogenic bioassays of hGHs containing mutations of residues in the coupling motif, there were also significant decreases in the ED50 values of the mutant proteins when compared with parallel assays containing endogenous zinc. The zinc-induced increases in activities of the mutant proteins were larger than that produced in wild-type hGH, varying from 11- to 65-fold (Table I). Again, the proximity of the mutation to site 1 was associated with a larger zinc-induced increase in activity. For several of the mutated hGHs, the addition of zinc largely eliminated the effect of the mutation (L93E, Y160E and L163F hGHs). Unlike dose–response curves with endogenous zinc concentrations, dose–response studies performed in the presence of exogenous zinc increased the sensitivities (produced left-shifts) of both agonist and antagonist portions of the dose–response curves (Figure 1). This parallel shift of both agonist and antagonist responses is compatible with zinc affecting site 1 and not influencing the functional coupling of sites 1 and 2, as explored by these mutations (observed as a shift in the agonist phase only).

Lactogenic bioassays: site 2 mutation

With endogenous zinc concentrations, a G120R mutation reduced the maximum activity of the dose–response curve to ~22% of the activity of wild-type hGH without shifting either the agonist or antagonist portions of the curve.

The effect of exogenous zinc on hGHs with a mutation in site 2 (G120R) was different than the effects of mutations in the coupling motif (Figure 2). As observed in the preceding experiment, the addition of 25 µM zinc to the biological assay for wild-type hGH left-shifted both the agonist and antagonist activities by approximately an order of magnitude. In contrast, the addition of 25 µM zinc to biological assays treated with G120R hGH further reduced the activity to <10% of the activity of wild-type hGH. A shift in the dose–response curve could not be calculated due to the low activity of G120R hGH (Figure 2). Although zinc ameliorates the effect of mutations in the coupling motif, it enhances the effects of a mutation in site 2.



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Fig. 2. Effect of zinc on the lactogenic activity of the site 2 mutant G120R. Dose–response studies were performed either in the presence of endogenous zinc (–Zn) or with the addition of 25 µM zinc (+Zn). The y-axis represents the normalized scale indicating the reduction of Alamar Blue.

 
Effects of zinc on the folded structure of hGH

The two residues in hGH that bind zinc are located in helices 1 and 4. Zinc binding will constrain the articulation of these helices and restrict the ensemble of conformers available to hGH. If these effects are large, then spectroscopic methods may be able to detect changes in the averaged structures of the ensembles. The presence or absence of zinc did not affect the fluorescence spectra of wild-type hGH. Zinc-induced changes in the circular dichroism spectrum were very modest, not affecting the 222 nm signal and marginally diminishing the 208 nm signal (data not shown). Thus, zinc binding to hGH does not produce measurable changes in hydrophobic packing or large changes in secondary structure.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
hGH binds two lactogenic or somatotrophic receptors at the plasma membrane of target tissues by a sequential binding mechanism (Wells et al., 1993Go). Structural changes within the first receptor-binding site affect site 1 affinity by steric hindrance and shift both the agonist and antagonist phases of the dose–response curve of biological assays (Ilondo et al., 1994Go; Goffin et al., 1996Go). This type of shift occurs because both phases of the curve are dependent on site 1 affinity. Mutations within site 2 reduce the maximal activity in biological assays (Ilondo et al., 1994Go; Goffin et al., 1996Go), presumably reducing the site 2 affinity of the ligand for receptors by steric hindrance. Mutations of residues that are outside the two binding surfaces produce other functional changes in biological assays. We have identified a motif in hGH, external to both binding sites, that transmits a site 1 binding-induced conformation change to site 2 (Duda and Brooks, 2003Go). Mutation of residues that constitute the coupling motif have little or no influence on site 1 binding, but inhibit the function of site 2. In biological assays these mutations shift the agonist phase of the dose–response curve (dependent on both sites 1 and 2) while not influencing the antagonist phase (only site 1 dependent) (Duda and Brooks, 1999Go, 2003).

In the FDC-P1 bioassay, zinc increases the lactogenic activity of hGH by increasing the sensitivities of both arms of the dose–response curve, pointing to an increase in site 1 lactogenic receptor affinity. These data are reasonable based on the location of the zinc-binding site that bridges helices 1 and 4 and will reduce their movement relative to one another. These bioassay data are consistent with the effects of zinc on hGH/hPRLbp binding (Cunningham et al., 1990Go) and both Nb-2 and FDC-P1 lactogenic bioassay data (Fuh et al., 1993Go). Both Fuh et al. (Fuh et al., 1993Go) and our studies observed an ~10-fold increase in the sensitivity of these bioassays. But, these data are dissimilar to those of Dattani et al. (Dattani et al., 1993Go), where the addition of 50 µM zinc to an Nb-2 bioassay (Tanaka et al., 1980Go) did not affect the agonist phase of the dose–response curve for pituitary isolates of hGH.

One type of hGH mutation can provide a steric hindrance within a receptor-binding topology. G120R hGH places a bulky arginine within the site 2 binding surface (PDB no. 1A22) (Clackson et al., 1998Go). Our data and that of others (Ilondo et al., 1994Go) show that this mutation reduces the maximal biological activity without shifting the dose–response curve. We observed that the addition of zinc further reduced the maximum biological activity of G120R hGH. Similar reductions in maximal biological activities have also been noted by Fuh et al. (Fuh et al., 1993Go). Since G120R hGH most likely functions by reducing the affinity for the second hPRLbp, then zinc must reduce biological activity by further increasing the difference in affinities between the two binding sites, thus shifting the equilibrium away from a 1:2 active trimeric complex and towards an inactive 1:1 complex bound only at site 1. This finding is in sharp contrast to our data for wild-type hGH where the addition of zinc did not affect the maximal biological activity but increased the sensitivity of the dose–response curve.

A second category of hGH mutations affects the functional coupling of sites 1 and 2 (Duda and Brooks, 2003Go). This class of mutations influences site 2 performance indirectly, decreasing the sensitivity (right-shift) of the agonist phase of the dose–response curve without influencing either the antagonist phase or the maximal biological activity (Duda and Brooks, 1999Go). The addition of zinc to bioassays for this class of mutant hGHs increases the sensitivity of the dose–response curve, nullifying the effect of the mutations. This response to zinc is unique and again serves to illustrate that the structure of hGH is highly cooperative, where distant structural features can interact to either reduce or enhance biological activities. Interestingly, the measured ED50 values from hGHs in this project were generally reduced (by an average of ~2-fold) from those we measured previously (Duda and Brooks, 2003Go). Two factors could account for these differences: first, the proteins could actually have different proportions of fold variants; or, second, the FDC-P1 cells used in the biological assay could have lost sensitivity during the time between these two sets of assays. Spectroscopic characterization of each batch of proteins did not reveal significant differences in the ensemble of folds. Therefore, we believe that modest changes in the cells, brought about by subtle differences in culture conditions, most likely account for this change.

The zinc-induced enhancement of the biological activity of wild-type hGH has been interpreted to indicate that zinc limits the number of potential conformers at site 1 to improve the affinity of site 1 and that zinc actively participates in the hGH/hPRLbp binding interface. Because of this site 1 promotion of activity, the effect of zinc on mutants already deficient in binding at the site 2 interface is the opposite, suppressing hGH activity. Finally, zinc counteracts the amelioration of hGH lactogenic activity brought about by a set of non-binding site mutations.

The cell culture media in which we conducted the biological assays contained an estimated 3 µM zinc that was supplemented with 25 µM zinc. These concentrations bracket the physiological concentrations of total blood zinc. The changes in hGH activities between the endogenous and supplemented zinc concentrations are consistent with the hypothesis that zinc excess and deficiency may have physiological effects mediated by its association with hGH.

During the last decade, experimental data and theory have significantly changed our understanding of proteins. Previously, proteins were viewed as relatively rigid structures that might be induced to undergo changes in conformation. Now proteins are viewed as dynamic molecules best represented by an ensemble of conformers. We provide evidence that binding of co-factors or other proteins will influence the probability of specific conformers being present in the ensemble and will influence the activities of proteins. In these studies, we have shown that zinc binding can have dramatically different consequences in the context of various categories of mutagenic modification of hGH.


    Acknowledgements
 
This work was supported by grant no. R01 DK56117 from the National Institutes of Health, US Department of Health and Human Services.


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 Materials and methods
 Results
 Discussion
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Received April 10, 2003; revised June 7, 2003; accepted June 8, 2003.





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