Structural Characterization of the Stem-Stem Dimerization Interface between Prolactin Receptor...

doi:10.1016/j.jmb.2010.09.036 J. Mol. Biol. (2010) 404, 112–126

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Journal of Molecular Biologyj ourna l homepage: ht tp : / /ees .e lsev ie r.com. jmb

Structural Characterization of the Stem–StemDimerization Interface between Prolactin ReceptorChains Complexed with the Natural Hormone

Jan van Agthoven1,2, Chi Zhang3,4, Estelle Tallet3,4, Bertrand Raynal5,6,Sylviane Hoos5,6, Bruno Baron5,6, Patrick England5,6,Vincent Goffin3,4 and Isabelle Broutin1,2⁎1CNRS UMR 8015, Laboratoire de cristallographie et RMN biologiques, F-75006 Paris, France2Université Paris Descartes, Faculté de Pharmacie, F-75006 Paris, France3Inserm, U845, Centre de Recherche "Croissance et Signalisation", Equipe "Physiopathologie des hormones PRL/GH",Paris F-75015, France4Université Paris Descartes, Faculté de Médecine, site Necker, F-75015 Paris, France5Institut Pasteur, Plateforme de Biophysique des Macromolécules et de leurs Interactions,Département de Biologie Structurale et Chimie, F-75015 Paris, France6CNRS URA 2185, F-75015 Paris, France

Received 12 July 2010;received in revised form15 September 2010;accepted 16 September 2010Available online25 September 2010

Edited by I. Wilson

Keywords:prolactin;growth hormone;receptor dimerization;structure;surface plasmon resonance

*Corresponding author. E-mail addAbbreviations used: PRL, prolacti

ECD, extracellular domain; GHR, G

0022-2836/$ - see front matter © 2010 E

The most promising approach to targeting the tumor-growth-promotingactions of prolactin (PRL) mediated by its autocrine/paracrine pathway hasbeen the development of specific PRL receptor (PRLR) antagonists.However, the optimization of such antagonists requires a thoroughunderstanding of the activation mechanism of PRLR. We have thusconducted a systematic X-ray crystallographic study in order to visualizethe successive steps of PRLR activation by PRL.We report here the structureat 3.35 Å resolution of the 1:2 complex between natural PRL and two PRLRchains (PRLR1 and PRLR2), corresponding to the final activated state ofPRLR. Further than our previously published structure involving anaffinity-matured PRL variant, this structure allowed to visualize for the firsttime the loop L5 spanning PRLR2 residues Thr133-Phe140, revealing itscentral implication for the three intermolecular interfaces of the complex.We equally succeeded in obtaining a comprehensive picture of the PRLR–PRLR dimerization interface, also called stem–stem interface. Site-directedmutagenesis was conducted to probe the energetic importance of stem–stem contacts highlighted by the structure. Surprisingly, in spite ofsignificant structural differences between the PRL/PRLR2 complex andthe 1:2 growth hormone/growth hormone receptor complex, our muta-tional data suggest that hot-spot residues that stabilize the receptordimerization interface are equivalent in the two complexes. This studyprovides a new overall picture of the structural features of PRLR involved instabilizing its complex with PRL.

© 2010 Elsevier Ltd. All rights reserved.

ress: [email protected]; PRLR, PRL receptor; GH, growth hormone; PL, placental lactogen; wt, wild type;H receptor; SPR, surface plasmon resonance; PDB, Protein Data Bank.

lsevier Ltd. All rights reserved.

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http://dx.doi.org/10.1016/j.jmb.2010.09.036

1131:2 Natural Prolactin / Receptor Complex

Introduction

Prolactin (PRL) is a single-chain protein hormonethat is closely related to growth hormone (GH) andplacental lactogen (PL).1 The anterior pituitary is themajor source of PRL, but the hormone is synthesizedand secreted in many other tissues.2 Overall, severalhundred different functions have been reported forPRL in various species, but its main actions arerelated to pregnancy and lactation.3,4 Its predomi-nant role in the proliferation and differentiation ofthe mammary gland rapidly suggested that it couldbe involved in breast cancer.5,6 Several in vivoexperiments performed using genetically modifiedmice confirmed this hypothesis.7–9 In humans, theprotumoral role of PRL also seems to involve anautocrine/paracrine pathway independent of thepituitary source.10,11 In contrast to the latter,extrapituitary PRL expression is not regulated bydopamine.2 Consequently, dopamine agonists clas-sically used for treating hyperprolactinemia areinefficient for targeting the autocrine/paracrinePRL loop. This led several teams to investigatenew strategies aimed at targeting PRL actions ratherthan PRL expression, which resulted in the devel-opment of PRL analogs acting as competitiveantagonists of the PRL receptor (PRLR).12–14 Unfor-tunately, although these antagonists are promisingtherapeutic candidates, their affinity for PRLR isweaker than that of wild-type (wt) PRL. To improvethe affinity and efficiency of these molecules, weneed to understand the precise mechanism of PRLRactivation, a question on which our team hasfocused for several years using a crystallographicapproach.15,16

PRL shares the canonical fold of the othermembers of the cytokine family, which is composedof four helices bundled in an up–up down–downorientation and linked to each other by one shortloop and two long loops.17 PRLR is composed of (i)one N-terminal extracellular domain (ECD) com-prising two subdomains termed D1 and D2, each ofwhich adopts the fibronectin type III fold;18 (ii) onetransmembrane helix; and (iii) a C-terminal cyto-plasmic domain of unknown structure that trans-duces the signal triggered by PRL binding throughvarious signalling cascades, including the canonicalJak2/Stat5 pathway.3 The mechanism by whichPRL induces PRLR activation has been largelystudied by our group using several experimentalapproaches.1,19 These studies led to the proposal ofa sequential activation model in which PRL firstbinds to one PRLR chain with a nanomolar affinity,forming the site 1 interface, and then to a secondPRLR chain with a micromolar affinity, forming thesite 2 interface. However, recent studies havesuggested that preformed PRLR homodimers likelyexist at the membrane prior to PRL binding,20–22although uncertainties remain as to whether this

applies to the entire receptor population or only toa part of it. Depending on whether PRL meetsPRLR monomers or inactive preformed homodi-mers, binding at site 1 would be the first step totrigger either PRLR dimerization19 or, alternatively,a conformational change(s) within the preformeddimer allowing binding of PRL at site 2, finallyleading to downstream PRLR signaling. These twomechanisms are not mutually exclusive, as bothreceptor stoichiometries may coexist at the cellsurface. In this process of receptor assembly andactivation, the role of the PRLR–PRLR dimerizationinterface, called site 3 or stem–stem interface, iscompletely unknown.A large part of the current knowledge about the

molecular mechanisms of PRLR activation has beenextrapolated from studies on GH and the GHreceptor (GHR),23,24 which have suggested thatGH binding induces extracellular structural mod-ifications of GHR that are then transmitted insidethe cell, tuning precisely the different transductionpathways.25 In order to broaden the analysis to thePRL/PRLR system and to improve current antago-nists, we have to obtain an atomic-scale insight intothe different steps of PRLR activation and, inparticular, to determine the structure of the differentforms of 1:1 and 1:2 PRL/PRLR complexes involv-ing either wt PRL or PRL variants.One of the recurrent problems encountered in

determining the PRL/PRLR2 structure has been thepoor stability of this ternary complex in vitro.Although the formation of 1:2 complexes has beendemonstrated by surface plasmon resonance (SPR),site 2 interaction appeared to be too transient forcrystallographic studies.15,16,26 To circumvent thisproblem, we first aimed at generating an affinity-matured hPRL (h for human) variant that is able toform stable 1:2 complexes: this approach allowed usto solve the crystal structure of the 1:2 complexinvolving the most potent affinity-matured hPRL(hPRLFull-oPL) and the ECD of rPRLR (rPRLR-ECD; rfor rat).16 This structure mimics the final state ofactivation of PRLR by PRL, even if it does not involvethe natural hormone. In an attempt to characterizethe intermediate 1:1 complex, we decided to cocrys-tallize rPRLR-ECD with the wt hPRL, as it wasexpected that no stable 1:2 complex involving thesetwo proteins would be isolated. However, weobtained conflicting gel-filtration and native-gelresults on the nature of the hPRL/rPRLR-ECDcomplex formed in vitro, an inconsistency thatcould be solved by a thorough characterization ofthe stoichiometry of the complex in solution byanalytical ultracentrifugation. The wt hPRL/rPRLR-ECD 1:2 complex structure at 3.35 Å resolution thatwe report here highlights the natural site 2 PRL/PRLR interface and allows a meticulous parsing ofthe site 3 stem–stem interface, supported by site-directed mutagenesis studies.

114 1:2 Natural Prolactin / Receptor Complex

Results and Discussion

Characterization of the stoichiometry of the wthPRL/rPRLR-ECD complex in solution

We analyzed the wt hPRL/rPRLR-ECD complex,later called hPRL/rPRLR, on a size-exclusion chro-matography column, calibrated using monomerichPRL and rPRLR-ECD, the hPRLFull oPL/rPRLR2complex, and different globular protein molecularmass standards (Fig. 1a). The elution volume of thehPRL/rPRLR complex was lower than that ofunliganded hPRL or rPRLR-ECD, but higher thanthat of the well-characterized 1:2 hPRLFull oPL/rPRLR2 complex, whose structure was recentlysolved.16 With the calibration of the column takeninto account, the elution volume of the hPRL/rPRLRcomplex corresponded to an apparent molecularmass of ∼50 kDa, close to that expected for the 1:1complex. This was further confirmed by the size-exclusion analysis of the 1:1 complex formed byrPRLR-ECD and the antagonist Del1-9-G129R-

Fig. 1. Characterization of PRL/PRLR stoichiometry in solu(wt, or variant hPRLFull-oPL or Del1-9-G129R-hPRL), rPRLR-ECto facilitate their comparison. The molecular masses (kDa) oindicated at their respective elution volumes (67: albumin; 43:(b) Analytical ultracentrifugation analysis of hPRL/rPRLR mix1:4, broken curve). Continuous sedimentation coefficient dcomparison. Sedimentation coefficients are expressed in svedb

hPRL, whose mutated site 2 is unable to bindPRLR.15 However, the hPRL/rPRLR complex wasalso controlled for homogeneity on a native acryl-amide gel prior to crystallization after being concen-trated to 5 mg ml−1. Under these conditions, weobserved surprisingly that its migration appeared tobe similar to that of the hPRLFull oPL/rPRLR2complex, which was used as a mass marker (Fig. S1).In view of these conflicting results, we conducted

analytical ultracentrifugation sedimentation velocityexperiments to further characterize the hPRL/rPRLR complex in solution (Fig. 1b). In a firstexperiment, in which rPRLR was mixed with hPRLin a 4:1 ratio, two species were observed: one with asedimentation coefficient s20,w of 2.3 ± 0.2 S(corresponding to excess monomeric rPRLR) andthe other with a s20,w of 4.7±0.2 S, compatible with a1:2 hPRL/rPRLR2 complex (Fig. 1b, broken curve)and similar to that found for the hPRLFull oPL/rPRLR2 complex.16 On the contrary, when hPRLwas in 4-fold excess over rPRLR, we observed twoother species—the first with a s20,w of 2.5±0.3 S(excess hPRL) and the second with a s20,w of 3.9±

tion. (a) Size-exclusion chromatography analysis of hPRLD, and their complexes. Elution profiles were normalizedf the globular proteins used for column calibration areovalbumin; 25: chymotrypsinogen; 17.7: ribonuclease A).tures at different ratios (4:1, plain curve; 1:1, dotted curve;istribution profiles were normalized to facilitate theirergs (1 S=10−13 s).


0.3 S—that are compatible with a 1:1 hPRL/rPRLRcomplex (Fig. 1b, plain curve). Finally, when hPRLand rPRLR were mixed in equimolar proportions,we observed a composite peak with a s20,w of 4.3±0.3 S correspondingmost likely to amixture of the 1:1and 1:2 complexes (Fig. 1b, dotted curve). Thus, insolution, the hPRL/rPRLR complex appears toundergo a fast equilibrium between the 1:1 stateand the 1:2 state, which can be shifted towards thelatter either by increasing the total protein concen-tration (as in the native-gel experiment) or by addingrPRLR in molar excess over hPRL (as in the firstanalytical ultracentrifugation experiment).

Global description of the structure of the hPRL/rPRLR2 complex and comparison with that of theaffinity-matured hPRLFull‑oPL/rPRLR2 complex

Consistent with the native-gel and analyticalultracentrifugation results, the X-ray structurerevealed a 1:2 hPRL/rPRLR-ECD complex, latercalled hPRL/rPRLR2 (Fig. 2a). Its hydrodynamiccharacteristics were calculated from the crystalstructure, giving a sedimentation coefficient of4.66 S, in excellent agreement with the analyticalultracentrifugation experimental value (4.7±0.2 S).The dimensions and shape of the crystallizedcomplex are therefore consistent with those of thecomplex in solution.As expected, the structure of the hPRL/rPRLR2

complex is globally similar to that of thehPRLFull oPL/rPRLR2 complex, which we recentlydetermined.16 The root-mean-square deviation(RMSD) between the two structures, calculated onthe common main-chain atoms, is 1.54 Å for thehormone, and 0.54 Å and 0.98 Å for the two rPRLR-ECD chains, respectively. While the superimposi-tion is excellent at the level of site 1 interface(involving PRL and chain PRLR1) and at the level ofthe glycine cavity of PRL involved in site 2 interface(with chain PRLR2), with only a few differencesmainly localized in noninteracting regions andexternal loops, the higher resolution of the hPRL/rPRLR2 complex structure (3.35 Å against 3.8 Å)allowed several additional details to be constructed.In hPRL, the long loop connecting the first two

helices is now complete. The first part of this loop(residues Arg43-Thr52) was never built in totality inthe other complex structures involving PRLR, evenin those solved at high resolution such as oPL (o forovine)/rPRLR2 (2.3 Å)27 and hPRL/hPRLR1(2.5 Å).28 This loop may be of functional relevanceto the functional coupling of sites 1 and 2, as reflectedby the drastically altered biological properties of theΔ41–52-hPRL mutant.14 Another significant addi-tional detail concerning hPRL is the presence of thedisulfide bond at the C-terminus. Only three of thesix Protein Data Bank (PDB) structures of PRL or PL,alone or involved in a complex, reveal this disulfide

bond that is supposed to be characteristic of thesehormones, and this presence cannot be correlatedwith the pH of the crystallization conditions. On thecontrary, this disulfide bondwas always observed inGH structures.PRLR1 does not present any differences with the

equivalent moiety in the hPRLFull-oPL/rPRLR2 struc-ture, except for two residues (Asp118-Lys119)missing in the structure described herein. Of interest,no difference could be detected in the regioninteracting with the C-terminus of PRL (Val135-Gly138PRLR1) in spite of the presence of the above-mentioned disulfide bond. As for PRLR2, the loopbetween the second β-sheet and the third β-sheet(Thr28-Gly31) is now closed, and the neighboringloop (Asn83-Ser87) is shifted by more than 3 Å.The main differences between the two structures

are localized at the hormone N-terminal interfaceand at the upper part of the PRLR–PRLR interface,also called stem–stem interface or site 3, with theconstruction of loop Thr133-Phe140 in PRLR2 thathad never been seen in any other 1:2 complexinvolving PRLR (Fig. 2; Fig. S2). It is not surprisingthat the structure of the N-terminus of hPRL differsfrom that of the affinity-matured variant hPRLFull oPL

as, in the latter, the natural PRL N-terminus(LPICPGGAARCQVT) was replaced with that ofoPL (AQHPPYCRNQPGKCQIP). Divergences werelocalized ahead of the first cysteine (numbered Cys4in hPRL). In hPRLFull oPL, there were threeN-terminalcontacts with rPRLR involving the main chain ofHis0Full oPL (see Fig. 2a) and the side chains ofPro1Full oPL and Tyr3Full oPL, while in wt hPRL, there isonly one van der Waals contact involving Pro2hPRL.16Furthermore, the presence of the oPL N-terminus inhPRLFull oPL pushed Pro186-His188PRLR2 towardsThr141PRLR2,16,27 while in the wt hPRL/rPRLR2 com-plex, the Pro186-His188PRLR2 loop is closer to theN-terminus of hPRL, resulting especially in a differentside-chain orientation of His188. Of special interest, themovement of the equivalent loop in GHR (Gln216-Ser219) has been shown to switch its signaling partnerfrom Jak2 to an Src family kinase by regulating thetransmembrane domain realignment.25 The move-ment of the Pro186-His188PRLR2 loop towards thePRL N-terminus provides a space that becomesavailable for loop Thr133-Phe140PRLR2 (Fig. 2a,zoom-in). As can be seen in Fig. 2, thisThr133-Phe140PRLR2 loop, also called loopL5PRLR2, is localized at a central position in thecomplex, participating in all three interfaces, andforms a sort of stretcher on which PRL helix 1 lies.This position of loop L5 forces us to revisit the

definition of the different binding sites. To date, PRLsite 1 was thought to correspond to residuesinteracting only with PRLR1, and site 2 was thoughtto correspond to those interacting only with PRLR2.This definition was based on the structural analysisof the different PRL (and GH) complexes, together

Fig. 2. The hPRL/rPRLR2 complex structure. (a) Global cartoon representation of the complex structure. Residuespresented in ball-and-stick correspond to the N-terminus of PRL and to loop Thr133-Phe140PRLR. A highlight of the N-terminal region, superimposed on the hPRLFull-oPL/rPRLR2 structure (dark blue; PDB code 3EW316), is presented in theblowup. The N-terminal sequence alignment of hPRL and oPL is indicated for clarity. (b) Zoom on loop L5. The tablesummarizes the involvement of loop L5 in the different interfaces, as described in the text. The figure illustrates theenvironment of loop L5 (dark brown), with all the residues mentioned in the text presented as sticks.


with alanine scanning mutagenesis. It had thus beendetermined that PRL site 1 was composed of helix 1residues (Val23, His27, His30, Asn31, and Phe37),

loop Lys53-Ser57, residue His59, loop Pro66-Lys69,helix 4 residues (Tyr169, His173, Arg176, Arg177,His180, Lys181, Asp183-Tyr185, Lys187, Leu188,

image of Fig. 2


and Cys191), and the C-terminus. Similarly, PRL site2 had been shown to be composed of the N-terminus(Leu1-Val13), helix 1 residues (Asp17, Arg21, Leu25,and Tyr28), and helix 3 residues (Arg125, Glu128,Gly129, and Leu132). However, our hPRL/rPRLR2structure now shows that loop L5 from PRLR2 isengaged in interactions with PRL residues that hadbeen attributed to site 1. However, for the sake ofsimplicity, the classical definition of the two differentPRL sites will be retained in the following sections toallow for an easy comparison with previous reportsdescribing homologous complexes (Fig. 2a).At “site 1,” the loop L5PRLR2 residues engaged in

the larger number of contacts with PRL areThr137PRLR2 and Trp139PRLR2, which interact withresidues from PRL helix 1 (Val23, Val24, His27, andAsn31) andwithLys187PRL, aswell aswithThr141PRLR1

from loop L5PRLR1 (Fig. 2b). It should be noticed thatthe side chain of Lys187PRL is stacked between that ofThr141PRLR1 and that of Asp183PRL. The GH residueequivalent to the latter (Glu174GH) is involved in thebinding of Zn2+ and plays a crucial role in the hGH/hPRLR1 interface, as itsmutation in alanine results ina 350-fold reduction of the affinity of hGH forhPRLR-ECD.29 This connects loop L5PRLR2 to theGH/PRLR Zn binding pocket, highlighting itsimportance at “site 1.” At the opposite extremity ofloop L5PRLR2, Thr133PRLR2 is directly involved in the“site 2” interface, interacting with Arg125PRL, aresidue of major importance for the site 2 hydrogen-bond network.16 Finally, Phe140PRLR2 is involved inthe “site 3” interface, which will be described in itstotality in Description of the Stem–Stem Interface(Site 3).The stabilization of loop L5PRLR2 in our 3. 35-Å-

resolution structure is quite amazing, as it had neverbeen built previously in any 1:2 complex involvingPRLR. In addition to the displacement of the Pro186-His188PRLR2 loop, an explanation can be provided bythe comparison of the buried areas at the differentinterfaces. At the hormone N-terminus, the buriedarea decreases from 690 Å2 in hPRLFull oPL/rPRLR2to 500 Å2 in hPRL/rPRLR2. The buried areas for thePRL site 2 glycine cup and site 1 are equivalent in thetwo complexes, accounting for 850 Å2 and 2370 Å2,respectively. However, the constructed Thr133-Phe140PRLR2 loop contributes 584 Å2 of additionalburied surface to the site 1–site 2 interfaces, and315 Å2 to the site 3 interface. This increase ininteracting surface presumably balances the loss atthe N-terminus compared to the hPRLFull oPL/rPRLR2 complex. This is in accordance with the“three-pin plug” hypothesis described by Broutin etal., which states that the formation of a stable 1:2PRL/PRLR2 complex requires the integrity of threeconstitutive elements of PRL site 2: the glycine cuparea, a group of hydrogen bonds surrounding thiscup, and the N-terminal region.16 In the structuredescribed herein, the first two elements are con-

served, while the N-terminal interaction is reducedwith respect to the hPRLFull-oPL/rPRLR2 complex,with this reduction being compensated for by thesupplemental contacts provided by the stabilizedThr133-Phe140PRLR2 loop.

Description of the stem–stem interface (site 3)

We decided to pay special attention to thePRLR–PRLR stem–stem interface, as the nature ofthe interactions governing its formation hasnever been described in detail previously. Thereare now three different 1:2 complex structuresinvolving the rPRLR: oPL/PRLR2 (PDB code1F6F27), hPRLFull oPL/PRLR2 (PDB code 3EW316),and the one described here. The backbones of the D2domains of the three complexes, as well as the sidechains of the residues localized in the core of D2(between its two fibronectin type III β-barrels), areperfectly superimposable. In particular, this is thecase for the residue Ile146PRLR, whose mutation intoleucine has been shown to confer constitutiveactivity to the receptor,30 as well as for itssurrounding residues. Ile146PRLR is situated at thecore of the D2 domain; however, modeling itssubstitution into leucine does not allow one toanticipate any significant structural consequence ofthis mutation. Nevertheless, it cannot be completelyruled out that small alterations induced by the I146Lmutation could have repercussions on the stem–stem interface.A detailed comparison of the stem–stem interac-

tions observed in the three complex structures ispresented in Fig. 3a. In consideration of all theresidues involved in at least one of the threestructures, site 3 is composed of 15 residues fromPRLR1 and 16 residues from PRLR2, with 15residues out of the total being aromatic. Thisinterface is not symmetrical, as there is a 12-Åslide between the two PRLR chains. Consequently,the amino acids involved in the interface are not thesame for PRLR1 and PRLR2 (Fig. 3a). Only seven ofthem (His159, Phe160, His163, Phe167, Lys168,Phe170, and Asp171) are common to both; however,even for these residues, the nature of the interactionsand the partners involved are not identical. As canbe seen in Fig. 3a, the bundle of contacts involvingPhe170PRLR1 and Asp171PRLR1 on the PRLR1 side,and the bundle of contacts involving Gln164PRLR2 onthe PRLR2 side suggested that these three residuesplayed a central role in the topology of the PRLR–PRLR interface. Phe170PRLR1 and Asp171PRLR1 circlea depression with Glu157PRLR1, His159PRLR1, andPhe167PRLR1 inside, and with Val169PRLR1 at thebottom (Fig. 3b). Gln164PRLR2 enters into thisdepression, interacting with almost all its residues.Among these contacts, only two could correspond tohydrogen bonds—one with Asp171PRLR1 and an-other with the main chain of Phe170PRLR1, the latter

Fig. 3. PRLR residues involved in stem–stem interfaces. (a) Exhaustive list of PRLR stem residues situated at a distancelower than 4.2 Å in the three 1:2 complex structures involving rPRLR. This high cutoff was chosen due to the lowresolution of the two PRL/PRLR structures. The orange lines connect two noncarbon atoms. PRLR1 residues belonging tothe interface are shown in blue, and PRLR2 residues are shown in brown. (b) Stereo view of the interface. PRLR1 ispresented as a semitransparent surface with a color gradient ranging from light blue to dark blue, in view of the number ofinteractions presented in (a). Only contact residues are presented for PRLR2. The color gradient ranges from yellow to red,following the same criteria. The two lonely hydrogen bonds are indicated as yellow broken lines. Residue numbers areindicated in blue for PRLR1 and underlined in red for PRLR2. D171, shown in white for clarity, is part of PRLR1.


image of Fig. 3


being the only hydrogen bond common to the threestructures. Another singularity of the stem–steminterface is the presence of a negatively chargedsurface on PRLR1 formed by two glutamic acids(Glu155PRLR1 and Glu157PRLR1) and Asp171PRLR1,which forms a favorable cup for Lys168PRLR2.The principal differences in the binding schemes

of the three structures of 1:2 complexes involvingPRLR are mainly due to the presence of the Thr133-Phe140PRLR2 loop L5, which is only visible in thedensity of the structure described herein. This loopextends the stem–stem region, which is no longerlimited to the bottom of the D2 domain (Fig. 3b).Consequently, the interacting surface area is en-larged and reaches 1410 Å2 for this complex against1060 Å2 for hPRLFull oPL/PRLR2 and against 1110 Å2

for oPL/PRLR2. The hot spot of this additionalinteracting region is localized at Trp139PRLR2, whichcaps His163PRLR1, and at Phe140PRLR2, which entersa hydrophobic pocket formed mainly of aromaticresidues (Phe160PRLR1, His163PRLR1, Phe167PRLR1,Pro129PRLR2, Val135PRLR2, and His163PRLR2) (Fig.2b). Nevertheless, the principal region involved insite 3 is still the bottom of the D2 domain aroundresidues Glu157-Asp171PRLR (Fig. 4).

Comparison with the hGH/GHR2 complex

The stem–stem interactions observed for the PRLRand GHR complexes are quite different (Fig. 4) evenif two common regions are involved. In the GHRcomplex, most of the interacting residues are part ofthe Leu142GHR-Asp152GHR loop (equivalent toLys114PRLR-Tyr122PRLR). There are six hydrogenbonds at the GHR–GHR interface, three of whichinvolve residues from this loop on both ECDs(Ser145GHR1-Asp152GHR2, Thr147GHR1-Asp152GHR2,and His150GHR1-Asn143GHR2), while two othersinvolve residues from this loop on GHR1 only(Leu146GHR1-Ser201GHR2 and Asp152GHR1-Tyr200GHR2). The equivalent loop in PRLR, whichhas no density at the level of residues 116–118PRLR2

in our structure, is two residues shorter (Fig. S3) andtherefore unable to get involved in PRLR–PRLRinteractions. As a result, it is not stabilized in PRLR2,explaining why it is not visible in the electron densitymap. Due to the absence of this loop at the interface,the two PRLRD2 domains are closer than in the GHRcomplex. Consequently, the interface is completelydifferent in the PRLR complex, even if one commonregion is involved in the stem–stem interface in thetwo complexes (residues 155–173PRLR2, corres-ponding to 185–203GHR2). None of the “Val depres-sion” residues is involved in GHR–GHR contacts,limiting the interacting surface area in this region to afew residues. Most of those residues, even whentopologically equivalent, do not interact in the samemanner, resulting in a different relative position ofthe two receptor's D2 domains. When visualized

perpendicularly to the hormone helices with the twoD1 domains aligned, the two D2 domains are alignedbut shifted vertically with respect to each other in theGHR complex,23 while in the PRLR complex, inaddition to the vertical movement, the two D2domains are twisted with respect to each other(Fig. 4b). This is emphasized when the superimpo-sition of the complexes is performed using thehormone+receptor 1 region as reference (Fig. 4c).The angle between the two domains D1 and D2 doesnot seem to be an intrinsic crucial feature of thesecytokine receptors27,31–34 but more a consequence ofthe twist between the two D2 domains, which coulditself be considered as a signature of each receptor. Insummary, the hot-spot residues in the GHR stem–stem interface are all localized at its bottom in thetwo abovementioned regions. Elsewhere, the dis-tance between the GHR chains is higher than 10 Å,leaving a hole at the middle of the three interactingpartners (GH and the two GHR chains). In thehPRL/rPRLR2 complex, this hole is filled by thenewly built Thr133-Phe140PRLR2 loop. It forms anadditional stem–stem region that has no equivalentin the GHR family. Trp139PRLR1 and Trp139PRLR2 areboth involved in the same “site 1” interface (Fig. 2a),while in GHR, the equivalent Trp169 from GHR1and Trp169 from GHR2 are involved exclusivelyeither in site 1 or in site 2 (Fig. S4).

Site-directed mutagenesis

The energetics governing PRLR homodimeriza-tion has never been investigated. Our structuralanalysis led to the proposal of a list of potential hot-spot residues, and a site-directed mutagenesisanalysis was performed in order to determinetheir functional importance. Loop L5, which couldplay a central role in PRL/PRLR interactions assuggested above, was not considered for mutagen-esis in this work as it is involved simultaneously inall three binding interfaces. Among the residuesinvolved in stem–stem contacts, we focused ourattention on a subset of those that appeared tobe essential on a structural basis: Gln164PRLR,Lys168PRLR, Phe170PRLR, and Asp171PRLR. Theseresidues were mutated into alanine to disrupt oneor several interreceptor contacts while attemptingnot to disturb the global structure of PRLR-ECD.Wewere able to produce and purify all mutated ECDs,except for D171A, which suggests that this residuemay play a central role in the proper folding of ECDs.We verified, by size-exclusion chromatographycoupled with static light-scattering detection, thatthe wt and the three mutant ECDs were allmonomeric in solution and that no significantaggregation could be detected (data not shown).Furthermore, we analyzed the secondary folding ofthe different ECDs by far-UV circular dichroismspectroscopy. All the spectra exhibited a positive

Fig. 4. Buried areas at the stem–stem interface. (a) Comparison of the buried areas for residues involved in thereceptor–receptor interacting region for all of the 1:2 complexes of the family: wt hPRL/rPRLR2 (this work), hPRLFull-oPL/rPRLR2 (PDB code 3EW3), oPL/rPRLR2 (PDB code 1F6F), and hGH/hGHR2 (PDB code 1HWG). Residues indicated areinvolved in close noncarbon interactions. (b) Side view (perpendicular to the hormone helix 1) of the complexes, with thereceptors presented as surfaces and with the hormones presented as cartoons. The shift between the two receptor moietiesis sketched by arrows. (c) Superimposition of the two complexes based on a hormone +receptor 1 three-dimensionalalignment, presented perpendicularly to the (b) view. The GH/GHR complex is shown in yellow. The large shift (∼10 Å)between the two PRLR2 D2 domains is materialized by a dotted line.


ellipticity at 230 nm and a broad negative ellipticityat 210–213 nm (Fig. S5). This profile is a character-istic signature of the unliganded ECDs of PRLR andseveral other cytokine receptors, composed of twoanti-parallel β-sheet fibronectin III domains and aTrp-Ser-X-Trp-Ser motif in the D2 domain.35 The

affinities of hPRL for the four PRLR1-ECDs (site 1interface), as well as the affinities of the fourPRLR2-ECDs for the four hPRL/PRLR1 1:1 com-plexes (site 2+site 3 interfaces), were determinedusing the real-time SPR assay that we hadpreviously set up.15,16 As expected, the mutations

image of Fig. 4


had a negligible effect on the site 1 interactionparameters. On the contrary, the impact of several ofthe mutations on site 2+ site 3 affinities wassignificant (Fig. 5 and Table 1). The most importantdecreases in affinity (about 10-fold) were obtainedfor the F170APRLR2 and K168APRLR2 mutations. Incontrast, the F170APRLR1 and Q164APRLR2 muta-tions, which were expected to have a major impactbased on the structural analysis of the site 3interface, led to a 2-fold decrease and a 2-foldincrease in site affinity, respectively. This suggestseither that the bundle of contacts established byPhe170PRLR1 and Gln164PRLR2 (Fig. 3a) has nosignificant importance for site 3 interface stabilityor that the effect of the side-chain mutationsinvolving these residues can be compensated forby the establishment of alternative contacts betweenthe two PRLR-ECDs. Interestingly, it was only in thecontext of the F170APRLR1 mutant that theQ164APRLR2 mutation led to a slight decrease insite 2+site 3 affinity, suggesting a certain degree ofcooperativity between the two mutations. As thePRLR–PRLR binding hot spots did not appear tocorrespond to those suggested by the structure,except for Lys168PRLR, we compared our muta-genesis results with those of alanine scanningmutagenesis performed on the GHR–GHR inter-

Fig. 5. Real-time SPR characterization of site 2+site 3 intmutated rPRLR-ECDs were immobilized on a Ni-NTA sursensorgrams corresponding to (a) the injection of wt rPRLR-ECmutated rPRLR-ECDs. (b) The injection of wt or mutated rPRLcomplexes. The PRLR2-ECD concentration dependence of threported in Table 1 were determined, is shown to the right of

face. This study had led to the identification ofresidues Tyr200GHR-Ser201GHR and loop Asn143-Asp152GHR2 as energetic hot spots of the GHR–GHR interface.36 The equivalent loop in PRLR(Gln115-Tyr122PRLR) is shorter and does notappear to be involved in the PRLR–PRLR struc-tural interface. As for residue Ser201GHR

(corresponding to Asp171PRLR), it had beenshown to be required for proper signal transduc-tion using a c-fos promoter-driven luciferasereporter assay.37 Unfortunately, its role for PRLRcould not be experimentally demonstrated due tothe fact that the mutation of Asp171PRLR intoalanine resulted in an insoluble PRLR-ECD. As forresidueLys168PRLR2,which is responsible for a 10-folddecrease in site 2+site 3 affinity, its equivalent residue,Pro198GHR2, was never considered as a potential hotspot in the GHR–GHR interface and thus was nevermutated, rendering impossible a comparison betweenthe two families. Nevertheless, in GHR, Pro198GHR2 isdirectly surrounded by Tyr200GHR1, Ser201GHR1, andTyr200GHR2; it can therefore be hypothesized that itcould play an important role in the GHR–GHRinterface. In PRLR, Lys168PRLR2 is in direct contactwith Asp171PRLR1. As a lysine is a long-chargedresidue compared to a proline, this residue mayinduce some local rearrangements to prevent steric

eractions involving wt and mutated rPRLR-ECDs. wt orface and saturated with PRL (250 nM). RepresentativeD (8.75 μM) into preformed 1:1 complexes involving wt orR-ECDs (≈3 μM) onto preformed 1:1 PRL/wt rPRLR-ECDe steady-state SPR responses, from which the Kd valuesthe figure.

image of Fig. 5

Table 1. Site-directed mutagenesis analysis of the PRLR1–PRLR2 interface

PRLR2

PRLR1

Wt Q164A K168A F170A

wt 3.15±0.16 (1) 3.14±0.26 (1) 2.12±0.11 (0.7) 7.93±0.39 (2.5)Q164A 1.92±0.35 (0.6) 2.24±0.14 (0.7) ND 14.3±1.3 (4.5)K168A 33.4±2.2 (10.6) ND 28.9±2.0 (9.2) 55.0±5.9 (17.5)F170A 35.9±2.5 (11.4) 37.3±2.1 (11.8) 22.2±1.7 (7.0) 52.6±1.0 (16.7)

ND, not determined.The site 2+site3 affinities were determined by SPR. Equilibrium Kd values and corresponding standard errors are shown. The valuesnormalizedwith respect to the Kd of the wt–wt PRLR-ECD interaction (Kd mut/Kd wt) are indicated in parenthesis. PRLR1-ECDmutationsare listed horizontally, and PRLR2-ECD mutations are listed vertically.


and charge repulsions and, thus, be partly responsiblefor the particular twist between the two PRLR D2domains. Finally, the affinity decreases resulting frommutations F170APRLR1 and F170APRLR2 (2-fold and10-fold, respectively) can be correlated with the effectof the equivalent mutations in GHR (20-fold forY200AGHR1 and 300-fold for Y200AGHR2). Tyr200GHR

has also been shown to be important for proper signaltransduction induced by GH binding.37Interestingly, most of these hot-spot residues had

been predicted by Kossiakoff et al. to play animportant role in the hPRLR–PRLR stem–steminterface.31 This assumption was based on a modelof the hPRLR dimerization interface obtained byhomology with the hGH/hGHR2 structure38 inwhich hGH/hGHR1 was replaced by the hGH/hPRLR1 structure18 and GHR2 was replaced by aPRLR molecule. The relative orientation of thereceptor D2 domains is important in determiningthe structural importance of the different residuesinvolved in the stem–stem interface. The crystalstructure we report herein corresponds to aninstantaneous static snapshot of the PRL/PRLR2complex. Taken together with our mutagenesisresults, our study could suggest that, in a first“encounter” step, the two D2 domains might adopta relative position similar to that seen in the GH/GHR complex, and that, in a second step, a relativemovement of the two domains might occur,minimizing steric hindrance and resulting in thefinal locking of the dimerization domains that can bevisualized in our crystal structure. To furtherexplore this two-step mechanism hypothesis, wewill extend our preliminary mutational study to theother residues of the stem–stem interface and wewill have to assess their implication for signaltransduction in functional assays.

Conclusion

For many years, tremendous effort has beeninvested to optimizemolecules that are able to inhibitPRLR activation. At first, the strategy followed wasbased on that which was successful in developingGHR antagonists. As the PRLR antagonists obtained

this way did not fulfill all their expectations, astructural approach was envisaged in order to obtainprecise knowledge of the different binding sitesinvolved in the PRL/PRLR complex. Further thanthe first structure of a 1:2 PRL/PRLR complex thatinvolved an affinity-matured PRL, we now providethe crystallographic structure of the 1:2 complexbetween PRLR andwt PRL. This structure allows, forthe first time, to determine the precise position ofloop L5PRLR2 that had never been visualized in anyother 1:2 complex involving PRLR. This loop islocalized at the center of the PRL/PRLR2 complexand is thus involved in all three binding interfaces.This position of loop L5PRLR2 is specific for the wthPRL/PRLR2 complex and forces us to revisit thedefinition of the different binding sites: indeed, thisloop is part of PRLR2 but interacts with certain PRLresidues previously thought to be involved exclu-sively in the interaction with PRLR1 (and thusascribed to PRL site 1). In order to determine theexact role of this loop at the different binding sites,we need to perform a complete mutagenesis studyinvolving each of the loop residues individuallyand the PRL residues with which they interact.Such athorough structure–function study clearly fellbeyond the scope of the present report.Finally, an analysis of the site 3 (or stem–stem)

interface, highlighting the structural importance ofseveral residues in PRLR1 and PRLR2, was carriedout. The results obtained with two of the site 3alanine mutants were not in accordance with thestructural predictions, but were similar to thoseobtained by mutating the equivalent residues inGHR, in spite of the differences observed in thecrystal structures of the PRLR–PRLR and GHR–GHR stem–stem interfaces. This might be explainedby assuming that the PRL/PRLR and GH/GHRbinding processes involve similar initial recognitionsteps that can be explored by SPR. These encountercomplexes might then undergo different structuralrearrangements, resulting in distinct final lockedstructures that are visualized in their respectivecrystal structures. Despite their global similarities,the PRL and GH complexes reveal a large number ofstructural and functional differences, which justifythe efforts made to dissect the respective activation


mechanisms of both hormone receptors. The struc-ture presented here, with the particular position ofPRLR loop L5 revealed, could be the basis for thedevelopment of a new class of antagonists that arenot limited to the classical approach targeting site 2.

Materials and Methods

Expression plasmids and site-directed mutagenesis

The pQE-70 expression plasmid encoding rPRLR-ECDwas available from previous studies.15 This constructencodes the ECD with a His6-tag at the C-terminal end. Itwas used as template for generating mutated ECDs,using the QuikChange II Mutagenesis kit (Stratagene, LaJolla, CA). The sequences of the forward and reverse(complementary) primers are as follows:

rPRLR Q164A for 5′-gatccattttacaggtcatgcaacacagtt-taaagtttttg-3′rPRLR Q164A rev 5′-caaaaactttaaactgtgttgcatgacctg-taaaatggatc-3′rPRLR K168A for 5′-cacagtttgcagtttttgacctatatccagggc-3′rPRLRK168A rev 5′-gccctggatataggtcaaaaactgcaaactgtg-3′rPRLR F170A for 5′-cacagtttaaagttgctgacctatatccagggc-3′rPRLR F170A rev 5′-gccctggatataggtcagcaactttaaactgtg-3′rPRLR D171A for 5′-cacagtttaaagtttttgccctatatccagggc-3′rPRLR D171A rev 5′-gccctggatatagggcaaaaactttaaactgtg-3′

For all steps, we strictly followed the recommendations ofthe manufacturer. After transformation, Escherichia coliBL21(DE3) colonies were analyzed for their DNA content.Plasmids were sequenced to verify the presence of theexpected mutations.

Production of recombinant proteins

Recombinant wt hPRL and rPRLR-ECDs were over-expressed in 0.5-L to 1 -L cultures of E. coli BL21(DE3) andpurified as described previously.15 Briefly, proteins wereoverexpressed as insoluble inclusion bodies, which weresolubilized in 8 M urea (for 5 min at 55 °C, then for 2 h atroom temperature) and refolded by continuous dialysis(for 72 h at 4 °C) against 100 vol of 25 mM NH4HCO3(pH 8.6). Solubilized proteins were then loaded onto aHiTrapQ anion-exchange column (GE Healthcare) equili-brated in 25 mM NH4HCO3 (pH 8.6). PRL and receptorECDs were eluted along a NaCl gradient (0–500 mM), andthe major peak was collected, quantified, and kept frozenuntil use. The purity of the various hormone/receptor ECDbatches was N95%, as judged from SDS-PAGE analysis.The complex between hPRL and rPRLR-ECD was pre-pared by mixing (in an equimolar ratio) the two purifiedproteins concentrated at 2 mg ml−1 in 25 mM NH4HCO3and 150 mM NaCl (pH 8.6) buffer. The complex waspurified by gel filtration in the same buffer using aSuperdex 75 column (GE Healthcare) and concentratedto a final protein concentration of 5 mg ml− 1. Thestoichiometry and stability analyses of the differentcomplexes and proteins were performed by rerunning

the eluted peaks on the same column. The loaded quantitywas 10 μg in 1 ml for each run, except in the case of rPRLR-ECD for which 25 μg was loaded.

Analytical ultracentrifugation

Sedimentation velocity experiments were carried out at25 °C in an XL-I analytical ultracentrifuge (BeckmanCoulter) equipped with double-UV and Rayleigh interfer-ence detection. Samples were prepared in 25 mMNH4HCO3 and 150 mM NaCl (pH 8.6) and spun usingan An60Ti rotor and 12-mm double-sector aluminumcenterpieces. The partial specific volumes of hPRL(0.733 ml g−1), rPRLR-ECD (0.728 ml g−1), and theircomplexes (0.730 ml g−1) were estimated from their aminoacid sequences using the software Sednterp 1.09 (availableonline from The Boston Biomedical Research Institute).The same software was used to estimate buffer viscosity(η=1.002 cP) and density (ρ=1.0032 g ml−1). The hPRL/rPRLR-ECD mixture (400 μ l at 5 μM:20 μM,12.5 μM:12.5 μM, or 20 μM:5 μM) was spun at36,000 rpm. Absorbance and interference profiles wererecorded every 5 min. Sedimentation coefficient distribu-tions c(s) were determined using the software Sedfit 11.8.39

Theoretical sedimentation coefficients of the complexeswere calculated from the PDB file using Hydropro 7c,40

with a hydrated radius of 2.8 Å for atomic elements.

Crystallization and X-ray diffraction data collection

Initial crystallization screening was performed on 96-well sitting-drop crystallization plates (Greiner Bio-One)using a Cybi-Disk robot (Cybio). Crystallization screenswere set up using several commercially available high-throughput crystallization screening kits (Hampton Re-search). Crystals appeared as either small lozenges orpoint-ended sticks after 2 weeks at 18 °C under condition13 of the MemFac crystallization kit. The crystallizationcondition was optimizedmanually on Limbro plates usingthe hanging-drop method. Crystals appeared in 1 weekand reached a final crystal size of 50 μm×80 μm×100 μmafter an additional month. The reservoir was composed of100 mM Na3-citrate (pH 5.6), 14% polyethylene glycol4000, 100 mM LiSO4, and 12% glycerol. The drop wasformed by mixing 1 μl of protein at 5 mg ml−1 with 1 μl ofreservoir. Crystalswere flash frozen in liquid nitrogen, and180° of reciprocal space was collected on beamline ID14 ofthe European Synchrotron Radiation Facility (Grenoble,France); however, as crystal diffraction suffers fromradiation, only the first 150 images were kept forprocessing. The images were reduced, scaled, and mergedwith the programsMOSFLM and SCALA,41,42 leading to adata set at 3.35 Å resolution with a completeness of 98%.The intensities were then converted into structure factoramplitudes with TRUNCATE.41,42 Crystals belonged tospace group P432121 with unit cell dimensions ofa=93.37 Å, b=93.37 Å, and c=216.13 Å.

Phase determination and structure refinement

The crystal structure was solved by molecular replace-ment using the program Phaser,43 with the 1:2 complexstructure of the recently determined structure of

Table 2. Data collection and refinement statistics

hPRL/rPRLR2

Data collectionBeamline ID14 (European Synchrotron

Radiation Facility)Space group P43212Unit cell parameters a, b, c (Å) 92.17, 92.17, 216.07Resolution (Å) 3.35–84.82 (3.35–3.53)Total number of reflections 160,854 (23,547)Number of unique reflections 13,695 (1938)Completeness (%) 97.8 (97.5)Rmerge

a (%) 13.1 (38.8)I/σ(I) 22.7 (7.6)Multiplicity 11.7 (12.2)

RefinementResolution (Å) 3.35–84.78Number of reflections used (%) 13,675 (97.0)Rcryst/Rfree (%)b 23.4/29.3Estimated coordinate error (Å) 0.44Number of observed

residues/total residues587/627

Mean B value (Å2): global 60.8for each chain A/B/C 56.1/53.2/73.7

RMSD bond lengths (Å) 0.01RMSD bond angles (°) 1.31Ramachandran plot (%)

Most favored regions 88.4Allowed regions 8.9

Values in parentheses are for the highest-resolution shell.a Rmerge=∑h∑j|⟨I⟩h− Ih,j|∑h∑jIh,j, where ⟨I⟩h is the mean

intensity of symmetry-equivalent reflections.b Rcryst=∑|Fobs−Fcalc|/∑Fobs. The formula for Rfree is the

same as that for Rcryst, except that it is calculatedwith a portion (inour case, 5%) of the structure factors that had not been used forrefinement.

†http://pymol.sourceforge.net/‡http://www.rcsb.org/pdb/home/home.do


hPRLFull-oPL/rPRLR2 complex16 used as searchmodel. Thecomplete complexwas easily positioned in one block in theunit cell, and the position of each domain was refined byrigid-body refinement using PHENIX.44 Refinement of thestructure was carried out by multiple cycles of manualrebuilding using the programs Coot45 and PHENIX,resulting in a final model with an Rcryst of 23.4% and anRfree of 29.3%. As the structure is of low resolution, onlyone atomic displacement parameter was refined perresidue. A summary of crystallographic data and refine-ment statistics is given in Table 2. The refined structurewasvalidated both by the MolProbity46 and by the Polygon47

validation tools of PHENIX. The geometry is of goodquality, with 88.4% and 8.9% of the residues in the mostfavored and allowed regions of Ramachandran plots,respectively. The Polygon shows a good agreement of therefined structure with the 606 selected PDB entries ofsimilar resolution (Fig. S6).Most of the amino acids have been positioned in the

electron density; nevertheless, the structure presents somemissing parts. The missing regions correspond to residuesPro5PRL-Arg10PRL, Glu143PRL-Ile146PRL, Gln157PRL-Met158PRL, Asp118PRLR1-Lys119PRLR1, Asp205PRLR1-Ser214PRLR1, Leu116PRLR2-Asp118PRLR2, Glu152PRLR2-Ala153PRLR2, and Asp205PRLR2-Ser214PRLR2 (PRLR1 andPRLR2 refer to receptor molecules interacting with hPRLsite 1 and site 2, respectively). In addition, Met105PRL wasreplaced by an alanine, as only the main chain was definedin the electron density.

Figure panels showing three-dimensional structureswere generated using the PyMOL Molecular GraphicsSystem† (DeLano Scientific).

Circular dichroism

Far-UV circular dichroism spectra (195–260 nm) wereacquired with an Aviv 215 spectropolarimeter (AvivBiomedical) at 25 °C using a cylindrical cell with a 0.02-cm path length and an averaging time of 1 s per 0.5-nmstep. The triplicate spectra of the samples (0.8 mg ml−1),corrected using buffer baselines measured under the sameconditions, were normalized to the molar peptide bondconcentration and path length (1 cm) as mean molardifferential extinction coefficient per residue (Δɛ).

Surface plasmon resonance

The wt and mutant rPRLR-ECDs were immobilizedcovalently onto nitrilotriacetic-acid-derivatized NTA sen-sor chips, as described previously.15,16 Surface densitiesranging from 300 resonance units to 450 resonance units(resonance unit≈pg mm−2) were obtained. Binding assayswere performed at least in triplicate, as previouslydescribed.15 For site 1 characterization, hPRL (1–500 nM)was injected onto the rPRLR-ECD surfaces. For site 2+site3 characterization, the PRLR-ECD surfaces were firstsaturated with hPRL (250 nM). rPRLR-ECDs (0.4–50 μM)were then injected for 3 min into the preformed PRLR-ECD/PRL complexes, followed by a 5-min dissociationperiod. The association and dissociation profiles weredouble referenced using the Scrubber 2.0 software (Bio-Logic Software) [i.e., both the signals from the referencesurface (ethanolamine derivatized) and the signals fromblank experiments (using running buffer instead of protein)were subtracted]. The site 2+site 3 steady-state SPRresponses (Req) were plotted against the concentration ofrPRLR-ECD (site 2) and fitted using the following equation:

Req = RmaxCð Þ = Kd + Cð Þwhere Kd is the equilibrium dissociation constant and Rmaxis the maximal binding capacity of the surface.

PDB accession number

The coordinates and structure factors for the hPRL/rPRLR2 complex have been deposited in the PDB‡ underaccession number 3NPZ.

Acknowledgements

We acknowledge the European Synchrotron Ra-diation Facility for providing access to beamlineID14. This work was supported, in part, by

http://pymol.sourceforge.net/

http://pymol.sourceforge.net/


University Paris Descartes and the AgenceNationalede la Recherche (grant ANR-07-PCVI-0029). J.v.A.,C.Z., and E.T. acknowledge the Ministère del'Education Nationale de la Recherche et de laTechnologie for fellowship support.

Supplementary Data

Supplementary data associated with this articlecan be found, in the online version, at doi:10.1016/j.jmb.2010.09.036

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