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Monomeric dark rhodopsin holds the molecular determinants for transducin recognition: Insights from computational analysis
FEBS Letters 581 (2007) 944–948
Monomeric dark rhodopsin holds the molecular determinants for transducin recognition: Insights from computational analysis Daniele Dell’Orcoa,b, Michele Seebera,b, Francesca Fanellia,b,* a
Department of Chemistry, University of Modena and Reggio Emilia, via Campi 183, 41100 Modena, Italy b Dulbecco Telethon Institute, via Campi 183, 41100 Modena, Italy Received 21 December 2006; revised 15 January 2007; accepted 25 January 2007 Available online 6 February 2007 Edited by Robert B. Russell
Abstract In this computational study, we have investigated the implications of rhodopsin (Rho) oligomerization in transducin (Gt) recognition. The results of docking simulations between heterotrimeric Gt and monomeric, dimeric and tetrameric inactive Rho corroborate the hypothesis that Rho and Gt can be found coupled already in the dark. Moreover, our extensive computational analysis suggests that the most likely Rho:Gt stoichiometry is the 1:1 one. This means that the essential molecular determinants for Gt recognition and activation are contained in one Rho monomer. In this respect, the complex between one Rho molecule and one heterotrimeric Gt should be considered as the functional unit. Ó 2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: Molecular recognition; Docking; GPCRs; G proteins
1. Introduction The G protein-coupled receptor (GPCR) rhodopsin (Rho) is the rod-cell visual pigment responsible for the dim light vision, which acts as a trigger of the visual cascade in all vertebrates. The absorption of a photon by the receptor results in the alltrans isomerization of the 11-cis retinal, the chromophore covalently linked to Rho by a protonated Schiff base (PSB) [1]. Light-induced fast isomerization of retinal (200 fs) triggers a cascade of early-photointermediate states, characterized by nano to microsecond relaxation kinetics, resulting in the active state of the photoreceptor, i.e. Meta II (MII). MII formation from MI follows Schiff base deprotonation and takes about 1 ms to occur [1]. So far, MII has been considered the unique Rho state able to recognize the cognate G protein, Gt or transducin [1–3]. The consequent MII-induced Gt activation trig* Corresponding author. Address: Dulbecco Telethon Institute, Department of Chemistry, via Campi 183, 41100 Modena, Italy. Fax: +39 059 37353. E-mail address: [email protected] (F. Fanelli).
Abbreviations: Gt, transducin; GPCRs, G protein-coupled receptors; Rho, rhodopsin; PSB, protonated Schiff base; MII, Meta II; MI, Meta I; H, helix; AFM, atomic force microscopy; ZD, ZDOCK; PDB, Protein Data Bank; Ca-RMSD, Ca-atom root mean square deviation; I2, second intracellular loop; E2, second extracellular loop; I1, first intracellular loop; I3, third intracellular loop; PWR, plasmon-waveguide resonance
gers the intracellular signaling pathway, ultimately resulting in the visual response [1]. To date, the structure of Rho in its dark-inactive state has ˚ [4]. Very recently, a photobeen completely resolved at 2.2 A activated deprotonated intermediate of bovine Rho, reminis˚ [5]. Comparisons of cent of MII, has been resolved at 4.15 A the dark and photo-activated structures reveal that the changes that accompany photo-activation are smaller than previously predicted for MII and consist in modest increases in solvent accessibility of selected amino acids essentially located at the cytosolic extensions of helices 3 and 6 (H3 and H6). The significant structural similarity between the cytosolic domains of dark and activated Rho suggests that both the receptor forms are able to recognize Gt. This is in line with the inferences from our previous computational experiments that disagree with the commonly accepted wisdom that MII is the unique Rho state intended for recognizing Gt [3,6,7]. Indeed, following computational analyses of the shape and electrostatic complementarities between the crystal structures of dark Rho and heterotrimeric Gt, we inferred that Rho has the potential to recognize Gt even in its dark state [6,7]. The evidence that GPCRs exist and function as constitutive dimers/oligomers seems to be true also for dark Rho. In fact, atomic force microscopy (AFM) images showed that dark Rho forms paracrystalline arrays of dimers in mouse disc membranes [8]. AFM measurements led to the building of a semiempirical structural model of oligomeric Rho (PDB code: 1N3M) [9]. In this study, we have extended to dimeric and tetrameric dark Rho the computational analysis of Rho–Gt complementarity previously done on the monomeric form [6]. The aim is to explore the implications of Rho oligomerization in Gt recognition. The results of this study strengthen the hypothesis that Rho can be pre-coupled to Gt in the dark, suggesting also that the molecular determinants for Gt recognition and activation are held by the monomeric form of the photoreceptor. In this respect, the complex between monomeric Rho and heterotrimeric Gt should be considered as the functional unit, consistent with in vitro evidence [10,11].
2. Methods The analysis of the structural complementarity between the cytosolic domains of dark Rho and heterotrimeric Gt was done by exhaustively sampling the roto-translational space of one protein (probe) with respect to the other (target). The rigid-body docking algorithm ZDOCK
0014-5793/$32.00 Ó 2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2007.01.074
D. Dell’Orco et al. / FEBS Letters 581 (2007) 944–948 (ZD) was employed [12]. The crystal structure of dark Rho in its monomeric (PDB code: 1U19 [4]), dimeric, and tetrameric forms was used as a fixed protein (i.e. target), whereas heterotrimeric Gt was allowed to explore all the possible orientations around the cytosolic domains of the target (i.e. probe). Dimeric and tetrameric Rho models were achieved by fitting the highest resolution structure, i.e. 1U19 ˚ ), into each monomer in the semi-empirical oligomeric model (2.2 A of Rho released as 1N3M [9]. Such fitting was aimed at improving the resolution level of the oligomer. In fact, in 1N3M, each Rho mono˚ structure 1HZX completed in the missing cytosolic mer is the 2.8 A domains by molecular modeling [9]. Thus, dimeric and tetrameric Rho employed in this study are assemblies of two and four 1U19 structures, respectively. The Gt structure employed as a probe was the one named as Gt_mut3 in our previous study [6]. This structure was a mutated form of the crystal structure of Gtab1c1 (i.e. a Gta/Gia1 chimera, PDB code: 1GOT [13]) holding the bovine Gta sequence. Other modifications made to the original 1GOT were the addition of the last ten amino acids of Gta from the NMR structure (i.e. PDB code: 1AQG [14]), and the last 10 amino acids of Gtc, modeled in a-helix. To improve sampling efficiency, the Rho portions 1–59, 76–130, 157–220, and 252–308, corresponding to the transmembrane and extracellular domains, were not taken into account in docking simulations. A rotational sampling interval of 6° was employed, and the best 4000 solutions were retained and ranked according to the ZD score. To filter the most reliable solutions among the 4000 best scored ones, the low resolution information from in vitro experiments on MII–Gt interactions [2,15–19] were translated into broad Ca–Ca intermolecular dis˚ between Rho-S240 and tance constraints, i.e. (a) lower than 15.0 A ˚ between Rhoboth Gta-N343 and Gta-D311; (b) lower than 20.0 A ˚ between Rho-Q312 R135 and Gta-F350; and (c) lower than 20.0 A and Gta-K345 (see Ref. [6] for more detail). Distance-based filtering was carried out by means of the FIPD software [20]. Filtering of solutions concerning oligomeric receptors, i.e. dimers and tetramers, was carried out on each Rho monomer. For each docking run, the solutions fulfilling at least one of the three distance constraints were collected and subjected to cluster analysis followed by visual inspection of the cluster centers (i.e. the solution representative of each clusters). A Ca-atom root mean square deviation ˚ was employed as a threshold for clustering (see (Ca-RMSD) of 3.0 A Ref. [6] for details). Energy minimization of the selected complexes between dimeric or tetrameric Rho and Gt was done by means of CHARMM [21], by employing the IMM1 implicit water/membrane model [22]. Minimizations were carried out by using 1500 steps of steepest descent followed by a conjugate gradient minimization, until the root mean square gra˚ . The adjustable parameter ‘‘a’’ dient was less than 0.001 kcal/mol A ˚, and the non-polar core thickness were set equal to 0.85 and 32 A respectively. The ‘‘united atom approximation’’ was employed. All
945 the backbone atoms except those of the last ten amino acids of Gta were kept fixed during minimization. Side chain minimization concerned the amino acids at the receptor-G protein interface.
3. Results Our previous docking simulations between different structural models of heterotrimeric Gt and monomeric Rho (i.e. 1U19) proved the good tolerance of the ZD software to changes in the side chain conformations of Gtab, as well as of the length and side chain and main chain conformations of Gtc [6]. In fact, all the different docking simulations converged into very similar predictions, i.e. the Ca-RMSDs of the best scored solutions from each run, which passed the distance-based filter and fulfilled the membrane topology requirements for the N-terminal a-helix of Gta (i.e. aN), were close to zero [6]. Importantly, such high scored realistic solutions fell among the first 23 out of the 4000 output solutions from each run [6]. In particular, the best realistic solution achieved with the same Gt employed in this study is the 10th out of 4000 (i.e. S10, ZD score = 60.18, Table 1, Figs. 1a and 2a). In such complex, the N-terminal a-helix of Gta is almost parallel and close enough to the membrane surface to allow the hydrophobic N-acyl and farnesyl modifications of the a- and c-subunits,
Table 1 Best docking solutions and docking parameters MODELa
Sol. N.b
ZD scorec
Rank N.d
˚ )e Ca-RMSD (A
Monomer Dimer Tetramer
604 400 385
60.18 60.53 67.94
10 37 30
– 0.79 1.51
a Oligomeric state (i.e. monomer, dimer, or tetramer) of the rhodopsin molecule employed as a target in each rigid-body docking run. b Number of solutions that passed the distance-based filters. c ZDOCK score of the best docking solution from each run carried out by employing monomeric, dimeric and tetrameric rhodopsin. d Rank order of the best docking solution. e ˚ ) of the best docking solution for dimeric and tetrameric Ca-RMSD (A rhodopsin with respect to the best solution for monomeric rhodopsin.
Fig. 1. Best complex between heterotrimeric Gt and the cytosolic half of (a) monomeric (ZD score = 60.18), (b) dimeric (ZD score = 60.53), and (c) tetrameric (ZD score = 67.94) dark Rho, seen in a direction parallel to the putative membrane surface. In panels (b) and (c), only the Rho monomer that participates in the interface with Gt is shown. The Gt a-, b- and c-subunits are colored in violet, orange and blue, respectively, whereas Rho is colored in green. Sticks represent the side chains of R135 and E247, from Rho, and of K345, from Gta. Drawings were done by means of the software PYMOL 0.97 (http://pymol.sourceforge.net/).
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D. Dell’Orco et al. / FEBS Letters 581 (2007) 944–948
Fig. 2. Views in a direction perpendicular (top) and parallel (bottom) to the membrane surface of the best predicted complexes between heterotrimeric Gt and (a) monomeric (ZD score = 60.18), (b) dimeric (ZD score = 60.53), and (c) tetrameric (ZD score = 67.94) Rho. All these complexes are characterized by a 1:1 Rho:Gt stoichiometry. The Gt a-, b- and c-subunits are colored in violet, orange and blue, respectively, whereas Rho monomers A, B, C and D are colored in green, gray, magenta, and cyan, respectively. Drawings were done by means of the software PYMOL 0.97 (http://pymol.sourceforge.net/).
respectively, to insert into the membrane (Figs. 1a and 2a). Furthermore, the C-term of Gta makes contacts with the second intracellular loop (I2), the cytosolic extensions of H3 and H6, as well as with H8 of Rho [6]. The functionally important R135 of the E/DRY motif is almost accessible to the C-term of Gta already in the dark state. Indeed, a limited energy minimization of the Rho–Gt complex revealed the possibility for R135 to switch from an intramolecular salt bridge with E247 to an intermolecular interaction with the backbone carboxylate of Gta, i.e. that of F350 (Fig. 1a). This is concurrent with the establishment of a salt bridge between E247 of Rho and K345 of Gta (Fig. 1a). The semiempirical model of tetrameric Rho can be considered as the weak aggregation of two rows of dimers [9]. The inter-monomer interface in each dimer is characterized by contacts between (a) the extracellular loop 2 (i.e. E2), from both monomers, (b) I2, from both monomers, and (c) H4 and H5, from both monomers. In contrast, contacts between dimers are few and involve I1, I3, and the C-tail [9]. The number of docking solutions that passed the distancebased filter in docking simulations using dimeric Rho as a target is lower compared to docking simulations with monomeric Rho (i.e. 400 and 604 solutions, respectively, Table 1). This is suggestive of a diminished propensity of the cytosolic surface
of dimeric Rho to recognize Gt, compared to the cytosolic surface of the monomeric form. In line with these results, the most reliable complex between dimeric Rho and Gt, i.e. S37 (ZD score = 60.53, Table 1), holds a 1:1 Rho:Gt stoichiometry (Figs. 1b and 2b). In fact, in this complex, only the cytosolic domains of the Rho monomer A participate in the Rho–Gt interface (Figs. 1b and 2b). This architecture is essentially the same as that of the best complex achieved by using monomeric Rho as a target (Figs. 1a, b and 2a, b). In fact, the Ca-RMSD (computed over Rho and Gta) between the best complexes involving monomeric ˚ (Table 1). Such deviation and dimeric Rho is equal to 0.79 A does not change following energy minimization of the two complexes. An alternative complex characterized by a 2:1 Rho:Gt stoichiometry (i.e. with both the Rho monomers involved in the interface with Gt) was also found though with a lower ZD score compared to the 1:1 complex (i.e. S153, ZD score = 56.92, Fig. 3). The 2:1 complex is characterized by the docking of the Gta C-term on the cytosolic extension of H3 of Rho monomer A, whereas Gtb docks on the C-tail of Rho monomer B (Fig. 3). It is worth noting that the ZD score concerning the 1:1 complex is higher than that of the 2:1 complex, in spite of the larger Rho-Gt interface in the latter (i.e.
D. Dell’Orco et al. / FEBS Letters 581 (2007) 944–948
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a 1:1 Rho:Gt stoichiometry and is the 30th one out of 4000 (i.e. S30, ZD score = 67.94, Table 1 and Figs. 1c and 2c). This solution is very similar to the best ones achieved by using monomeric and dimeric Rho as targets, i.e. the Ca-RMSDs ˚ and 0.82 A ˚ , respectively (Fig. 1). Such deviations are 1.51 A do not change following energy minimization of the complexes. We could not find any alternative high scored and reliable Rho:Gt stoichiometry involving the tetrameric model of the photoreceptor. We cannot exclude, however, that these results depend on the low resolution of the oligomeric Rho model.
4. Discussion
Fig. 3. Views in a direction perpendicular (top) and parallel (bottom) to the membrane surface of an alternative complex between Gt and dimeric Rho (ZD score = 56.92). This complex is characterized by a 2:1 Rho:Gt stoichiometry. The Gt a-, b- and c-subunits are colored in violet, orange, and blue, respectively, whereas Rho monomers A and B are in green and gray, respectively. Drawings were done by means of the software PYMOL 0.97 (http://pymol.sourceforge.net/).
ZD scores of 60.53 and 56.92 versus buried solvent accessible ˚ 2 and 4195 A ˚ 2, respectively). This is surface areas of 3430 A suggestive of an overall better complementarity achieved with the 1:1 Rho:Gt stoichiometry compared to the 2:1 one. Docking simulations by using tetrameric dark Rho as a target (i.e. a supramolecular assembly made of inter-dimeric contacts between A–B and C–D dimers) led to further decrease in the number of filtered solutions compared to docking simulations with dimeric Rho (i.e. 385 and 400 solutions, respectively, Table 1). The only reliable docking solution is again that with
It is perceived wisdom that Rho recognizes Gt only in its active state [1–3]. Our recent study, based on robust integration between computational modeling and in vitro experiments, suggested that also inactive Rho has the potential to recognize heterotrimeric Gt [6]. Indeed, the predictions of a number of rigid body docking simulations all converged into the same high scored dark Rho–Gt complex. In this complex, the functionally important R135 of the E/DRY motif of Rho makes contacts with the C-terminal carboxylate of Gta, indicating that the highly conserved amino acid residue of Rho is accessible to Gt already in the dark state (Fig. 1) [6]. Although their physiological implications in phototransduction await clarification, the inferences from our study are supported by ever increasing evidence from in vitro experiments. One is that proton uptake from the cytosol, associated with MII formation, requires the presence of Gt to occur [23]. Other evidence comes from surface modification and mass spectrometry [24] as well as from plasmon-waveguide resonance spectroscopy (PWR) determinations [25,26]. PWR spectroscopy experiments, in fact, showed that the affinity of dark Rho for heterotrimeric Gt is 64 nM [25]. The hypothesis that dark Rho can recognize Gt is also supported by recent observations that Gta can be activated by Rho from red crystals in the dark, though at slower activation rate compared to photoactivation [5]. Further support to the hypothesis comes from the striking structural similarities between the crystal structures of dark and photoactivated deprotonated Rho [5]. The results of docking simulations done in this study between oligomeric Rho and heterotrimeric Gt further corroborate the predictions achieved with monomeric Rho. In fact, the most reliable and best scored complexes achieved by using dimeric or tetrameric Rho as a target are very similar to each other and to the best complex achieved by using monomeric Rho (Figs. 1 and 2). Even if these results could be consequences of the low resolution of the oligomeric Rho model, they, however, strongly suggest that the molecular determinants for recognizing Gt are held by one Rho monomer. In this respect, the relevant Rho–Gt interface is predicted to be made by residues from the cytosolic extensions of H3 and H6 and from I2 of one Rho monomer and by residues from the a3/b5 and a4/b6 loops as well as the C-term of Gta (Figs. 1 and 2). Docking simulations between dimeric Rho and Gt provided also an alternative complex characterized by a 2:1 Rho:Gt stoichiometry, i.e. with both Rho monomers involved in the interface with Gt (Fig. 3). However, both the ZD score and the topology of the Gta N-term significantly disfavor this complex compared to the one characterized by a 1:1 Rho:Gt
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stoichiometry. Furthermore, docking simulations by using tetrameric Rho could not find any high scored and reliable solution with 2:1, or 3:1, or 4:1 Rho:Gt stoichiometry, i.e. with two, or three or four Rho monomers contributing to the interface with Gt. These results disagree with a computational model proposed by Filipek and co-workers, concerning the complex between heterotrimeric Gt and a tetrameric Rho model holding the same architecture as that employed in our study [27]. In the study by Filipek and co-workers, the tetramer is made by three Rho monomers in an inactive conformation and one monomer mimicking the active state. Furthermore, the Rho:Gt stoichiometry is 4:1, i.e. all the four Rho monomers participate in the interface [27]. In conclusion, the results of our computational analysis strengthen the hypothesis that dark Rho has the potential to recognize Gt or, in other words, that Rho and Gt can be found coupled already in the dark. Moreover, simulations with oligomeric Rho support the hypothesis that the most likely Rho:Gt stoichiometry is the 1:1 one. This means that the essential molecular determinants for Gt recognition and activation are held by one Rho monomer. In this respect, the complex between one Rho molecule and one heterotrimeric Gt should be considered as the functional unit, consistent with in vitro evidence [10,11]. Acknowledgement: This study was supported by a Telethon–Italy Grant No. TCP00068 (To FF).
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Monomeric dark rhodopsin holds the molecular determinants for transducin recognition: Insights from computational analysis Daniele Dell’Orcoa,b, Michele Seebera,b, Francesca Fanellia,b,* a
Department of Chemistry, University of Modena and Reggio Emilia, via Campi 183, 41100 Modena, Italy b Dulbecco Telethon Institute, via Campi 183, 41100 Modena, Italy Received 21 December 2006; revised 15 January 2007; accepted 25 January 2007 Available online 6 February 2007 Edited by Robert B. Russell
Abstract In this computational study, we have investigated the implications of rhodopsin (Rho) oligomerization in transducin (Gt) recognition. The results of docking simulations between heterotrimeric Gt and monomeric, dimeric and tetrameric inactive Rho corroborate the hypothesis that Rho and Gt can be found coupled already in the dark. Moreover, our extensive computational analysis suggests that the most likely Rho:Gt stoichiometry is the 1:1 one. This means that the essential molecular determinants for Gt recognition and activation are contained in one Rho monomer. In this respect, the complex between one Rho molecule and one heterotrimeric Gt should be considered as the functional unit. Ó 2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: Molecular recognition; Docking; GPCRs; G proteins
1. Introduction The G protein-coupled receptor (GPCR) rhodopsin (Rho) is the rod-cell visual pigment responsible for the dim light vision, which acts as a trigger of the visual cascade in all vertebrates. The absorption of a photon by the receptor results in the alltrans isomerization of the 11-cis retinal, the chromophore covalently linked to Rho by a protonated Schiff base (PSB) [1]. Light-induced fast isomerization of retinal (200 fs) triggers a cascade of early-photointermediate states, characterized by nano to microsecond relaxation kinetics, resulting in the active state of the photoreceptor, i.e. Meta II (MII). MII formation from MI follows Schiff base deprotonation and takes about 1 ms to occur [1]. So far, MII has been considered the unique Rho state able to recognize the cognate G protein, Gt or transducin [1–3]. The consequent MII-induced Gt activation trig* Corresponding author. Address: Dulbecco Telethon Institute, Department of Chemistry, via Campi 183, 41100 Modena, Italy. Fax: +39 059 37353. E-mail address: [email protected] (F. Fanelli).
Abbreviations: Gt, transducin; GPCRs, G protein-coupled receptors; Rho, rhodopsin; PSB, protonated Schiff base; MII, Meta II; MI, Meta I; H, helix; AFM, atomic force microscopy; ZD, ZDOCK; PDB, Protein Data Bank; Ca-RMSD, Ca-atom root mean square deviation; I2, second intracellular loop; E2, second extracellular loop; I1, first intracellular loop; I3, third intracellular loop; PWR, plasmon-waveguide resonance
gers the intracellular signaling pathway, ultimately resulting in the visual response [1]. To date, the structure of Rho in its dark-inactive state has ˚ [4]. Very recently, a photobeen completely resolved at 2.2 A activated deprotonated intermediate of bovine Rho, reminis˚ [5]. Comparisons of cent of MII, has been resolved at 4.15 A the dark and photo-activated structures reveal that the changes that accompany photo-activation are smaller than previously predicted for MII and consist in modest increases in solvent accessibility of selected amino acids essentially located at the cytosolic extensions of helices 3 and 6 (H3 and H6). The significant structural similarity between the cytosolic domains of dark and activated Rho suggests that both the receptor forms are able to recognize Gt. This is in line with the inferences from our previous computational experiments that disagree with the commonly accepted wisdom that MII is the unique Rho state intended for recognizing Gt [3,6,7]. Indeed, following computational analyses of the shape and electrostatic complementarities between the crystal structures of dark Rho and heterotrimeric Gt, we inferred that Rho has the potential to recognize Gt even in its dark state [6,7]. The evidence that GPCRs exist and function as constitutive dimers/oligomers seems to be true also for dark Rho. In fact, atomic force microscopy (AFM) images showed that dark Rho forms paracrystalline arrays of dimers in mouse disc membranes [8]. AFM measurements led to the building of a semiempirical structural model of oligomeric Rho (PDB code: 1N3M) [9]. In this study, we have extended to dimeric and tetrameric dark Rho the computational analysis of Rho–Gt complementarity previously done on the monomeric form [6]. The aim is to explore the implications of Rho oligomerization in Gt recognition. The results of this study strengthen the hypothesis that Rho can be pre-coupled to Gt in the dark, suggesting also that the molecular determinants for Gt recognition and activation are held by the monomeric form of the photoreceptor. In this respect, the complex between monomeric Rho and heterotrimeric Gt should be considered as the functional unit, consistent with in vitro evidence [10,11].
2. Methods The analysis of the structural complementarity between the cytosolic domains of dark Rho and heterotrimeric Gt was done by exhaustively sampling the roto-translational space of one protein (probe) with respect to the other (target). The rigid-body docking algorithm ZDOCK
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D. Dell’Orco et al. / FEBS Letters 581 (2007) 944–948 (ZD) was employed [12]. The crystal structure of dark Rho in its monomeric (PDB code: 1U19 [4]), dimeric, and tetrameric forms was used as a fixed protein (i.e. target), whereas heterotrimeric Gt was allowed to explore all the possible orientations around the cytosolic domains of the target (i.e. probe). Dimeric and tetrameric Rho models were achieved by fitting the highest resolution structure, i.e. 1U19 ˚ ), into each monomer in the semi-empirical oligomeric model (2.2 A of Rho released as 1N3M [9]. Such fitting was aimed at improving the resolution level of the oligomer. In fact, in 1N3M, each Rho mono˚ structure 1HZX completed in the missing cytosolic mer is the 2.8 A domains by molecular modeling [9]. Thus, dimeric and tetrameric Rho employed in this study are assemblies of two and four 1U19 structures, respectively. The Gt structure employed as a probe was the one named as Gt_mut3 in our previous study [6]. This structure was a mutated form of the crystal structure of Gtab1c1 (i.e. a Gta/Gia1 chimera, PDB code: 1GOT [13]) holding the bovine Gta sequence. Other modifications made to the original 1GOT were the addition of the last ten amino acids of Gta from the NMR structure (i.e. PDB code: 1AQG [14]), and the last 10 amino acids of Gtc, modeled in a-helix. To improve sampling efficiency, the Rho portions 1–59, 76–130, 157–220, and 252–308, corresponding to the transmembrane and extracellular domains, were not taken into account in docking simulations. A rotational sampling interval of 6° was employed, and the best 4000 solutions were retained and ranked according to the ZD score. To filter the most reliable solutions among the 4000 best scored ones, the low resolution information from in vitro experiments on MII–Gt interactions [2,15–19] were translated into broad Ca–Ca intermolecular dis˚ between Rho-S240 and tance constraints, i.e. (a) lower than 15.0 A ˚ between Rhoboth Gta-N343 and Gta-D311; (b) lower than 20.0 A ˚ between Rho-Q312 R135 and Gta-F350; and (c) lower than 20.0 A and Gta-K345 (see Ref. [6] for more detail). Distance-based filtering was carried out by means of the FIPD software [20]. Filtering of solutions concerning oligomeric receptors, i.e. dimers and tetramers, was carried out on each Rho monomer. For each docking run, the solutions fulfilling at least one of the three distance constraints were collected and subjected to cluster analysis followed by visual inspection of the cluster centers (i.e. the solution representative of each clusters). A Ca-atom root mean square deviation ˚ was employed as a threshold for clustering (see (Ca-RMSD) of 3.0 A Ref. [6] for details). Energy minimization of the selected complexes between dimeric or tetrameric Rho and Gt was done by means of CHARMM [21], by employing the IMM1 implicit water/membrane model [22]. Minimizations were carried out by using 1500 steps of steepest descent followed by a conjugate gradient minimization, until the root mean square gra˚ . The adjustable parameter ‘‘a’’ dient was less than 0.001 kcal/mol A ˚, and the non-polar core thickness were set equal to 0.85 and 32 A respectively. The ‘‘united atom approximation’’ was employed. All
945 the backbone atoms except those of the last ten amino acids of Gta were kept fixed during minimization. Side chain minimization concerned the amino acids at the receptor-G protein interface.
3. Results Our previous docking simulations between different structural models of heterotrimeric Gt and monomeric Rho (i.e. 1U19) proved the good tolerance of the ZD software to changes in the side chain conformations of Gtab, as well as of the length and side chain and main chain conformations of Gtc [6]. In fact, all the different docking simulations converged into very similar predictions, i.e. the Ca-RMSDs of the best scored solutions from each run, which passed the distance-based filter and fulfilled the membrane topology requirements for the N-terminal a-helix of Gta (i.e. aN), were close to zero [6]. Importantly, such high scored realistic solutions fell among the first 23 out of the 4000 output solutions from each run [6]. In particular, the best realistic solution achieved with the same Gt employed in this study is the 10th out of 4000 (i.e. S10, ZD score = 60.18, Table 1, Figs. 1a and 2a). In such complex, the N-terminal a-helix of Gta is almost parallel and close enough to the membrane surface to allow the hydrophobic N-acyl and farnesyl modifications of the a- and c-subunits,
Table 1 Best docking solutions and docking parameters MODELa
Sol. N.b
ZD scorec
Rank N.d
˚ )e Ca-RMSD (A
Monomer Dimer Tetramer
604 400 385
60.18 60.53 67.94
10 37 30
– 0.79 1.51
a Oligomeric state (i.e. monomer, dimer, or tetramer) of the rhodopsin molecule employed as a target in each rigid-body docking run. b Number of solutions that passed the distance-based filters. c ZDOCK score of the best docking solution from each run carried out by employing monomeric, dimeric and tetrameric rhodopsin. d Rank order of the best docking solution. e ˚ ) of the best docking solution for dimeric and tetrameric Ca-RMSD (A rhodopsin with respect to the best solution for monomeric rhodopsin.
Fig. 1. Best complex between heterotrimeric Gt and the cytosolic half of (a) monomeric (ZD score = 60.18), (b) dimeric (ZD score = 60.53), and (c) tetrameric (ZD score = 67.94) dark Rho, seen in a direction parallel to the putative membrane surface. In panels (b) and (c), only the Rho monomer that participates in the interface with Gt is shown. The Gt a-, b- and c-subunits are colored in violet, orange and blue, respectively, whereas Rho is colored in green. Sticks represent the side chains of R135 and E247, from Rho, and of K345, from Gta. Drawings were done by means of the software PYMOL 0.97 (http://pymol.sourceforge.net/).
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Fig. 2. Views in a direction perpendicular (top) and parallel (bottom) to the membrane surface of the best predicted complexes between heterotrimeric Gt and (a) monomeric (ZD score = 60.18), (b) dimeric (ZD score = 60.53), and (c) tetrameric (ZD score = 67.94) Rho. All these complexes are characterized by a 1:1 Rho:Gt stoichiometry. The Gt a-, b- and c-subunits are colored in violet, orange and blue, respectively, whereas Rho monomers A, B, C and D are colored in green, gray, magenta, and cyan, respectively. Drawings were done by means of the software PYMOL 0.97 (http://pymol.sourceforge.net/).
respectively, to insert into the membrane (Figs. 1a and 2a). Furthermore, the C-term of Gta makes contacts with the second intracellular loop (I2), the cytosolic extensions of H3 and H6, as well as with H8 of Rho [6]. The functionally important R135 of the E/DRY motif is almost accessible to the C-term of Gta already in the dark state. Indeed, a limited energy minimization of the Rho–Gt complex revealed the possibility for R135 to switch from an intramolecular salt bridge with E247 to an intermolecular interaction with the backbone carboxylate of Gta, i.e. that of F350 (Fig. 1a). This is concurrent with the establishment of a salt bridge between E247 of Rho and K345 of Gta (Fig. 1a). The semiempirical model of tetrameric Rho can be considered as the weak aggregation of two rows of dimers [9]. The inter-monomer interface in each dimer is characterized by contacts between (a) the extracellular loop 2 (i.e. E2), from both monomers, (b) I2, from both monomers, and (c) H4 and H5, from both monomers. In contrast, contacts between dimers are few and involve I1, I3, and the C-tail [9]. The number of docking solutions that passed the distancebased filter in docking simulations using dimeric Rho as a target is lower compared to docking simulations with monomeric Rho (i.e. 400 and 604 solutions, respectively, Table 1). This is suggestive of a diminished propensity of the cytosolic surface
of dimeric Rho to recognize Gt, compared to the cytosolic surface of the monomeric form. In line with these results, the most reliable complex between dimeric Rho and Gt, i.e. S37 (ZD score = 60.53, Table 1), holds a 1:1 Rho:Gt stoichiometry (Figs. 1b and 2b). In fact, in this complex, only the cytosolic domains of the Rho monomer A participate in the Rho–Gt interface (Figs. 1b and 2b). This architecture is essentially the same as that of the best complex achieved by using monomeric Rho as a target (Figs. 1a, b and 2a, b). In fact, the Ca-RMSD (computed over Rho and Gta) between the best complexes involving monomeric ˚ (Table 1). Such deviation and dimeric Rho is equal to 0.79 A does not change following energy minimization of the two complexes. An alternative complex characterized by a 2:1 Rho:Gt stoichiometry (i.e. with both the Rho monomers involved in the interface with Gt) was also found though with a lower ZD score compared to the 1:1 complex (i.e. S153, ZD score = 56.92, Fig. 3). The 2:1 complex is characterized by the docking of the Gta C-term on the cytosolic extension of H3 of Rho monomer A, whereas Gtb docks on the C-tail of Rho monomer B (Fig. 3). It is worth noting that the ZD score concerning the 1:1 complex is higher than that of the 2:1 complex, in spite of the larger Rho-Gt interface in the latter (i.e.
D. Dell’Orco et al. / FEBS Letters 581 (2007) 944–948
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a 1:1 Rho:Gt stoichiometry and is the 30th one out of 4000 (i.e. S30, ZD score = 67.94, Table 1 and Figs. 1c and 2c). This solution is very similar to the best ones achieved by using monomeric and dimeric Rho as targets, i.e. the Ca-RMSDs ˚ and 0.82 A ˚ , respectively (Fig. 1). Such deviations are 1.51 A do not change following energy minimization of the complexes. We could not find any alternative high scored and reliable Rho:Gt stoichiometry involving the tetrameric model of the photoreceptor. We cannot exclude, however, that these results depend on the low resolution of the oligomeric Rho model.
4. Discussion
Fig. 3. Views in a direction perpendicular (top) and parallel (bottom) to the membrane surface of an alternative complex between Gt and dimeric Rho (ZD score = 56.92). This complex is characterized by a 2:1 Rho:Gt stoichiometry. The Gt a-, b- and c-subunits are colored in violet, orange, and blue, respectively, whereas Rho monomers A and B are in green and gray, respectively. Drawings were done by means of the software PYMOL 0.97 (http://pymol.sourceforge.net/).
ZD scores of 60.53 and 56.92 versus buried solvent accessible ˚ 2 and 4195 A ˚ 2, respectively). This is surface areas of 3430 A suggestive of an overall better complementarity achieved with the 1:1 Rho:Gt stoichiometry compared to the 2:1 one. Docking simulations by using tetrameric dark Rho as a target (i.e. a supramolecular assembly made of inter-dimeric contacts between A–B and C–D dimers) led to further decrease in the number of filtered solutions compared to docking simulations with dimeric Rho (i.e. 385 and 400 solutions, respectively, Table 1). The only reliable docking solution is again that with
It is perceived wisdom that Rho recognizes Gt only in its active state [1–3]. Our recent study, based on robust integration between computational modeling and in vitro experiments, suggested that also inactive Rho has the potential to recognize heterotrimeric Gt [6]. Indeed, the predictions of a number of rigid body docking simulations all converged into the same high scored dark Rho–Gt complex. In this complex, the functionally important R135 of the E/DRY motif of Rho makes contacts with the C-terminal carboxylate of Gta, indicating that the highly conserved amino acid residue of Rho is accessible to Gt already in the dark state (Fig. 1) [6]. Although their physiological implications in phototransduction await clarification, the inferences from our study are supported by ever increasing evidence from in vitro experiments. One is that proton uptake from the cytosol, associated with MII formation, requires the presence of Gt to occur [23]. Other evidence comes from surface modification and mass spectrometry [24] as well as from plasmon-waveguide resonance spectroscopy (PWR) determinations [25,26]. PWR spectroscopy experiments, in fact, showed that the affinity of dark Rho for heterotrimeric Gt is 64 nM [25]. The hypothesis that dark Rho can recognize Gt is also supported by recent observations that Gta can be activated by Rho from red crystals in the dark, though at slower activation rate compared to photoactivation [5]. Further support to the hypothesis comes from the striking structural similarities between the crystal structures of dark and photoactivated deprotonated Rho [5]. The results of docking simulations done in this study between oligomeric Rho and heterotrimeric Gt further corroborate the predictions achieved with monomeric Rho. In fact, the most reliable and best scored complexes achieved by using dimeric or tetrameric Rho as a target are very similar to each other and to the best complex achieved by using monomeric Rho (Figs. 1 and 2). Even if these results could be consequences of the low resolution of the oligomeric Rho model, they, however, strongly suggest that the molecular determinants for recognizing Gt are held by one Rho monomer. In this respect, the relevant Rho–Gt interface is predicted to be made by residues from the cytosolic extensions of H3 and H6 and from I2 of one Rho monomer and by residues from the a3/b5 and a4/b6 loops as well as the C-term of Gta (Figs. 1 and 2). Docking simulations between dimeric Rho and Gt provided also an alternative complex characterized by a 2:1 Rho:Gt stoichiometry, i.e. with both Rho monomers involved in the interface with Gt (Fig. 3). However, both the ZD score and the topology of the Gta N-term significantly disfavor this complex compared to the one characterized by a 1:1 Rho:Gt
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stoichiometry. Furthermore, docking simulations by using tetrameric Rho could not find any high scored and reliable solution with 2:1, or 3:1, or 4:1 Rho:Gt stoichiometry, i.e. with two, or three or four Rho monomers contributing to the interface with Gt. These results disagree with a computational model proposed by Filipek and co-workers, concerning the complex between heterotrimeric Gt and a tetrameric Rho model holding the same architecture as that employed in our study [27]. In the study by Filipek and co-workers, the tetramer is made by three Rho monomers in an inactive conformation and one monomer mimicking the active state. Furthermore, the Rho:Gt stoichiometry is 4:1, i.e. all the four Rho monomers participate in the interface [27]. In conclusion, the results of our computational analysis strengthen the hypothesis that dark Rho has the potential to recognize Gt or, in other words, that Rho and Gt can be found coupled already in the dark. Moreover, simulations with oligomeric Rho support the hypothesis that the most likely Rho:Gt stoichiometry is the 1:1 one. This means that the essential molecular determinants for Gt recognition and activation are held by one Rho monomer. In this respect, the complex between one Rho molecule and one heterotrimeric Gt should be considered as the functional unit, consistent with in vitro evidence [10,11]. Acknowledgement: This study was supported by a Telethon–Italy Grant No. TCP00068 (To FF).
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