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
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Abstract |
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Keywords: cytokine hormonereceptor complex/human growth hormone/proteinprotein interaction/zinc
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Introduction |
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Human growth hormone (hGH) binds either lactogenic or somatotrophic receptors to create heterotrimeric complexes (Wells et al., 1993). 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., 1994
). Subsequently, the second receptor binds in the surfaces between helices 1 and 3 (site 2) (Goffin and Kelly, 1997
). 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, 2003
). 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., 1994
; Chantalat et al., 1995
).
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., 1990). Similarly, zinc increases the activity of hGH biological assays (Dattani et al., 1993
; Fuh et al., 1993
). 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., 1994
). 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., 1990; Dattani et al., 1993
; Fuh et al., 1993
). Doseresponse studies using extended concentration ranges of lactogens describe agonist and antagonist phases (Fuh et al., 1993
). 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 doseresponse 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., 1994
; Goffin et al., 1996
). 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, 1999
); the agonist phase of the doseresponse 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 doseresponse curve; the uncoupling is observed as a decrease in sensitivity of the agonist phase of the doseresponse curve, which is influenced by the function of both sites 1 and 2.
In previous mutagenic studies (Duda and Brooks, 1999, 2003) interpreted with the aid of available structural data (Somers et al., 1994
; Chantalat et al., 1995
), 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, 2003
). 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.
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Materials and methods |
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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, RZ1032 and BL21 (DE3) were used for cloning, production of ssDNA and protein expression, respectively. This system was previously described (Peterson et al., 1999
).
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., 1999). Proteins were evaluated for size and purity by 15% SDSPAGE 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 doseresponse curves were obtained as previously described (Peterson et al., 1999). 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., 1985
).
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Results |
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Yields of purified proteins were >20 mg/l of bacterial fermentation and purity >95% as judged by SDSPAGE. 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). Doseresponse curves showed that, with endogenous zinc, each mutation within the coupling motif shifted the agonist phase of the doseresponse 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, 1999
, 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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Lactogenic bioassays: site 2 mutation
With endogenous zinc concentrations, a G120R mutation reduced the maximum activity of the doseresponse 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 doseresponse 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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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.
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Discussion |
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In the FDC-P1 bioassay, zinc increases the lactogenic activity of hGH by increasing the sensitivities of both arms of the doseresponse 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., 1990) and both Nb-2 and FDC-P1 lactogenic bioassay data (Fuh et al., 1993
). Both Fuh et al. (Fuh et al., 1993
) 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., 1993
), where the addition of 50 µM zinc to an Nb-2 bioassay (Tanaka et al., 1980
) did not affect the agonist phase of the doseresponse 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., 1998). Our data and that of others (Ilondo et al., 1994
) show that this mutation reduces the maximal biological activity without shifting the doseresponse 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., 1993
). 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 doseresponse curve.
A second category of hGH mutations affects the functional coupling of sites 1 and 2 (Duda and Brooks, 2003). This class of mutations influences site 2 performance indirectly, decreasing the sensitivity (right-shift) of the agonist phase of the doseresponse curve without influencing either the antagonist phase or the maximal biological activity (Duda and Brooks, 1999
). The addition of zinc to bioassays for this class of mutant hGHs increases the sensitivity of the doseresponse 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, 2003
). 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.
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Acknowledgements |
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References |
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Received April 10, 2003; revised June 7, 2003; accepted June 8, 2003.