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Originally published In Press as doi:10.1074/jbc.M801202200 on May 8, 2008

J. Biol. Chem., Vol. 283, Issue 27, 19085-19094, July 4, 2008
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Crystal Structure of a Prolactin Receptor Antagonist Bound to the Extracellular Domain of the Prolactin Receptor*Formula

L. Anders Svensson, Kent Bondensgaard, Leif Nørskov-Lauritsen, Leif Christensen, Peter Becker, Mette D. Andersen, Morten J. Maltesen1, Kasper D. Rand2, and Jens Breinholt3

From the Protein Engineering, Novo Nordisk A/S, Novo Nordisk Park, DK-2760 Måløv, Denmark

Received for publication, February 13, 2008 , and in revised form, April 4, 2008.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The crystal structure of the complex between an N-terminally truncated G129R human prolactin (PRL) variant and the extracellular domain of the human prolactin receptor (PRLR) was determined at 2.5Å resolution by x-ray crystallography. This structure represents the first experimental structure reported for a PRL variant bound to its cognate receptor. The binding of PRL variants to the PRLR extracellular domain was furthermore characterized by the solution state techniques, hydrogen exchange mass spectrometry, and NMR spectroscopy. Compared with the binding interface derived from mutagenesis studies, the structural data imply that the definition of PRL binding site 1 should be extended to include residues situated in the N-terminal part of loop 1 and in the C terminus. Comparison of the structure of the receptor-bound PRL variant with the structure reported for the unbound form of a similar analogue ( Jomain, J. B., Tallet, E., Broutin, I., Hoos, S., van Agthoven, J., Ducruix, A., Kelly, P. A., Kragelund, B. B., England, P., and Goffin, V. (2007) J. Biol. Chem. 282, 33118-33131[Abstract/Free Full Text] ) demonstrates that receptor-induced changes in the backbone of the four-helix bundle are subtle, whereas large scale rearrangements and structuring occur in the flexible N-terminal part of loop 1. Hydrogen exchange mass spectrometry data imply that the dynamics of the four-helix bundle in solution generally become stabilized upon receptor interaction at binding site 1.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Prolactin (PRL)4 is a protein hormone secreted by the anterior pituitary in vertebrates and possesses physiological functions of remarkable diversity, including effects on reproduction, lactation, and growth. PRL belongs to a family of homologous proteins comprising PRL, growth hormone (GH), and placental lactogen (PL). The biological effects associated with this cytokine family are mediated by two distinct classes of cell surface receptors, the PRL receptors (PRLR) and the GH receptors (GHR). The PRL/PL/GH biology is governed by a delicate balance between receptor cross-reactivity and selectivity; PRL and PL bind selectively to PRLR, whereas GH is capable of binding both PRLR and GHR.

After being proposed more than a decade ago (1), hormone-induced receptor dimerization became generally accepted as the model for cytokine receptor activation. For the PRL family members, the model describes the signaling molecular entity as a ternary complex between one hormone molecule and a receptor homodimer assembled in a strictly sequential and hormone-dependent fashion; first the hormone ligand engages via binding site 1 (BS1) in high affinity binding to one receptor chain forming a 1:1 hormone-receptor complex. This complex constitutes the template for binding a second, identical receptor molecule via binding site 2 (BS2), resulting in the active 1:2 complex. However, the model has been challenged by an increasing body of experimental evidence, initially reported for the homologous human erythropoietin receptor (2) and later for GHR (3) and PRLR (4). These studies suggest that preformed, inactive dimers exist in the absence of hormone. Thus, receptor dimerization is a necessary but not sufficient event for receptor activation and, notably, not strictly ligand-dependent. For both human erythropoietin receptor (5) and GHR (3), mechanistic models have been proposed, where receptor activation involves relative rotations and movements of receptor subunits induced by hormone binding. Allosteric reorganization of the intracellular receptor domains brings associated JAK2 kinases into close proximity, allowing their activation by cross-phosphorylation. This initial activation step triggers a cascade of molecular events leading to the functional receptor response (6).

The receptor activation mechanism involving two distinct binding sites on the hormone forms the functional basis for a class of antagonists that are characterized by possessing high BS1 and impaired BS2 binding affinities. Such molecules will occupy the receptor by forming 1:1 complexes via BS1 but fail to elicit a functional response, which requires productive interactions between BS2 and the second receptor chain. Examples of such antagonists forming hormone-receptor complexes with a 1:1 stoichiometry are GH-G120R (7) and the corresponding PRL analogue, PRL-G129R (8). Since wild type PRL (wtPRL) is able to stimulate proliferation of tumor cells, molecules capable of antagonizing the effect of wtPRL are potentially anti-cancer agents (9) and have received considerable attention.

Detailed structural information about the interaction between GH and the extracellular domain (ECD) of its receptor has been available since 1992, when de Vos et al. (10) reported the x-ray structure of the 1:2 complex between GH and GHR-ECD, providing evidence for the receptor activation mechanism involving a ternary complex between one hormone and two receptor molecules. Subsequently, several experimentally determined structures of GH and variants thereof, both free and in complex with receptor molecules, have been reported. This abundant structural information has formed the basis of the successful rational design of GH analogues with desired properties, including antagonists and superagonists (11). The ensemble of published GH complex structures includes a crystal structure of a BS2-inhibited variant (GH-G120R) in a 1:1 complex with PRLR-ECD (12). The only other structure in the Protein Data Bank including PRLR molecules is that of the complex between ovine PL and two molecules of the extracellular domain of the rat PRLR (13). Corresponding 1:1 or 1:2 complex structures of the PRL/PRLR system have not been reported, and experimental structures of free PRL molecules became available only quite recently. The NMR derived solution structure of wtPRL (14, 15) and the recently published x-ray structure of a PRLR antagonist, {Delta}1-9-PRL-G129R (16), represent major contributions to the structural characterization of the PRL molecule.

Due to the limited structural characterization of the PRL/PRLR system, design of improved PRLR antagonists has relied on homology models of the hPRL·PRLR complex, built using the available GH and PL complex structures as templates. The present characterization of the PRL·PRLR-ECD complexes is a significant contribution to the molecular description of the interaction between PRL and PRLR, with the crystal structure serving as an important tool in the design and development of novel PRL molecules.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Expression and Purification of wtPRL, PRL Variants, and PRLR-ECD—In addition to wtPRL, two PRL variants, PRLv1 and PRLv2, were produced for hydrogen exchange mass spectrometry (HX-MS), NMR, and x-ray studies: PRLv1 (Met-PRL-(1-199) G129R) and PRLv2 (PRL-(12-199) Q12S/G129R). PRLv3 (PRL-(10-199) C11S/G129R) refers to the variant crystallized by Jomain et al. (16). PRLR-ECD refers to Ser-hPRL receptor-(1-210). Proteins were produced in E. coli as inclusion bodies, refolded, and purified by standard methods (supplemental material). Proteins were characterized by various analytical methods, including reversed phase HPLC, MS, and tryptic digest, and further analyzed for the presence of multimers using SDS-PAGE (reduced and nonreduced) and size exclusion HPLC. The presence of deamidation species was measured by isoelectric focusing gels and ion exchange HPLC.

HX-MS—Buffer change and concentration in HX-MS and NMR experiments were conducted using Amicon Ultra centrifugal filter devices (cut-off 10,000 Da; Millipore). Reported pH values for buffers containing 2H2O are uncorrected for the deuterium effect. Amide hydrogen (1H/2H) exchange was initiated by a 10-fold dilution of PRLv2 in the absence or presence of PRLR-ECD into deuterated buffer (20 mM Tris, 150 mM NaCl, 99% 2H2O, pH 7.4). All exchange reactions were carried out at 30 °C and contained 6 µM PRLv2 in the absence or presence of 12 µM PRLR-ECD. At appropriate time intervals, aliquots of the exchange reaction were quenched by the addition of an equal volume of ice-cold quenching buffer (1.25 M tris(2-carboxyethyl)phosphine hydrochloride, adjusted to pH 2.0 using NaOH), resulting in a final pH of 2.5.

Quenched samples were run on a cooled HPLC-mass spectrometry system for rapid desalting and mass analysis, as described previously (17). Peptic peptides were identified in separate experiments using standard MS/MS methods on a Q-TOF2 instrument (Waters Inc.). The hydrogen exchange time course of 27 peptides, covering 90% of the primary sequence of PRLv2, was monitored. Average masses of peptide isotopic envelopes were determined from lockmass-corrected centroided data (processed using MassLynx software; Waters Inc.). Complete deuteration of control samples was achieved by incubation for 6 h at 90 °C. Average back-exchange (i.e. deuterium loss) was measured to be ~15-20% for the analyzed peptides. However, since only the relative levels of deuterium incorporation were compared for all samples, no correction was made for this deuterium loss.

Preparation of NMR Samples—Samples of [2H,15N]PRLv1 and [2H,13C,15N]PRLv1 were prepared in 5 mM ammonium bicarbonate, 1 mM sodium azide, and 10% (v/v) 2H2O, pH 8.0 (NMR buffer). The complex between [2H,15N]PRLv1 (or [2H,13C,15N]PRLv1) and PRLR-ECD was prepared by mixing 1 mg/ml solutions (5 mM ammonium bicarbonate, 50 mM NaCl, pH 8.0) of [2H,15N]PRLv1 (or [2H,13C,15N]PRLv1) and PRLR-ECD in a molar ratio of 1:1.2. Excess PRLv1 was removed from the binary complex by gel filtration in ammonium bicarbonate buffer using a Superdex 75 column (GE Healthcare). Finally, the complex was exchanged into the NMR buffer and concentrated to a final protein concentration of ~200 µM. For the cross-saturation experiments, the NMR buffer contained 90-95% 2H2O in order to quench potential amide proton-mediated spin diffusion in [2H,15N]PRLv1.

NMR Experiments—NMR experiments were recorded at 35 °C on a Bruker Avance 600 MHz spectrometer equipped with a room temperature triple resonance probe. Two-dimensional 1H,15N-HSQC, two-dimensional 1H,15N-TROSY, 15N-edited three-dimensional NOESY-HSQC, three-dimensional HNCO, three-dimensional HNCA, three-dimensional HNCOCA, three-dimensional HNCACB, and three-dimensional HNCOCACB spectra were recorded using standard Bruker pulse sequences. Cross-saturation experiments were recorded essentially as described previously (18).

X-ray Crystallography—The 1:1 complex between PRLv2 and PRLR-ECD was prepared and purified as described above for preparation of the PRLv1 complex for NMR studies, followed by concentration to 9.5 mg/ml (2 mM ammonium bicarbonate, pH 7.9). The PRLv2·PRLR-ECD complex was crystallized by the hanging drop method at 22 °C using a precipitant solution containing 3.5 M NaCl and 0.1 M Hepes buffer, pH 7.5. Hexagonally shaped crystals appeared in a few days and grew to dimensions of ~200 x 200 x 150 µm. Crystals were directly flash-frozen in liquid nitrogen. Crystallographic data were collected at 100 K using beam line BLI911-3 (19) at MAX-lab (Lund, Sweden). Diffraction data were processed by the XDS program (20), and the structure was determined by the molecular replacement method and refined as detailed in the supplemental material. Data collection and refinement statistics are seen in Table 1. Data were initially used to 2.0 Å resolution, based on a 2.0I/{sigma}(I) cut-off or better, but subsequently limited to 2.5 Å resolution based on Rcryst and refinement statistics. A total of 91.8% of the residues in the complex are in the favored region of the Ramachandran plot, 6.6% in the allowed region and 1.4% in the outlier region as calculated by the program RAMPAGE (21). The outliers are Arg-16 and Asp-17 in PRLv2 and Glu-45, Cys-51, and Pro-203 in PRLR-ECD, of which all, except for Cys-51, are situated in flexible regions. Coordinates for the PRLv2·PRLR-ECD structure have been deposited in the Protein Data Bank with the accession code 3D48. Graphical representations of molecular structures were created by Pymol (W. L. DeLano; available on the World Wide Web).


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  		TABLE 1
Crystal, refinement, and model statistics

 


Figure 1
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  		FIGURE 1.
Outline of HX-MS data. A, sequence of PRLv2 (using wtPRL numbering) and secondary structure (as defined by Protein Data Bank 1RW5) are displayed above the HX-MS-analyzed peptides (shown as horizontal bars). Peptides with reduced deuterium content (>0.4 deuteriums) in the presence of PRLR-ECD after 1000 s of exchange or earlier are colored in red, whereas peptides displaying reduced deuterium contents in the presence of PRLR-ECD only after 10,000 s of exchange are colored in orange. Localization of exchanging amides in both N- and C-terminally overlapping peptides was performed by subtraction, assuming complete back-exchange of the N-terminal amide hydrogen in each peptide. B, hydrogen exchange time plots of representative peptides of PRLv2. The deuterium content is plotted against time on a logarithmic scale in the presence (red) and absence (blue) of PRLR-ECD.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Three different experimental techniques, HX-MS, NMR spectroscopy, and x-ray crystallography, were applied to study the interaction between PRL variants and PRLR-ECD. Although the crystal structure contains by far the most detailed information, the solution state techniques (HX-MS and NMR) provide measures of dynamic properties in solution in terms of NMR chemical shifts and amide proton exchange rates, which are modulated by receptor binding. Thus, HX-MS and NMR provide important complementary information in linking the free and bound states of the hormone.

HX-MS Analyses of PRLv2 in the Absence and Presence of PRLR-ECD—HX-MS is a sensitive method for probing protein structure and dynamics in solution. In particular, protein-protein complex formation is readily detected by comparing deuterium incorporation in the absence and presence of ligand (23). We investigated the hydrogen exchange profile of PRLv2 in the absence and presence of PRLR-ECD by monitoring 27 peptides, covering 90% of the primary sequence of PRLv2.

Receptor binding resulted in a pronounced decrease in exchange of several peptides of PRLv2 (Fig. 1A). Since PRLv2 exclusively binds PRLR-ECD via BS1, the regional reductions in exchange report on peptides that comprise or are conformationally linked to BS1. Decreased deuterium incorporation was primarily confined to peptides covering helix 1 and helix 4 and with a particularly pronounced protection from exchange observed in peptides of the short helix 1'' and the C-terminal Cys-191-Cys-199 segment (Fig. 1B). Remarkably, the exchange of peptides covering helix 2 and 3 also exhibited moderate protection upon complex formation, however only after prolonged exchange (10,000-100,000 s; Fig. 1B). This exchange behavior implies a ligand-induced conformational stabilization occurring outside the binding interface (23). We rule out binding of a second PRLR-ECD molecule at BS2 as an explanation for the altered exchange properties of the helix 2 and 3 residues, because the effects were also observed without an excess of PRLR-ECD (data not shown). We conclude that the complex truly is of 1:1 stoichiometry.


Figure 2
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  		FIGURE 2.
Comparing free and bound states. A, backbone amide chemical shift differences ({Delta}CS) observed between free PRLv1 and PRLv1 bound to PRLR-ECD are displayed by black bars against residue number (proline residues are marked by open bars). The chemical shift differences are expressed as {Delta}CS = (({Delta}{delta}H)2 + (0.1 x{Delta}{delta}N)2)0.5, where {Delta}{delta}H and {Delta}{delta}N are amide proton and nitrogen chemical shift differences measured between the free and bound forms. The horizontal bars above the residue number scale mark positions where the amide proton was observed neither in the free nor in the bound form (red), was observed in the free form only (yellow), or was observed in the bound form only (green). B, differences (C{alpha} RMSD) between free PRLv3 (Protein Data Bank code 2Q98) and bound PRLv2. The horizontal blue bars below the residue number scale indicate helical segments in bound PRLv2.

 
Furthermore, we compared the exchange kinetics of PRLv2 with that of wtPRL, both in the absence of PRLR-ECD. The exchange profiles were largely identical; however, wtPRL showed significantly reduced exchange, relative to PRLv2, in a short segment (residues 16-22) of the N-terminal part of helix 1 (data not shown). This indicates that PRLv2 is destabilized in this region, which is important for BS2.

NMR Analyses of PRLv1 in the Absence and Presence of PRLR-ECD—The solution structure of wtPRL determined by NMR spectroscopy was first reported by Keeler et al. (14). However, the accuracy of the proposed structure was questioned, and an improved model was established by Teilum et al. (15). Due to the biophysical properties of PRL, NMR data were recorded at pH 8 and 37 °C in the work by Teilum et al. Similar conditions (pH 8, 35 °C) were applied in the present study, and high quality NMR data were obtained for PRLv1 both in its unbound state and complexed with PRLR-ECD. Backbone assignments for free PRLv1 and PRLv1 complexed with PRLR-ECD were established using 2H,13C,15N-labeled PRlv1 by standard assignment experiments. The extent of assignment for the complex structure was as follows: 84% for backbone amide proton, nitrogen, and {alpha}-carbon; 73% for β-carbon; and 82% for backbone carbonyl.

A 1H,15N correlation spectrum was recorded for the 1:1 complex between [2H,15N]PRLv1 and PRLR-ECD, and a reference spectrum was acquired for free [2H,15N]PRLv1 under identical conditions. The resonance assignments served for calculation of chemical shift differences between backbone amide protons in PRLv1 in the free and receptor-bound states. The NMR data are presented in Fig. 2A and compared with x-ray data in Fig. 2B. Under the high pH and high temperature conditions applied, NMR signals for amide groups were absent in several flexible stretches of the polypeptide chain due to fast, base-catalyzed exchange of the amide protons. Save for Ile-3,5 no amide resonances were observed for residues 1-15 in wtPRL (15). This is also the case for PRLv1, both free and in complex with PRLR-ECD, which indicates that the intrinsically flexible N terminus is not involved in or significantly influenced by BS1 binding. Also included in the NMR silent regions in PRLv1 are a significant part of loop 1 between helix 1 and helix 2 (Gly-47-Lys-69), a part of the loop connecting helix 3 and helix 4 (Ser-151-Gln-157), and two residues in the C-terminal segment (Asn-196-Asn-197). As shown in Fig. 2A, several residues, for which the amide proton signal is absent in the free state, display amide proton resonances in PRLv1 complexed with PRLR-ECD, indicating that these regions become shielded, structurally stabilized, or, by some other means, protected from solvent exchange upon complex formation. Collectively, the observed changes in chemical shifts and amide proton exchange rates point particularly to two dynamic regions, Ile-51-Ser-57 and Asn-197-Cys-199, as being important for receptor interaction. Due to fast amide proton exchange in the free state, chemical shift differences are not available for Lys-53, Ala-54, Asn-56, Ser-57, Cys-58, Asn-196, and Asn-197, but neighboring residues, in particular Thr-52, Ile-55, Asn-198, and Cys-199, exhibit strong perturbations. Of note, the backbone amide proton of Asn-56 appears in the complex markedly deshielded, exhibiting an unusual downfield chemical shift of 10.4 ppm.


Figure 3
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  		FIGURE 3.
Crystal structure of the PRLv2·PRLR-ECD complex. Shown is a schematic representation of the overall structure of the PRLv2·PRLR-ECD complex. PRLv2 is shown in cyan, with the binding interface marked in red. The four main helices are marked H1-H4, and the minor helices are marked H1b, H1', H1'', and H3'. The D1 and D2 domains and the interdomain linker segment of PRLR-ECD are shown in yellow, dark blue, and purple, respectively. Selected side chains (His-27, Asn-31, Asn-56, Ser-61, Lys-69, Arg-177, Trp-72R, and Trp-139R) are represented by sticks and balls. Two structured water molecules, Wa and Wb, are shown by magenta spheres.

 
For a more direct mapping of the contact surface between PRLv1 and PRLR-ECD the NMR cross-saturation method (18) was applied. The data (not shown) confirmed the importance of the Ile-51-Ser-57 and C-terminal segments, as suggested by the NMR chemical shift perturbation and HX-MS data.

Crystal Structure of PRLv2 Bound to PRLR-ECD—Hexagonally shaped crystals of the complex between PRLv2 and PRLR-ECD were obtained using the hanging drop method. The structure was determined by the molecular replacement method, and the overall crystal structure of the 1:1 complex is shown in Fig. 3. The secondary structural elements found in the crystal structure of receptor bound PRLv2 are similar to those of free PRLv3 (Protein Data Bank code 2Q98) (16). Present are the characteristic four main {alpha}-helices, denoted helix 1-4 (Leu-15-Phe-40, Gln-77-Val-102, Ile-112-Gln-136, and Glu-161-Ile-193, respectively) and connected by loops 1, 2, and 3. Similar to PRLv3, three short helices, helix 1', 1'', and 3' (residues His-59-Ser-62, Lys-69-Ala-72, and Leu-153-Leu-156, respectively), are present in loop 1 and loop 3. In the structure of the complex, an additional short helix segment, denoted helix 1b, is formed in the N-terminal part of loop 1 (Ile-51-Ala-54). Due to weak or absent electron density, part of the N-terminal segment of loop 1 (Arg-43-Arg-48), the short loop 2 (Gly-104-Glu-110), the N-terminal part of the long loop 3 (His-138-Glu-145), and Cys-199 in PRLv2 have been omitted from the model of the complex. In contrast, for free PRLv3, the complete sequence was modeled into the electron density (16). However, in the crystal structure of PRLv3, more than half of the residues (100 residues) are within a 5-Å distance of symmetry related molecules, resulting in stabilization of the flexible loops. Loop-stabilizing crystal packing interactions are less abundant for PRLv2 in the complex structure, and apparent differences in the flexibility of the loop segments between the free and bound forms can be explained mainly by crystal packing effects.

The structure of PRLR-ECD (Fig. 3) reveals the characteristic tandem repeat of fibronectin type III modules, D1 and D2 (residues 1-98 and 104-210, respectively), connected by a short interdomain linker (residues 99-103). The relative domain orientation is very similar to that observed for GHR-ECD in published 1:1 and 2:1 complexes with GH and variants but distinctively different from that of PRLR-ECD in complex with GH (12).

The surface that becomes buried at the interface between PRLv2 and PRLR-ECD covers an area of ~1200 Å2. Two essential tryptophan residues in PRLR-ECD, Trp-72R and Trp-139R, dock on PRLv2 in an extended hydrophobic groove lined by helix 4 and minihelix 1'' (Fig. 4). Besides the contact residues belonging to helix 1 (His-27, His-30, Asn-31), helix 4 (His-173, Arg-176, Arg-177, His-180, Lys-181, Asp-183, Asn-184, Tyr-185, Lys-187, Leu-188, Cys-191, and Arg-192) and the C-terminal half of loop 1 (Pro-66, Glu-67, Asp-68, Lys-69, Glu-70, Ala-72, and Gln-73), additional contact regions are situated in the N-terminal part of loop 1, including Ile-51, Thr-52, Ala-54, Ile-55, and Asn-56 (Fig. 5), and in the C terminus (Asn-197 and Asn-198).

Differences observed between the structures of free PRLv3 and PRLv2 bound to PRLR-ECD are displayed in Fig. 2B. The two structures were superimposed based on C{alpha} atoms in residues present in {alpha}-helical segments (root mean square difference of 0.54 Å for 108 C{alpha} atoms). Evidently, the most prominent backbone shifts are in the 49-57 loop segment, which in the free state appears largely unstructured and flexible but in the complex becomes structured by specific receptor interactions involving residues Ile-51, Thr-52, Ile-55, and Asn-56 (Fig. 5). In the complex structure, Ala-54-Ser-57 forms a type II β-turn, and the Ile-51-Ala-54 segment curls up in an {alpha}-helix (H1b). This structural arrangement exposes the side chains of Ile-51 and Ile-55 for hydrophobic interaction with Tyr-94R and Ile-76R on the receptor and spatially presents Asn-56, allowing for favorable receptor interactions by tight backbone and side chain hydrogen bonds with Glu-43R and Gly-44R (Fig. 5). In the context of comparing free and bound states of the hormones, it should be emphasized that the Ile-51-Ser-57 segment in the free form (represented by either the crystal or the NMR structure (Protein Data Bank code 2Q98 and 1RW5, respectively)) appears flexible and unstructured. In the crystal structure of PRLv3, the electron density corresponding to the Ile-51-Ser-57 segment is weak, and the Ramachandran plot corresponding to 2Q98 reveals that residues Lys-53, Ala-54, Ile-55, and Asn-56 all appear in disallowed regions. Thus, 2Q98 may not accurately represent the peptide chain in this particular region, and a detailed atom-to-atom comparison of the Ile-51-Ser-57 segment in PRLv3 and in PRLv2 makes no sense. Accordingly, the root mean square deviation values (Fig. 2B) derived for residues present in this region should only be interpreted qualitatively. The electron density corresponding to the receptor-interacting Ile-51-Ser-57 segment is well defined in the present structure of the complex. On the other hand, the immediate up-sequence segment appears highly flexible, particularly the Arg-43-Arg-48 stretch, which was excluded from the x-ray-derived model of PRLv2. The Arg-43-Arg-48 segment in the structure of PRLv3 appears well ordered, a difference that we, however, ascribe to stabilizing crystal contacts.


Figure 4
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  		FIGURE 4.
Topology of BS1 in the PRLv2·PRLR-ECD complex. Residues in PRLv2 within a distance of 5 Å from PRLR-ECD are represented by a gray surface, except for acidic and basic side chain groups, which are colored red and blue, respectively. Side chains of contact residues in PRLR-ECD are shown by sticks and colored according to the legend to Fig. 3. Central in the binding interface are the two essential tryptophan residues, Trp-72R and Trp-139R, docked in the hydrophobic groove lined by helix 4 and minihelix 1''.

 


Figure 5
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  		FIGURE 5.
Structure of the N-terminal part of loop 1 in PRLv2·PRLR-ECD. The Ile-51-Ser-57 segment of PRLv2 (cyan) interacts with residues Glu-43R, Gly-44R, Ile-76R, and Tyr-94R (yellow). Hydrogen bond interactions between Asn-56 and PRLR-ECD are indicated by red dashed lines. The green dashed lines indicate the hydrogen bonds stabilizing the β-turn (Ala-54-Ser-57) and the short helix 1b (Ile-51-Ala-54) induced by binding to PRLR-ECD.

 
Data in Fig. 2B imply that, upon receptor binding, large scale changes occur in the C terminus, containing the Cys-191-Cys-199 disulfide bond. However, caution is required when interpreting these differences, since both crystal structures exhibit structural irregularities in the C termini. In the structure of PRLv3 the Cys-191-Cys-199 disulfide bond is absent, leaving Cys-191 and Cys-199 with free thiol groups outside disulfide bonding range (16). Additionally, the spatial orientation of the C terminus appears to be governed by interactions that are specific for the crystalline state, since Cys-199 makes contact with a symmetry related molecule in the crystal (16). Furthermore, the electron density corresponding to the Asn-197-Asn-198-Cys-199 segment in PRLv3 is very weak. In contrast, the electron density in the complexed structure of PRLv2 is intense and well defined for the C-terminal segment, including Asn-198, but is completely absent for Cys-199. The distance between Asn-198 and Cys-191 is perfectly compatible with the presence of a Cys-191-Cys-199 disulfide bond, and apart from the missing density, there are no signs of structural damage. It was verified (mass spectrometry; data not shown) that the PRLv2 present in the solution, from which crystals of the complex were grown, was C-terminally intact. NMR chemical shifts recorded for Cβ atoms in cysteines carry diagnostic information with respect to the binding state (free thiol or disulfide bound) of the side chain sulfur atom (24). For PRLv1 in solution the NMR data unambiguously demonstrate that the C-terminal disulfide bond remains intact for weeks in samples of both free and complexed PRLv1.

It is well documented that synchrotron radiation can induce specific chemical damage to proteins (25), including both disulfide bond and main chain cleavage. Disulfide linkages were shown to differ substantially in their susceptibility toward radiation-induced damage, and the C-terminal disulfide bond in PRL may represent a particularly sensitive example.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Data from site-directed mutagenesis (26-28), identifying a set of PRL residues as being functionally important for receptor binding and activation, combined with the structural information represented by the NMR solution structure of free PRL, allowed Teilum et al. (15) to draw a tentative map at residue resolution of BS1. The bulk of the BS1 residues was mapped to the C-terminal part of helix 4 (Tyr-169, His-173, Arg-176, Arg-177, His-180, Lys-181, Asn-184, Tyr-185, Lys-187, and Leu-188), whereas fewer critical BS1 residues were implied to reside in helix 1 (His-27, His-30, and Phe-37) and in the C-terminal part of loop 1 (His-59, Pro-66, and Lys-69). The binding interface found in the crystal structure of the PRLv2·PRLR-ECD complex is generally in accordance with that defined by Teilum et al. (15) but includes additional contact residues in the N-terminal part of loop 1 and in the C terminus of the sequence.


Figure 6
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  		FIGURE 6.
Important BS1 contact residues. A, Lys-69 is stacking with Trp-139R and is part of an ion-pairing network comprising Lys-66R-Glu-18R-Lys-69-Asp-134R-Lys-136R. B, Arg-177 engages in complex ion and hydrogen bonding connectivities involving Glu-43R, Thr-74R, and Asp-96R and a water molecule (Wa). Coloring is according to the legend to Fig. 3, except for the side chains of Lys-69 and Arg-177, which are colored orange.

 
One part of the PRLv2 binding interface, comprising helix 1 and helix 4 residues, constitutes a relatively static and receptor complementary surface with a preshaped topology. For this part of BS1, only minor side chain adjustments are necessary to accommodate contact residues from the receptor chain, particularly Trp-72R and Trp-139R. Similar interactions involving Trp-72R and Trp-139R are essential in the binding of GH to PRLR-ECD, and the corresponding residues in GHR, Trp-104R and Trp-169R, are critically involved in binding GH, accounting for the majority of the free energy of receptor binding (29). Most likely, the primary energetic driving force in the formation of the PRLv2·PRLR-ECD complex comes from hydrophobic interactions involving Trp-72R and Trp-139R (Fig. 4). The other part of the PRLv2 binding interface, including loop 1 residues, appears considerably more plastic and acquires its final topology in the complex during the event of receptor binding.

The PRLv2·PRLR-ECD complex structure provides a rationale for most of the effects on receptor binding associated with the functionally important residues identified by mutagenesis studies (26-28). Particularly strong effects were observed by mutating Lys-69, Arg-177, and Lys-181. The Lys-69 side chain is in the PRLv2·PRLR-ECD complex stacking with Trp-139R and is further engaged in a tight, five-element ion pairing network comprising Lys-66R-Glu-18R-Lys-69-Asp-134R-Lys-136R (Fig. 6A). Similarly, Arg-177 engages in complex ion and hydrogen bonding connectivities involving Glu-43R, Thr-74R, and Asp-96R and a water molecule (Wa) (Figs. 3 and 6B). Such an interaction involving all three nitrogen atoms in the Arg-177 side chain conforms with the observation that even the conservative R177K mutation in the bovine system reduces the binding affinity by 90% (28). Finally, for Lys-181, the hydrophobic part of its side chain stacks tightly with the tryptophan ring of the essential Trp-72R. The side chain amino group in Lys-181 is not involved in receptor interaction but hydrogen-bonds intramolecularly with Thr-65 and might have an additional function in tethering loop 1 to helix 4. Provided that the BS1 interface found in PRLv2 is identical to that of the wild type hormone, the reduced BS1 binding affinity associated with some of the reported alanine mutants must be attributed to secondary effects, since the modified residues are not involved in direct contacts (i.e. are situated outside a 5-Å distance from receptor atoms). These functional residues include Val-23 and Phe-37 in helix 1, His-59 in loop 1, and Tyr-169 situated in helix 4.

Since the efficacy of a PRL antagonist based on the principle of impaired BS2 binding is in part defined by the affinity ratio between BS1 and BS2 (30), a key element in optimizing such antagonists, besides achieving efficient BS2 inhibition, is the maturation of BS1 binding affinity. In this context, the S61A and Q73A mutations attracted our attention. All alanine mutants reported for the Cys-58-Gln-74 segment, except for S61A and Q73A, exhibited impaired receptor affinity and lactogenic activity (26), indicating to us that positions 61 and 73 could be potential targets for affinity optimization. The effect of the S61A mutation was reported to be a marginally, and probably insignificant, increased biological activity compared with wtPRL (26). Surprisingly, we observe a robust 2-fold increase in receptor binding affinity (data not shown) when substituting Ser-61 with alanine in the PRL sequence. Using the crystal structure, a hypothesis explaining the increased affinity of S61A can be formulated. In the unbound form (PRLv3), the Arg-177 side chain is tied up internally by ion pairing with Asp-178 and further polar interactions involving Thr-60, Ser-61, and a water molecule (Wc) (Fig. 7). Wc is buried well below the protein surface, sandwiched between helix 4 and loop 1 and held in place by a polar interaction network, including the side chains of Arg-177, Asp-178, the backbone and the side chain of His-59, and the Cys-58-Cys-174 disulfide bond. Elimination of the hydrogen bonding capability of Ser-61 by substitution with alanine is hypothesized to weaken or abolish the intramolecular binding of the Arg-177 side chain. Following this line of argument, receptor binding is favored by reduction of the energy spent for reorientation of the Arg-177 side chain required for its interaction with receptor residues Glu-43R, Asp-96R, and Thr-74R (Fig. 6). A corresponding buried water molecule is absent in PRLv2 in the complex structure, implying that Wc present in the free form will have to be expelled upon binding. In the NMR-derived solution structure (Protein Data Bank code 1RW5) of wtPRL, the Arg-177 side chain is not tied up internally as it is in the crystal structure of PRLv3. However, no nuclear Overhauser effect-derived distance is constraining the position of the Arg-177 guanidinium group in the NMR structure, and adjustment of only the {chi}3 and {chi}4 angles of Arg-177 places the N{eta}1 nitrogen at a distance of 3.1 Å from the carboxyl carbon of Asp-178, optimally positioned for ion pairing (the corresponding distance in PRLv3 is 3.5 Å). Thus, the side chain conformation of Arg-177 defined by 2Q98 might be significantly populated in solution also.


Figure 7
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  		FIGURE 7.
Rationale for the effects of S61A. PRLv3 (Protein Data Bank code 2Q98) is shown in a gray schematic representation, with the side chains of Cys-58, Thr-60, Ser-61, Thr-60, Cys-174, Arg-177, and Asp-178 represented by green sticks. The tightly bound water molecule, Wc, is shown as a magenta sphere. The side chain of Arg-177 in PRLR-ECD-bound PRLv2, superimposed on PRLv3, is represented by cyan sticks.

 
Tightly bound water molecules are commonly found in protein crystal structures, either associated on the surface or buried. Such structured water molecules can be important in stabilizing protein structures and facilitate protein-protein contacts (31), as exemplified by the water (Wa)-mediated receptor interaction of Arg-177 in the present complex structure (Fig. 6B). In total, three buried water molecules, including Wc, are present in PRLv3, and of these only one (Wb) is conserved in the PRLv2·PRLR-ECD structure. Wb is positioned between helix 1 and helix 4 (Fig. 3), below the BS1 surface, and held in place solely by its polar interactions via main chain atoms, including the carbonyl oxygen of Ser-26 and Ser-179 and amide groups of His-30 and Asp-183. The influence of Wb on BS1 is not known, but its conservation in the complex structure implies that bridging of helix 1 and helix 4 by Wb might have an important stabilizing function. Thus, mutation of nearby residues could potentially affect BS1 binding indirectly by disrupting water binding. Notably, the structural rationale for the effects of mutating Ser-179 (32) adjacent to Wb has not previously been adequately accounted for.

Although the C-terminal part of loop 1 has been subject to a close mutational scrutiny (26), its N-terminal part has received less attention. Removal of the Cys-58-Cys-174 disulfide bond, tethering loop 1 to helix 4, essentially eliminates binding (26), and deletion of the amino acid sequence 41-52 (33) leads to a marked reduction in binding affinity, implying that the segment spanning the C terminus of helix 1 and Cys-58 is important for lactogenic activity. The present crystal structure demonstrates that residues (Ile-51, Thr-52, Ile-55, and Asn-56) situated in the N-terminal part of loop 1 are involved in receptor interaction and should be considered as belonging to BS1. Receptor-induced structuring of loop 1 in PRLv2 is similarly observed for GH. Thus, a single {alpha}-helical turn, denoted minihelix 1, is stabilized in the hGH molecule when it binds to GHR-ECD (12). In PRLv2, Asn-56 is engaged in tight hydrogen bond interaction with receptor residues, both via main chain and side chain amides. Interestingly, we have shown that Asn-56 is the primary site of deamidation in the hPRL sequence and that monodeamidated species (N56D mutants) exhibit markedly reduced receptor binding affinity (data not shown). This may have general implications for the design and development of PRL analogues as pharmaceuticals. In particular, for protracted analogues designed to circulate in the bloodstream for a prolonged period of time, inactivation by deamidation is an issue to consider.

The C-terminal disulfide bond breakage in the crystals of PRLv3 and probably in PRLv2·PRLR-ECD renders structural comparison of the C-terminal segments meaningless. For PRL free in solution, the C terminus appears to be relatively dynamic, but reductions in backbone amide proton exchange rates for C-terminal residues, observed by both NMR and HX-MS, demonstrate that the C-terminal segment becomes stabilized in the complex. Furthermore, marked NMR cross-saturation effects were observed for the C-terminal cysteine, demonstrating its intimate contact with the receptor chain.

It has been proposed (34) that PRL BS2 is functionally coupled to BS1 in such a fashion that receptor binding at BS1 increases the affinity at BS2 for a second receptor chain. Based on fluorescence resonance energy transfer data, an allosteric coupling of BS1 and BS2, involving receptor-induced conformational changes in PRL, was suggested (34). However, no detailed molecular mechanism was formulated, and the extent to which PRL actually undergoes significant structural rearrangements when interacting with the receptor chain via BS1 has been a matter of some debate. In the GH system, it has been demonstrated that BS1 and BS2 are allosterically coupled in the sense that mutations in BS1 can induce conformational changes in BS2 (35). The recent publication of the crystal structure of the PRLR antagonist, PRLv3 (16), in combination with the present determination of the receptor-complexed structure of the closely related variant, PRLv2, provides a unique opportunity to scrutinize the structural rearrangements associated with receptor binding at BS1. However, when comparing free and receptor-bound conformations using x-ray structures, the effects of crystal packing effects need to be carefully considered. To distinguish structural changes induced by crystal packing effects from changes directly associated with receptor interaction, solution state techniques, such as HX-MS and NMR, can provide important information. Several residues distributed throughout the primary sequence of PRLv1 exhibit significant amide proton chemical shift changes upon binding to PRLR-ECD (Fig. 2A). Chemical shifts can be affected either directly by contacts with the receptor at the binding interface or indirectly by conformational changes induced by binding. The perturbations observed in the N-terminal part of helix 4, which is not part of BS1, are in accordance with the structural changes in the long overhanging loop 1 implied by the crystal structures of free PRLv3 and receptor-bound PRLv2 (Fig. 2B). The NMR data thus indicate that the structural changes in loop 1 observed in the x-ray data are genuinely receptor-induced and not related to crystal packing effects.

Based on structural analysis, it was suggested for GH (and supported by mutagenesis data) (22) that a communication between BS1 and BS2 is transmitted by structural rearrangements through a contiguous hydrophobic motif comprising Phe-44, Leu-93, Tyr-160, Leu-163, and Tyr-164. The corresponding residues in the PRL sequence are Phe-50, Leu-98, Tyr-169, Leu-172, and His-173. Of these, only Phe-50 undergoes significant movements upon receptor interaction, whereas only subtle changes are observed for Leu-98, Tyr-169, Leu-172, and His-173. This implies that such a mechanism is most probably not operational in the PRL molecule.

As discussed above, receptor interaction of PRLv2 at BS1 induces part of the first loop to undergo large scale backbone movements, which appear to influence the stability of the C-terminal turn of helix 1. However, as evident from Fig. 2B, the relative position of the four-helix bundle backbone, including helix 3, harboring critical determinants of BS2 binding, remains essentially unaffected by BS1 binding. This is also reflected in the insignificant NMR chemical shift perturbations of helix 3 residues induced by receptor interaction (Fig. 2A). Thus, structural comparison of the free and bound states provides no evidence for an allosteric coupling of BS1 and BS2 in the G129R mutants. It is interesting, however, that the HX-MS data imply that receptor binding at BS1 modulates the backbone dynamics of the helix bundle, including helix 2 and 3, which are located on the opposite side of the cytokine from BS1 and in part harbor BS2. Recently, it was suggested that the intrinsic dynamic properties associated with PRL are essential for its ability to elicit a fully functional response through PRLR (16).

As shown by HX-MS comparison of free PRLv2 and free wtPRL, the N-terminal half of helix 1 in PRLv2 appears significantly destabilized relative to wtPRL, most likely as a consequence of the N-terminal deletion. This destabilization could be a major contributing factor to the attainment of full antagonistic properties of N-terminally deleted G129R-PRL variants. The influence of the dynamic properties of PRL variants on their receptor binding characteristics deserves further experimental investigation.

In summary, we have determined the crystal structure of a PRL variant bound to PRLR-ECD, the first structure reported for a PRL molecule bound to its cognate receptor. Additionally, we have characterized the PRL/PRLR-ECD interaction in liquid state using HX-MS and NMR, providing important insights in the solution structure and dynamics. The presented data adds important information to the structural characterization of the PRL system, improving the basis for further rational design and development of novel PRL molecules.


   FOOTNOTES
 
The atomic coordinates and structure factors (code 3D48) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. 1. Back

1 Present address: University of Copenhagen, Universitetsparken 2, Copenhagen DK-2100, Denmark. Back

2 Present address: University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark. Back

3 To whom correspondence should be addressed. Tel.: 45-44434318; E-mail: jbre{at}novonordisk.com.

4 The abbreviations used are: PRL, prolactin; hPRL, human PRL; PRLR, prolactin receptor(s); GH, growth hormone; GHR, growth hormone receptor(s); PRLR-ECD, PRLR extracellular domain; GHR-ECD, GHR extracellular domain; HPLC, high performance liquid chromatography; MS, mass spectrometry; HX-MS, hydrogen exchange mass spectrometry; PL, placental lactogen; BS1 and -2, binding site 1 and 2, respectively; wtPRL, wild type PRL. Back

5 Residue abbreviations without and with subscript R refer to the PRL and PRLR-ECD sequences, respectively. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Thomas Krogh and Dr. Per F. Nielsen for performing MS/MS analysis.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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