Structure-based Analysis of GPCR Function: Conformational Adaptation of both Agonist and Receptor upon Leukotriene B4 Binding to Recombinant BLT1

doi:10.1016/S0022-2836(03)00438-8

J. Mol. Biol. (2003) 329, 801–814

Structure-based Analysis of GPCR Function: Conformational Adaptation of both Agonist and Receptor upon Leukotriene B4 Binding to Recombinant BLT1 Jean-Louis Baneres*, Aime´e Martin, Pierre Hullot, Jean-Pierre Girard Jean-Claude Rossi and Joseph Parello UMR 5074 CNRS Chimie Biomole´culaire et Interactions Biologiques Faculte´ de Pharmacie 15 Avenue Ch. Flahault BP 14491, 34093 Montpellier Cedex 05, France

We produced the human leukotriene B4 (LTB4) receptor BLT1, a G-proteincoupled receptor, in Escherichia coli with yields that are sufficient for the first structural characterization of this receptor in solution. Overexpression was achieved through codon optimization and the search for optimal refolding conditions of BLT1 recovered from inclusion bodies. The detergent-solubilized receptor displays a 3D-fold compatible with a seven transmembrane (TM) domain with ca 50% a-helix and an essential disulfide bridge (circular dichroism evidence); it binds LTB4 with Ka ¼ 7.8(^ 0.2) £ 108 M21 and a stoichiometric ratio of 0.98(^ 0.02). Antagonistic effects were investigated using a synthetic molecule that shares common structural features with LTB4. We report evidence that both partners, LTB4 and BLT1, undergo a rearrangement of their respective conformations upon complex formation: (i) a departure from planarity of the LTB4 conjugated triene moiety; (ii) a change in the environment of Trp234 (TM-VI helix) and in the exposure of the cytoplasmic region of this transmembrane helix. q 2003 Elsevier Science Ltd. All rights reserved

*Corresponding author

Keywords: antagonistic effects; bacterial expression; conformational adaptation; leukotriene B4 receptor; recombinant GPCR

Introduction Leukotrienes (LTs) constitute a family of endogenous metabolites of arachidonic acid that are biosynthesized via the lipoxygenase pathway.1,2 Members of the group include potent bronchoconstrictors that play important roles in immediate hypersensitivity reactions and act as mediators of the inflammatory process, while other members are potent chemotactic agents.3 Leukotriene B4 (5S,12R-dihydroxy-6Z,8E,10E,14Z-eicosatetraenoic acid; LTB4) that derives from an unstable intermediate oxido-eicosatetraenoic acid LTA41 induces chemotaxis, chemokinesis, degranulation, superAbbreviations used: LTB4, human leukotriene B4; TM, transmembrane; GPCR, G-protein-coupled receptor; LDAO, lauryl dimethylamino oxide; PyMPO, or PyMPO-maleimide, N-(maleimidylethyl)-5-((4methoxyphenyl)oxazol-2-yl)pyridinium methane sulfonate. E-mail address of the corresponding author: [email protected]

oxide anion production and membrane adhesion enhancement in polymorphonuclear leukocytes and plays a central role in inflammation, immune response and host defense mechanisms.3 – 5 LTB4 activates inflammatory cells through binding to cell surface receptors (BLTs). The complementary DNA (cDNA) for a first human membrane LTB4 receptor, designated BLT1, was cloned by Yokomizo et al.6 This sequence encodes a 352residue protein that belongs to the family of the G-protein-coupled receptors (GPCRs). Recently, a second GPCR for LTB4, designated BLT2, has been identified.7,8 BLT1 is primarily involved in leukocyte activation,6 whereas BLT2 functions as a lowaffinity receptor with ligand recognition of various eicosanoids, and certainly mediates biological and pathophysiological roles distinct from BLT1.9 Although significant progress has been made within the last few years in dissecting GPCRmediated signal transduction pathways,10 understanding the mechanisms underlying ligand recognition and signal transduction through the membrane has been hampered by the lack of

0022-2836/03/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved

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information at the molecular level. This is largely due to the low abundance of most GPCRs in cellular membranes. Furthermore, few expression systems have proven satisfactory for producing these receptors in a functional state and sufficient yields (11 and references therein). Structural information on the GPCR family is therefore very sparse, with the exception of rhodopsin for which ˚ -resolution crystal structure has been a 2.8 A determined12 and which therefore appears as the structural prototype of the GPCRs with seven transmembrane (TM) helices. Our current knowledge about structure –function relationships in GPCRs is thus primarily based on the results from mutagenesis studies performed on several members of this receptor family. It is generally assumed that binding of the agonist to the receptor induces a set of changes in the tertiary structure of the receptor that are recognized by the associated G-protein.13 Present evidence suggests that such a conformational adaptation involves, among others, changes in the relative arrangement of the hydrophobic transmembrane domains, TM-III and TM-VI.14 – 16 Here we report on the production of human BLT1 in Escherichia coli in milligram quantities (per liter of bacterial culture) isolated as a functional protein in detergent solution. The method involves (i) a synthetic BLT1-coding sequence incorporating codons optimized for bacterial expression, (ii) procedures to optimize subsequent refolding of the protein present in insoluble inclusion bodies. This has allowed us to carry out the first structural characterization of this receptor in solution, as well as of its interactions with LTB4 and an antagonistic molecule synthesized in our laboratory.17 We demonstrate that formation of a stoichiometrically well-defined complex LTB4:BLT1 is associated not only with a conformational adaptation of the receptor but also with a change in the conformation of the agonist molecule that is likely to be essential for optimal binding to the receptor.

Results BLT1 cloning, expression and purification To overexpress human BLT1 in E. coli we devised a strategy in which the BLT1-coding nucleotide sequence was produced, using PCR, as a synthetic cDNA in which the codons of lowest abundance in the E. coli genome18 were replaced by the most frequent ones. This synthetic cDNA was efficiently expressed in the bacterium (Figure 1) but the recombinant BLT1 was found in the insoluble fraction after bacterial lysis, as part of misfolded proteins in inclusion bodies. We thus devised a purification scheme involving the recovery and refolding of BLT1 from the inclusion bodies. The C-terminally His-tagged protein was first purified in its unfolded state, i.e. in the presence of 6 M

Conformational Adaptation on LTB4 Binding to BLT1

Figure 1. Expression and purification of recombinant BLT1. SDS-PAGE analysis. Lanes 1 and 2, protein content of E. coli BL21(DE3) transformed with the pET21b-BLT1 vector before and after IPTG induction, respectively. Lane 3, purified BLT1, before thrombin cleavage. Markers, carbonic anhydrase A (31.5 kDa) and hen white egg ovalbumin (45.5 kDa).

urea, by immobilized metal affinity chromatography. No aggregated material was present at this stage of the purification as judged by the absence of scattering contribution to the UV spectrum; furthermore, based on circular dichroism (CD) evidence, BLT1 corresponds to a random coil polypeptide under these conditions (spectrum not shown). Based on previous refolding assays with matrix-bound proteins,19,20 refolding of chemically denatured BLT1 was carried out with the immobilized protein on the Ni-NTA support by progressively removing urea in the presence of detergent (see below). The protein was recovered from its solid support and aggregated material removed by centrifugation (see Materials and Methods). BLT1 refolding efficiency was assessed at the quantitative level (Figure 2(A)), as well as at the qualitative level by far-UV CD spectroscopy (Figure 2(B)) and by determining the affinity constant for LTB4 (Figure 2(C); Ka values inferred from CD-monitored titrations: see Figure 7). Refolding conditions were optimized as follows: (i) The recovery yield is highly dependent on the position of the His-Tag appendix: the highest yields were obtained when the receptor was immobilized through its C-terminal end. (ii) The recovery yield strongly depends on the quantity of protein immobilized; as previously observed with proteins of ca 250 residues,20,21 gel loading up to 0.6 mg ml21 of hydrated resin did not affect the recovery yield; above this threshold, the recovery yield rapidly decreased (data not shown), probably due to interactions between individual protein molecules on the solid matrix. (iii) The recovery yield is dependent on the nature and concentration of the detergent used. Different detergents were assayed and the best results were obtained in the presence of detergents with a sufficiently long alkyl chain such as lauryldimethylamino oxide (LDAO), as shown in Figure 3, at concentrations slightly above the critical micellar concentrations (cmc indicated in Figure 2). We used LDAO (C12) here, although other mild detergents such as n-dodecyl-b-D -maltoside can also be used favorably (results not shown). The fact that the DAO series included representatives with aliphatic

803

Conformational Adaptation on LTB4 Binding to BLT1

Figure 3. Influence of alkyl chain length (alkyldimethylamino oxide or alkyl-DAO series) on BLT1 refolding. Chemically denatured BLT1 (6 M urea), immobilized on a Ni-NTA matrix, was refolded as described in Materials and Methods. Alkyl-DAO detergents (C7 through C13) were used at critical micellar concentrations.

Secondary and tertiary fold of recombinant BLT1 We carried out a first characterization of refolded BLT1 in LDAO solution by CD.

Far-UV CD spectrum (Figure 4)

Figure 2. Role of detergent concentration on the recovery yield of native BLT1. Chemically denatured BLT1 (6 M urea), immobilized on a Ni-NTA matrix, was refolded as described in Materials and Methods at different LDAO concentrations: (A) recovery yield (defined as the ratio of the amount of refolded and functional protein obtained after dissociation from the matrix to that of protein initially immobilized; see Materials and Methods); (B) molar ellipticity at 222 nm; (C) affinity constant for LTB4 as a function of LDAO concentration (Ka values inferred from CD-monitored titrations; see Figure 7(A)). The critical micellar concentration (cmc) of LDAO is indicated by the arrow.

chains C7 through C13 allowed us to investigate the role of alkyl chain length on receptor refolding. LDAO also proved to be particularly useful for all the CD analyses (high transparency at low wavelength). We finally defined a set of refolding conditions combining the parameters defined above in an optimal manner (see Materials and Methods). A yield of about 20% of the protein initially solubilized in the 6 M urea buffer was thus achieved, giving 2 –3 mg of purified protein per liter of culture, properly folded in its native state (as assessed by its Ka value for LTB4 binding; see below in Results).

The 185– 250 nm spectrum is characteristic of a folded protein with a high content of secondary structure (ca 50% of a-helix and ca 15% of b-strand; see Materials and Methods and Table 1). We note a strong quantitative resemblance between

Figure 4. CD spectrum of LDAO-solubilized BLT1. Main Figure: far-UV spectrum of free BLT1. Inset: nearUV spectrum of free BLT1 (profile 1) and of the double mutant C90S, C168S (profile 2). The arrow indicates a broad negative band associated with the unique C90C168 disulfide bridge (see the text). Wavelength of extrema indicated by an arrow. Measurements in buffer D at 22 8C.

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Conformational Adaptation on LTB4 Binding to BLT1

Table 1. LTB4 affinity constants and secondary structure of different Cys-to-Ser BLT1 mutants

Ka (108 M21) % a-Helix % b-Strand

wt

C90S

C93S

C97S

C168S

C287S

C93S,C97S,C287S

7.9 47 16

0.0003 48 15

7.5 48 13

6.8 47 17

0.0002 42 13

8.1 44 14

6.7 51 16

The affinity constants for LTB4 were inferred from CD-monitored titrations (see Figure 7(A)). a-Helix and b-strand contents were inferred from the far-UV CD spectra, as described in Materials and Methods.

the CD profiles of BLT1 and rhodopsin (see Figure 4 of Fasman22) in the 200– 250 nm range. Near-UV CD spectrum (inset in Figure 4) It includes all chromophores from the aromatic residues in the protein, as well as a negative broad band at ca 265 nm corresponding to the unique disulfide chromophore, C90-C168 (profile 1). This disulfide band disappears in the presence of b-mercaptoethanol (not shown), as well as in the double mutant C90S,C168S (profile 2). Conformational adaptation of LTB4 upon binding to BLT1 We analyzed the interactions between BLT1 and its natural agonist LTB4 by UV and CD in the LDAO solution. The UV spectrum of free LTB4 gives rise to an intense electronic transition with a characteristic vibrational structure that is associated with the conjugated triene (profile 1 in Figure 5(A)), in agreement with previous data.23 Upon addition of LTB4 to BLT1 (at 1:1 molar ratio), a decrease of ca 25% in the molar absorptivity of all LTB4 triene absorption bands was observed (profile 2 in Figure 5(A)). Since the affinity constant of BLT1 for LTB4 is about 8 £ 108 M21 (see below), an equimolar agonist-receptor ratio at a protein concentration of ca 1026 M practically corresponds to full binding of LTB4 to BLT1. Profile 2 in Figure 5(A) corresponds to a difference spectrum in which the contribution of BLT1 was subtracted. Such a subtraction of free BLT1 assumes that the UV spectrum of BLT1 in the 250– 300 nm region remains sufficiently unperturbed upon LTB4 binding. In agreement with this assumption, we observe that the UV spectrum of BLT1 displays an intensity variation between the native and the denatured apoforms by no more than 4% at 278 nm (i.e. ca 900 l mol21 cm21; see Materials and Methods for the calculation of the receptor extinction coefficient) so that profile 2 in Figure 5(A) cannot be significantly affected by any subtle conformational effect. We then undertook a parallel CD analysis of LTB4 binding to BLT1. As shown in Figure 5(B), free LTB4 is characterized by a four-band CD spectrum in the 240 – 290 nm region (profile 1). All CD bands closely coincide with the UV absorption bands in Figure 5(A), in agreement with theoretical predictions in the case of strongly absorbing

bands.24 In the free state, all the resolved triene CD bands display relatively low dissymmetry factors D1/1 (for a definition, see Woody25), in the 1024 range (note that the CD absorption values in Figure 5(B) are given in molar ellipticities, as well as in D1 units; see Materials and Methods). In the presence of recombinant BLT1, at an input molar ratio of 1, an increase of about eightfold is observed in the intensity of all LTB4 dichroic bands (Figure 5(B), profile 2). Such an increase can be interpreted as a skewing of the time-averaged planar triene in free LTB4 (see Discussion). This property was extensively used for monitoring the binding of LTB4 to BLT1, as well as the competition of the antagonist 5ba with LTB4 for binding to BLT1 (see below in Results). Ligand-induced changes in the conformation of BLT1 We subsequently investigated whether BLT1 undergoes a conformational adaptation upon complex formation with LTB4. In the presence of the intense LTB4 CD bands, the weakly absorbing BLT1 aromatic chromophores (see inset in Figure 4) could not be used for monitoring possible changes in the receptor 3D-fold upon complex formation with its agonist. The human BLT1 amino acid sequence, however, displays several tryptophanyl residues that were used for monitoring changes in protein conformation by fluorescence spectroscopy. As shown in Figure 6(A), the BLT1 emission spectrum, in the absence of ligand, displays a single maximum at 339 nm. Such a value indicates that all the tryptophan (Trp) residues lie in a rather polar environment (see Discussion). Under saturation conditions by LTB4, a marked change is observed in the emission spectrum, with a large blue shift of the maximum to 319 nm. It is well known that Trp emission maxima in the fluorescence spectra of proteins vary from ca 315 to 350 nm depending on whether the indolyl moiety lies in a hydrophobic or polar environment, respectively.26,27 The blue shift observed here with BLT1 likely translates a decrease in the polarity of the immediate environment of one, or several, Trp fluorophores upon binding of LTB4. As expected, a fluorescence-monitored titration profile obtained by increasing the LTB4 concentration at constant BLT1 concentration gave the same Ka and stoichiometry values as those obtained in the CDmonitored titration given in Figure 7 (Ka and n

Conformational Adaptation on LTB4 Binding to BLT1

805

Figure 5. BLT1-induced changes in the conformation of LTB4. (A) UV absorption spectra of free (profile 1) and BLT1-bound (profile 2) LTB4. The latter was obtained after subtracting the UV spectrum of free BLT1 from that of the BLT1:LTB4 complex (see Results). (B) CD spectra of free (profile 1) and BLT1-bound (profile 2) LTB4.

Figure 6. Ligand-induced changes in BLT1 conformation monitored by fluorescence. Trp emission spectra of (A) wtBLT1 in the absence (profile 1) or presence (profile 2) of LTB4. (B) W41L,W83L,W142L,W161L mutant in the absence (profile 1) or presence (profile 2) of LTB4. (C) W234L mutant in the absence (profile 1) or presence (profile 2) of LTB4. (D) wtBLT1 in the absence (profile 1) or presence (profile 2) of 5ba. Wavelengths of maxima indicated by an arrow.

806

Conformational Adaptation on LTB4 Binding to BLT1

of this Trp emission spectrum, we mutated the BLT1 Trp residues to Leu. Only the mutation of the conserved Trp residue in TM-VI (W234L) decreased the affinity for the agonist moderately (1.7 £ 108 M21 to be compared with 7.8 £ 108 M21 for the wild-type receptor), in contrast to an alanine replacement at this position, for which the Ka value decreased by two orders of magnitude. The other Trp-to-Leu mutations resulted in no significant alteration of the Ka value. Upon binding to LTB4, a blue shift similar to that observed in the case of the wild-type receptor is observed with the all the Trp-to-Leu mutants (Figure 6(B)), with the exception of the W234L one where no shift in the emission spectrum was observed upon addition of LTB4 (Figure 6(C)). This indicates that the spectral effects observed with wild-type BLT1 upon LTB4 binding are exclusively due to a change in the polarity at the level of Trp234 in helix TM-VI (see Discussion). Finally, as shown in Figure 6(D), a blue shift of the BLT1 Trp emission spectrum is also observed upon binding of the 5ba antagonist molecule that shares common structural features with LTB4 (see Figure 9). However, the spectral changes are different in wavelength and intensity in comparison to those resulting from the binding of LTB4 (compare Figure 6(A) and (D)). These changes are likely to be associated with a close proximity between the hydrophobic tail of the antagonist molecule and Trp234, as inferred from labeling experiments with a 5ba photoactivatable analog (J.-L.B. et al., unpublished results). Figure 7. Ligand-binding properties of isolated BLT1. (A) CD-monitored titration of LTB4 by BLT1 (see Materials and Methods): normalized molar ellipticity variations, at 270.5 nm, plotted as a function of BLT1 concentration. Measurements in triplicate; the continuous line is the best hyperbolic fit through the mean experimental values; Ka ¼ 7.8(^ 0.2) £ 108 M21 and n ¼ 0:98ð^0:02Þ (stoichiometric ratio). Inset: Scatchard plot (errors are only indicated for the points at low r values). (B) Competition between LTB4 and the 5ba antagonist as monitored by CD at 270.5 nm (ordinates in % of LTB4: BLT1 complex) starting from the 1:1 BLT1:LTB4 complex. Inset: Scatchard plot for the binding of the antagonist 5ba to free BLT1 (fluorescence-monitored titration). The continuous line in the main Figure is a simulation (without any fit procedure) based on the affinity constants and stoichiometric ratios for LTB4 and 5ba binding, as inferred from insets in A and in B, respectively (PRISM software; see Materials and Methods). All measurements (in A and B) in buffer D at 22 8C; r (dimensionless) is the degree of binding and Lf the concentration of free ligand.

values determined by fluorescence measurements: 7.5 £ 108 M21 and 0.97, respectively). As apparent in Figure 6(A), the spectrum recorded in the presence of LTB4 displays a prolonged tail toward the long-wavelength region that suggests the occurrence of a composite emission spectrum. To have a clear understanding

Ligand-binding properties of recombinant BLT1 The aromatic chromophores from BLT1 contribute only in a negligible manner to the CD spectrum of the LTB4:BLT1 complex in the 250– 300 nm range (less than 1023 of the LTB4 signal; compare Figures 4 and 5), so that this region represents an intrinsic property of the bound agonist molecule with no real interference from the receptor. We therefore carried out a CD-monitored titration in the 250– 300 nm range to assess whether the increase in intensity of the LTB4 CD bands (Figure 5(B)) is due to the formation of a stoichiometrically well-defined complex. As shown in Figure 7(A), the intensity variations of the CD band at 270.5 nm resulted in a monophasic saturation profile (linear Scatchard plot: inset in Figure 7(A)). An affinity constant, Ka ¼ 7.8(^ 0.2) £ 108 M21, as well as a stoichiometric ratio of 0.98(^ 0.02) LTB4 molecule bound per receptor, is thus inferred (see Materials and Methods for the calculation of the stoichiometric ratio). The observation of a degree of binding of 0.98 at saturation, slightly lower than one, might reflect the fact that the LTB4 molar absorptivity was not corrected for solvent effects (see Materials and Methods). A first analysis of LTB4 binding to recombinant BLT1 by equilibrium dialysis using radiolabeled LTB4 gave affinity constant and stoichiometry values closely related

807

Conformational Adaptation on LTB4 Binding to BLT1

to those inferred from the CD or fluorescencemonitored titrations, i.e. 7.4 £ 108 M21 and 0.97 for Ka and n, respectively, indicating that the spectroscopically monitored titrations can be reliably used for analyzing LTB4 binding to recombinant BLT1. An extensive equilibrium dialysis-based study is currently being carried out, in particular at low degree of binding, to assess for the occurrence of any subtle cooperative effects (work in progress). We also analyzed the binding of 5ba to BLT1. This antagonist is known to bind to the LTB4 receptors in human leukocytes.17 Two different experiments were carried out as reported in Figure 7(B): (i) a CD-monitored titration (at 270.5 nm) in which the preformed 1:1 LTB4:BLT1 complex was subjected to competition by 5ba; the CD signal of bound LTB4 monotonously decreased upon addition of 5ba as the result of the competition between the agonist and antagonist molecules (no significant CD contribution of 5ba is to be considered for this chiral antagonist in the near UV region); (ii) a fluorescence-monitored binding study of 5ba to free BLT1 that allowed a Ka of 3.5 £ 106 M21, as well as a stoichiometric ratio of one 5ba molecule bound per receptor, to be measured (see Scatchard plot: inset in Figure 7(B)). Finally, we simulated the competition profile of Figure 7(B), using the binding parameters determined independently for the binding of LTB4, on the one side, and of 5ba, on the other side (see Materials and Methods for the simulation method used). A remarkable agreement is observed between the experimental data and the simulated competition curve (Figure 7(B)) which suggests that 5ba competes with LTB4 for binding to a common receptor site (or two mutually exclusive sites). Agonist-induced changes in the orientation of the TM-VI segment in BLT1 Recent proposals for the formation of functionally active GPCRs have placed critical importance on the selective displacement of the TM-VI helix (13 and references therein). We investigated whether such a displacement occurs in isolated BLT1 upon ligand binding (agonist or antagonist). The method used was initially described in the case of rhodopsin.16 It is based on the reactivity of receptor mutants containing a unique cysteine residue introduced by site-directed mutagenesis in the cytoplasmic end of TM-VI towards the cysteine-specific reagent, N-(maleimidylethyl)-5((4-methoxyphenyl)oxazol-2-yl)pyridinium methane sulfonate (PyMPO-maleimide, or PyMPO). Human BLT1 contains five cysteine residues in total. Cys93, Cys97 and Cys287 can be replaced by Ser without significantly affecting either the conformation or the ligand-binding properties of the receptor (Table 1). In contrast, when Cys90 or Cys168 was replaced by Ser, a dramatic decrease by as much as four orders of magnitude was observed for the affinity constant (Table 1), thus indicating that both cysteinyl residues

are essential for BLT1 function. Neither Cys90 nor Cys168, in the triply mutated receptor C93S,C97S,C287S, reacted with PyMPO-maleimide, suggesting that these residues form a disulfide bond. In agreement with this assumption, the nearUV CD spectra of both single mutants, C90S and C168S (not shown), as well as the spectrum of the double mutant C90S,C168S (inset in Figure 4) showed no contribution from the disulfide bond chromophore. The absence of reactivity of Cys90 and Cys168 to PyMPO does not reflect an intrinsic lack of accessibility since: (i) Cys93, that is located in close proximity to Cys90, fully reacts with PyMPO; (ii) a S166C mutant, where a novel Cys residue was introduced close to Cys168 in the second extracellular loop e2 also fully reacts with PyMPO (data not shown). Ten mutated proteins were then produced by introducing a single cysteine in each mutant, between positions 223 and 232 in the cytoplasmic end of TM-VI starting from the triple mutant C93S,C97,C287S. All these BLT1 mutants displayed a conformation (CD evidence) and an affinity for LTB4 (CD-monitored titrations) similar to those of the wild-type protein (Table 2). The accessibility of the different cysteine residues within the set of the ten mutants was first investigated in the ligandfree receptor using PyMPO, as described above. This bulky reagent demonstrated marked differences in its ability to react with the TM-VI cysteine residues (Figure 8(A)). While residues, C224, C225, C228, C229, C231 and C232, showed a near one-toone labeling efficiency, the remaining ones, i.e. C223, C226, C227 and C230, showed little reactivity in the free receptor. We subsequently analyzed the accessibility of these different cysteine residues in the presence of LTB4 (under agonist saturation conditions). A totally different distribution was observed with regard to the accessibility to PyMPO (Figure 8(B)). All Cys residues, including those initially inaccessible in the absence of agonist, now reacted fully. This result is indicative of a strong conformational change in the TM-VI region of the receptor upon agonist binding (see Discussion). We also analyzed the accessibility of the TM-VI cysteine residues to PyMPO, in the presence of the antagonist 5ba. The accessibility of the cysteine mutants was found to be very similar to that observed for free BLT1 (compare Figure 8(A) and (C)). Finally, the pattern observed in the presence of LTB4 with all the Cys residues accessible (Figure 8(B)) was reverted to the differential pattern initially observed with the free receptor (Figure 8(A)) by adding 5ba in excess (Figure 8(D)).

Discussion There are presently few reports of systems allowing recombinant GPCR expression at levels suitable for high yield isolation and purification (Ref. 11 and references therein). Here we describe

808

Conformational Adaptation on LTB4 Binding to BLT1

Table 2. LTB4 affinity constants and secondary structure of the TM-VI Cys BLT1 mutants

Ka (108 M21) % a-Helix % b-Strand

wt

V223C

V224C

L225C

I226C

I227C

L228C

T229C

F230C

A231C

A232C

7.9 47 15

7.8 45 14

7.7 48 16

7.5 47 14

7.9 42 13

8.1 44 15

7.5 45 14

7.6 46 16

8.1 48 18

7.4 47 16

7.6 45 15

Ka values, a-helix and b-strand contents determined as described in Table 1. The triply mutated receptor, C93S,C97S,C287S, was subsequently mutagenised by introducing a Cys residue at the different positions indicated in the amino acid sequence.

a strategy for high yield bacterial production (2 – 3 mg of purified detergent-solubilized receptor per liter of culture) of a structurally and functionally well-defined GPCR, the human LTB4 receptor BLT1, and we report the first structural characterization of this receptor in the detergent solution. In contrast to other approaches in which the GPCR has been produced as a functional protein inserted in the bacterial membrane,28 our strategy relies on the overexpression of BLT1 in insoluble inclusion

bodies and refolding of the recovered protein. Overexpression was only achieved by substituting most of the codons in the human BLT1 cDNA by codons preferentially used in E. coli (see Results). The search for convenient refolding conditions was the next critical step. It is only through a convergent analytical approach, using two distinct methods, one at the structural level (CD spectroscopy) and the other at the functional level (affinity constant of the isolated receptor for LTB4),

Figure 8. Accessibility to PyMPO-maleimide of the different TM-VI Cys mutants (Table 2). Molar ratio PyMPO:protein as a function of cysteine position (see reaction conditions in Materials and Methods): (A) free BLT1, (B) BLT1 in the presence of LTB4 under saturation conditions, (C) BLT1 in the presence of the 5ba antagonist under saturation conditions, (D) BLT1 after displacement of LTB4 (from the 1:1 complex LTB4:BLT1) by an excess 5ba.

Conformational Adaptation on LTB4 Binding to BLT1

that the quality of our refolding process, as well as its overall yield, could be properly assessed. This strategy has been successfully applied to the production of another GPCR, the serotonin 5-HT4a receptor. Our recombinant BLT1 receptor isolated in the detergent medium is characterized by a welldefined structural organization, as inferred from the far-UV and near-UV regions of its CD spectrum. Most CD information available so far on GPCRs derives from studies on rhodopsin and is restricted to the far-UV region.22 Deconvolution of the rhodopsin far-UV CD spectrum yields 53% a-helix, comparable to crystallographic data12 giving 57% of the residues in a-helices. Similar results (ca 50% a-helix) were obtained here with BLT1 through deconvolution of the far-UV CD spectrum. The quantitative resemblance of the CD spectra of rhodopsin and BLT1 in the 200 – 250 nm range (see Results) certainly reflects a high homology in secondary structure. Most of the GPCRs display two conserved Cys residues, likely forming a conserved disulfide bridge connecting the extracellular tip of helix TM-III (close to the extracellular loop e1) and the extracellular loop e2 (named here the “TM-III/e2” disulfide bridge). This conserved disulfide bridge in the GPCRs is crucial to receptor function.29 – 31 An interesting feature in our work with BLT1 is the identification of a broad negative CD band centered at ca 265 nm that represents the unique C90-C168 disulfide bridge in this receptor. The fact that this band is absent in the CD spectrum of the double mutant C90S,C168S unambiguously demonstrates its origin. It is well established that the sign of the CD bands associated with a disulfide chromophore, in the near-UV region, is directly related to the handedness of the C –S –S – C motif.32 However, attempts to correlate the sign and amplitude of the CD signal with the handedness of the disulfide bridge will require a comparative analysis of the CD spectra of several GPCRs (work in progress). A remarkable observation is the eightfold increase in intensity that is observed for all the CD bands originating from the LTB4 conjugated triene chromophore upon formation of the LTB4:BLT1 complex, as well as the concomitant decrease in the intensity of the same bands in the UV spectrum. This large increase of the CD spectrum can be unambiguously interpreted as the result of skewing effects at the level of the conjugated triene (see formula of LTB4 in Figure 9) by analogy with experimental and theoretical evidence in the case of constrained conjugated dienes.33,34 In the case of constrained conjugated dienes investigated in parallel by X-ray crystallography35,36 and by CD spectroscopy,33,34 it appears that even a moderate departure from planarity (dihedral angles about the central s-bond in the 10– 158 range, in absolute values) is responsible for intense CD spectra. In the case of LTB4, although our CD results unambiguously establish that the triene motif is

809

skewed, the exact topology (amplitude, handedness, number of s-bonds involved) adopted by LTB4 bound to its receptor BLT1 cannot be unambiguously inferred from the present data. Our CD observations are reminiscent of what has been previously observed with rhodopsin, for which a skewed topology of the retinal molecule in the receptor is inferred from a CD analysis.37 Based on X-ray crystallographic evidence,12 the extent of skewing of the retinylidene moiety is a moderate one. The decrease in UV absorptivity of LTB4 bound to BLT1, by ca 25% (Figure 5(A)), is also reminiscent of what is experimentally observed with constrained conjugated dienes with regard to their isomerism about their central single bond. In this case, 1s-trans is usually larger than 1s-cis by as much as two- to threefold.38 To our knowledge, there is no example in the literature of a systematic analysis of constrained conjugated trienes by UV (and/or CD) spectroscopy. While skewing of the LTB4 triene probably explains the large increase of the CD signal, the concomitant decrease in UV absorptivity can be viewed as the consequence of a loss of conjugation between the double bonds in the final skewed form. In any case, it appears that upon binding to BLT1, LTB4 undergoes a significant conformational adaptation, at least at the level of its triene moiety. The occurrence of two hydroxyl groups in LTB4 (at positions 5 and 12, respectively; Figure 9), as well as their absolute configurations, are essential for optimal LTB4 binding to the receptor.39 Both allylic hydroxyl groups are an integral part of the conjugated triene motif (Figure 9). Therefore, skewing of the conjugated triene might result in a subtle adjustment of the hydroxyl groups relative to the receptor matrix that may contribute to achieving optimal binding. As reported in Results, the formation of the 1:1 agonist:receptor complex is characterized by a conformational adaptation of the receptor itself. Agonist binding is associated with both a large blue shift of the receptor Trp234 fluorescence spectrum and an increase in its fluorescence intensity. This is likely to be associated with a change in the polarity of the immediate surrounding of this residue. Indeed, a decrease in the polarity of the Trp environment is generally associated to both an increase in the fluorescence yield and a blue-shift of the maximum emission wavelength.26 It is to be noted that the change in the fluorescence emission maximum wavelength is of significant range (20 nm). However, such a significant change in the emission maximum has already been observed associated with ligand-induced protein conformational changes. For example, a blue shift of ca 25 nm is observed upon calcium removal from annexin V.40 Examination of the position of homologous residues in the crystallographic structure of rhodopsin suggests that Trp234 is likely to be oriented inwards with regard to the 7TM domain (see Figure 10(A)). One explanation for the fluorescence maximum at 339 nm in free BLT1

810

Conformational Adaptation on LTB4 Binding to BLT1

Figure 9. Chemical structures of the natural agonist LTB4 and the synthetic antagonist 5ba. (A þ B) LTB4 agonist (with the exception of the conjugated triene motif itself no preferential conformer is adopted for the remaining parts of the molecule): (A) family of conformations with s-trans7,8 and s-trans;9,10 (B) family of conformations with s-cis7,8 and s-trans:9,10 the s-cis7,8 bond is likely skewed, in agreement with the CD observations in Figure 5(B), due to steric hindrance effects.48 (C) Synthetic antagonist 5ba: family of conformers that closely resembles the LTB4 conformation in B (cyclohexyl moiety in the chair conformation with the tertiary OH axial).

would be that the interior of the 7TM domain accommodates a given number of water molecules. The crystal structure of rhodopsin shows the occurrence of such inner water molecules.41 Displacement of water molecules by the hydrophobic LTB4 ligand might account for the blue shift in the Trp234 emission spectrum observed upon LTB4 binding (Figure 6(B)). We tested here the possibility that a conformational rearrangement of the TM-VI helix correlates with agonist binding to BLT1, as previously proposed for other GPCRs14 – 16 by introducing single Cys residues in the cytoplasmic end of TM-6. In the absence of LTB4, there is a marked differential accessibility of the different Cys residues to the PyMPO-maleimide reagent that follows an i/i þ 3 or i/i þ 4 accessibility pattern, in agreement with the expected topology of a regular a-helix for the TM-VI 223 –232 segment (Figure 10(D)). One explanation would be that, in the absence of LTB4, the internal face of TM-VI is close to other neighboring TM helices (see Figure 10(C)). The relatively bulky PyMPO reagent will thus have no access to any of the Cys residues that lie on this internal face while the Cys residues on the opposite face will be fully accessible. In contrast, in the presence of LTB4, all Cys residues become equally accessible (Figure 8(B)). This result, which appears somewhat puzzling, is discussed in

detail by Bane`res & Parello (accompanying paper). Binding of the 5ba antagonist to isolated BLT1 did not result in any increase of the accessibility of the TM-VI cytoplasmic region (see Figure 8(C) and (D)), thus indicating that the effects observed with LTB4 (Figure 8(B)) are likely to be specific to the binding of the natural agonist to its receptor and therefore might be related to receptor activation in vivo. The lack of any apparent receptor rearrangement in the presence of 5ba suggests that the binding of this antagonist molecule only requires a minimal adaptation of the receptor topology (fluorescence effects on Trp234; see Figure 6(D)) while efficiently competing with LTB4. In summary, the present study reports on the high yield bacterial production of a fully functional eukaryotic G-protein coupled receptor, the human BLT1 receptor of LTB4. This receptor has been isolated in detergent solution, as a structurally and functionally defined protein, thus allowing us to carry out a first structural characterization of this receptor in solution. The observation that this isolated receptor specifically recognizes its natural agonist, as well as a synthetic antagonist molecule, opens the way for analyzing the molecular mechanisms underlying BLT1 activation, as well as establishing an in vitro pharmacological approach for designing novel anti-inflammatory drugs.

Conformational Adaptation on LTB4 Binding to BLT1

811

˚ in thickness) parallel to the membrane plane through the 7TM domain of rhodopsin (X-ray Figure 10. (A) A slab (5 A crystal structure; pdb entry: 1f88) looking toward the cytoplasm. Amino acid side-chains omitted with the exception of Trp265 in TM-VI homologous of Trp234 in BLT1. The color code suggests a partition of the TM domain into a rigidly held cluster of helices (II through V) connected by the “TM-III/e2” disulfide bridge (see below in B) and the remaining helices that can be displaced from the cluster. (B) Conserved disulfide bridge (see arrow) connecting the TM-III helix (extracellular end) to the middle part of the external loop e2; the chirality (anti-clockwise dihedral angle) of the CSSC motif in rhodopsin is illustrated at the top right (Newman projection along the central S-S bond). (C) Helix contacts including TM-II, TM-III, TM-V, TM-VI and TM-VII (all amino acid side-chains included: horizontal cut as in A). (D) Helical wheel representation of the cytoplasmic 223– 232 region of TM-VI in BLT1; helix positioned as in rhodopsin (see C above); the positions substituted by Cys in the different mutants (see Table 2) are colored according to their accessibility to PyMPO-maleimide, in the absence of LTB4 (green, fully accessible; red, limited accessibility).

Materials and Methods Materials and buffers The antagonist molecule 5ba, (1S p,3S p)-1-hydroxy-3((3S pR p,E)-3-hydroxy-7-phenylhept-1-en-1-yl)cyclohexane1-N,N-dimethyl acetamide, was synthesized as described by Poudrel et al.17 LTB4 was purchased from BIOMOL laboratories. All detergents were from Calbiochem. All other reagents were purchased from Sigma. The oligonucleotides were synthesized by Sigma Genosys. Buffer A: 25 mM Tris – HCl (pH 7.8) containing 500 mM NaCl, 10 mg ml21 leupeptin, 10 mg ml21 aprotinin and 0.5 mM PMSF. Buffer B: 12.5 mM Na-borate, 10% (v/v) glycerol (v/v), 50 mM KCl, 2 mM LDAO, 2.5 mM reduced glutathione, 0.25 mM oxidized glutathione (pH 8.0). Buffer C: 25 mM K-phosphate, 2 mM LDAO (pH 6.0). Buffer D: 12.5 mM Na-borate, 50 mM KCl, 2 mM LDAO (pH 7.8). BLT1 cloning A set of 18 oligonucleotides, each approximately 70

nucleotides in length, corresponding to the sense and antisense strands of human BLT1 cDNA were constructed so that 21 bases were complementary to those of each of the two complementary strands from the opposite strand. The oligonucleotide sequence was designed using codons preferred for high-level expression in E. coli, i.e. the codons of lowest abundance in the bacterium genome were replaced by the most frequent ones.18 For cDNA synthesis, the 18 oligonucleotides were separated into four groups (four or six oligonucleotides per group) and 25 cycles of PCR were performed using Pfu polymerase (Stratagen). This step generated four small segments of the synthetic, or synBLT1, cDNA with complementary and overlapping ends. Equal amounts of each PCR mixture were combined and a second round of 25 cycles of PCR yielded the complete synBLT1 sequence. This sequence was then amplified using PCR (25 cycles) and two oligonucleotides (21 nucleotides in length each) corresponding to the 50 and 30 ends of the synBLT1 sequence, thus introducing a 50 Bam H1 restriction site and a 30 Eco R1 restriction site, as well as a thrombin cleavage site. After Bam H1 and Eco R1 digestion, the cDNA fragment was ligated into the similarly digested pET21b vector

812

(Novagen). The expression vector thus obtained was verified on the basis of restriction digestion mapping and nucleotide sequencing and named pET21b-BLT1. BLT1 expression and purification pET21b-BLT1 was introduced in the E. coli host strain BL21(DE3). The transformed bacteria were grown at 37 8C to an absorbance of 0.8 at 600 nm in 2YT medium containing 100 mg l21 ampicillin. Expression was induced by adding IPTG to a final concentration of 1 mM. Induction was allowed to proceed for four hours. The cells were harvested by centrifugation (4000g for 30 minutes), resuspended in buffer A and lyzed by sonication. Centrifugation (27,000g for 30 minutes) afforded an insoluble protein fraction that was solubilized in buffer A containing 6 M urea and loaded onto a NiNTA agarose column. After washing with buffer A þ 6 M urea, a linear imidazole gradient (0– 200 mM) in the same buffer was applied to elute the polyhistidine-tagged recombinant protein. Under these conditions, ca 10 mg of denatured BLT1 (His-tagged protein solubilized in buffer A þ 6 M urea) were obtained from one liter of bacterial culture. BLT1 refolding The urea-denatured His-tagged BLT1 was loaded again on a Ni-NTA matrix and immobilized at a protein-to-resin ratio of 0.6 mg of protein/ml of hydrated Ni-NTA agarose, and refolding was achieved by using a 6 – 0 M urea linear gradient in buffer B. Dissociation of BLT1 from the matrix was then achieved with buffer B containing 100 mM imidazole. The eluted protein was centrifuged (150,000g for one hour at 20 8C) to discard aggregated material. The efficiency of this centrifugation step was carefully monitored by UV spectroscopy until no scattering contribution at 320 nm was detected in the supernatant (usually after one hour). Beyond this time, we observed a continuous decrease in the protein absorbance at 278 nm that translates sedimentation of the detergent-solubilized receptor itself. The C-terminal His-tag appendix was removed by digestion with thrombin at 20 8C (one unit of thrombin/mg of protein) and the uncleaved protein was removed through a Ni-NTA chromatographic step. Cleaved BLT1 was then dialyzed in buffer C, loaded on an SP-Sepharose column (Amersham-Pharmacia), and eluted with a minimal volume of buffer C þ 50 mM KCl. About 2 – 3 mg of protein were obtained from one liter of bacterial culture. The integrity of recombinant BLT1 was assessed by electrospray mass spectrometry, as described for bacteriorhodopsin.42 Site directed mutagenesis All mutants (see Tables 1 and 2) were generated by PCR-mediated mutagenesis using Pfu polymerase and the pET21b-BLT1 vector as a template. Mutations were confirmed by nucleotide sequencing and mass spectrometry with a similar accuracy as observed with the wild-type receptor. UV and circular dichroism measurements UV spectra were recorded using spectrometers Cary 118 and Cary 400. CD spectra were recorded at 22 8C with a dichrograph CD6 (Jobin-Yvon). The CD spectra

Conformational Adaptation on LTB4 Binding to BLT1

are the average of five scans using a bandwidth of 2 nm, a step-width of 0.2 nm and a 0.5 second averaging time per point. Cell path lengths were 1.00(^ 0.01) mm and 50(^0.01) mm for measurements with BLT1 (far and near-UV measurements, respectively). In the presence of the strongly absorbing LTB4 molecule a common cell path length of 1.00 mm was used. LDAO-solubilized BLT1 concentrations were determined by UV using the molar absorptivity at 278 nm experimentally determined by the procedure of Gill & von Hippel,43 i.e. 2.17 £ 104 l mol21 cm21. A molar absorptivity of 5.0 £ 104 l mol21 cm21 at 270.5 nm (in methanol;23) was adopted for LTB4 in buffer D (without any correction for solvent effects). All ellipticities [u] are given in dmol of protein or LTB4 ligand (D1, the decadic molar CD, used in Figure 5(B), is given by [u] ¼ 3298, D1.25 The far-UV profile of free BLT1 was converted to a mean residue (Mr ¼ 115) ellipticity profile and deconvolution was carried out using the Convex Constraint Analysis procedure adapted to membrane proteins.44,45 Fluorescence measurements Tryptophan fluorescence emission spectra were recorded at 20 8C between 305 nm and 420 nm on a PT60 spectrofluorimeter (Cary Eclipse) with an excitation wavelength of 298 nm (bandwidth 2 nm). Buffer contributions were subtracted under the same experimental conditions. A protein concentration of 1027 M in buffer D was used in all measurements. LTB4 binding assays The LTB4-associated CD maximum at 270.5 nm (Figure 5(B)) was selected for monitoring the binding (titrations using other CD maxima gave identical results). Successive additions of BLT1 in buffer D were carried out whereas the LTB4 concentration remained nearly constant (usually 1028 M; dilution effects not exceeding 3%). Binding isotherms were obtained by plotting ([u] 2 [u]0)/([u]max 2 [u]0) as a function of BLT1 concentration, where [u]max, [u]0 and [u] correspond to the molar ellipticities at saturation, zero occupancy and intermediate occupancy, respectively. Data reduction (Scatchard plots) was performed as follows: ordinate (r/Lf) and abscissa (r) were equated to ([u] 2 [u]0)/ [Rt([u]max 2 [u]0)] and Lt([u] 2 [u]0)/[Rt([u]max 2 [u]0)], respectively, where r ¼ Lb =Rt and allowed the stoichiometry value to be calculated;46 the following symbols Lt, Lf, Lb, Rt, Rf, Rb were used for the concentrations with L, ligand; R, receptor; t, total; f, free; b, bound. The titration data were also analyzed using the PRISM software (Graphpad Inc, San Diego, CA, USA) by considering a set of usual models for describing the ligand:receptor interactions. The normalized CDmonitored titration profiles are not thermodynamic measurements per se (see also fluorescence titrations below); we assume here that the variations in optical properties are strictly proportional to the occupancy of the available ligand binding sites. 5ba binding assays Fluorescence-monitored titrations were carried out at 321 nm. Successive additions of 5ba in buffer D were carried out whereas the BLT1 concentration remained nearly constant (usually 1027 M; dilution effects not exceeding 3%). The corresponding binding isotherms

Conformational Adaptation on LTB4 Binding to BLT1

were obtained by plotting ðI 2 I0 Þ=ðImax 2 I0 Þ as a function of 5ba concentration, where Imax, I0 and I correspond to the emission intensity at saturation, zero occupancy and intermediate occupancy, respectively. The titration data were analyzed using the PRISM software (see above). LTB4 displacement assay Increasing amounts of 5ba were added to the LTB4: BLT1 complex (1:1 complex concentration: 1028 M) and the dissociation monitored by the decrease in the CD ellipticity intensity at 270.5 nm. The degree of complex dissociation was equated to [u] 2 [u]f/[u]b 2 [u]f, where [u]b corresponds to the molar ellipticity at 270.5 nm of BLT1-bound LTB4, [u]f to the molar ellipticity at 270.5 nm of free LTB4 and [u] to the molar ellipticity at 270.5 nm at a given 5ba concentration. The competition plot was simulated (without any fit procedure) by using a single site model and the affinity constants and stoichiometric ratios for LTB4 and 5ba binding as inferred from direct binding assays (PRISM software). PyMPO-maleimide labeling Experiments were carried out as described for rhodopsin.16 Briefly stated, the reactivity of His-tagged BLT1 Cys mutants (using the triply mutated receptor, C93S,C97S,C287S, as the initial receptor; this is the equivalent of the background mutant of rhodopsin containing no other reactive cysteine residues, as described by Resek et al.,47) in the absence or presence of ligand, was probed by reacting the samples in buffer D with a 50fold molar excess of PyMPO. The reaction was carried out for 16 hours at room temperature. The different mutants were then bound to a Ni-NTA agarose matrix to allow removal of unreacted PyMPO. The protein was eluted from the column (using imidazole), and the degree of labeling was determined from the absorbance at 380 nm using an extinction coefficient of 23,000 l mol21 cm21 for PyMPO.

Acknowledgements We thank CNRS (Programme “Physique-Chimie du Vivant”) and Ministe`re de la Recherche (Action Concerte´e Incitative “Mole´cules et Cibles The´rapeutiques”) for their financial support.

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Edited by R. Huber (Received 11 December 2002; received in revised form 17 March 2003; accepted 25 March 2003)