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The cycle of vision is a chain of biochemical reactions that occur after exposure of the pigments to the light. The known mechanisms of the transduction of the light pulse derive mainly from studies on bovine rhodopsin. The objective of this work is to construct molecular models of human rhodopsin and opsins, for which threedimensional structures are not available, to analyze the retinal environment and identify the similarities and differences that characterize the human visual pigments. One of the main results of this work is the identification of Glu102 as the probable second counterion of the Schiff base in M opsin (green pigments) and L opsin (red pigments). Further, the analysis of the molecular models allows uncovering the molecular bases of the different absorption maxima of M and L opsins with respect to rhodopsin and S opsin. These differences appear to be due to both an increase in the polarity of the retinal environment and specific electrostatic interactions, which determine a reorganization of the electronic distribution of retinal by selectively stabilizing one of the two resonance forms. Keywords: color vision; molecular modeling; opsin; rhodopsin; spectral tuning. Introduction The human retina has one type of rod cells for dim light vision and three types of cone cells that allow color discrimination. Rods and cones share the same principles of *Corresponding author: Fabio Polticelli, Department of Sciences, Roma Tre University, Viale G. Marconi 446, I-00146 Rome, Italy, E-mail: firstname.lastname@example.org. http://orcid.org/0000-00027657-2019; and National Institute of Nuclear Physics, Roma Tre Section, I-00146 Rome, Italy Francesca Centola: Department of Sciences, Roma Tre University, Rome, Italy phototransduction, the cellular mechanism of light detection. In vertebrates, there are five classes of visual pigments: RH1, RH2, SWS1, SWS2, and M/LWS. However, only three of these are present in the human eye: RH1, SWS1, and M/LWS . Human rhodopsin, characterized by a maximum absorption at 495 nm, belongs to the RH1 family . The cone opsin that absorbs at short wavelengths (at about 420 nm) belongs to the SWS1 class, whereas the human "green" and "red" cone pigments that absorb at medium and long wavelengths (at about 530 and 560 nm, respectively) belong to the M/LWS class . The color vision in humans is defined as trichromatic because the color perception relies upon this three cone opsins (called S, M, and L opsins). The rhodopsin and opsin chromophore is 11-cis-retinal covalently linked to a lysine residue (Lys296 in human rhodopsin) to form a Schiff base [4, 5]. The positive charge of the Schiff base is partially delocalized by alternative resonance structures (Figure 1). This delocalization is relevant for the spectral tuning of the chromophore. In fact, an increase in delocalization leads to a red shift of the maximum absorption, whereas a decrease in delocalization leads to a blue shift [6, 7]. The Schiff base local environment, and thus its protonation state, is another important factor influencing chromophore spectral properties. For instance, in bovine rhodopsin, the mutation of Glu113 to Gln determines a shift of the absorption peak from 500 to 380 nm [1, 2, 811]. In the M/LWS class of opsins, which display a maximum absorption higher than 500 nm, three main amino acid positions have been identified, which are thought to play a role in the spectral shift: 180, 277, and 285 (the numbering refers to human rhodopsin) [2, 6, 12]. The mutation of Ala180 to Ser is the most common for the red cone opsins, much less so for the green ones, as at least ~90% of them code for alanine in this position. Further experiments have shown that replacing the serine at position 180 with alanine in the red pigment makes it less sensitive to red light . Therefore, there is a much greater likelihood that visual transduction is initiated when, in the red pigment, serine rather than alanine is present in this position. The changes that occur in the 142Centola and Polticelli: Modeling of human visual pigments structural information on these proteins at the atomic level to analyze the relationship between their structure and function, with particular emphasis on the chromophore tuning mechanism. Methods The amino acid sequences of human rhodopsin and S, M, and L opsins were retrieved from the National Center for Biotechnology Information (NCBI) nonredundant protein sequence database (http://www.ncbi.nlm.nih. gov). The sequence accession numbers are the following: GI|4506527| human rhodopsin, GI|4502387| human S opsin, GI|187954663| human M opsin, and GI|9910526| human L opsin. These sequences were used in BLAST  searches against the Protein Data Bank (PDB)  subset of the NCBI nonredundant protein sequence database to identify suitable templates for structure modeling. This procedure identified the bovine rhodopsin three-dimensional structure (PDB code: 1F88 ) as the best template for all four proteins investigated. The molecular models were constructed using Iterative Threading Assembly Refinement (I-TASSER) , an online "workbench" for high-resolution modeling of the structure and function of proteins using the crystal structure of bovine rhodopsin as a template. The template-directed I-TASSER modeling pipeline consists of three general steps: (i) the sequence of interest is divided into template-aligned and templateunaligned regions. (ii) The fragments obtained from template-aligned regions are assembled by replica-exchange Monte Carlo simulations, whereas the structure of unaligned regions is built by ab initio folding simulations. (iii) The models obtained with this procedure are then refined in an iterative procedure in which the free energy, the global topology, and the hydrogen-bonding network of the model are optimized . The overall quality of the structural models is assessed by analyzing the values of C-score and TM-score provided by I-TASSER. C-score is a confidence score for estimating the quality of models generated by I-TASSER, which is calculated based on the significance of threading-template alignments and the convergence parameters of the structure assembly simulations. C-score values are typically in the range of (5, 2), where higher values characterize models with high confidence . TM-score is a scale for measuring the structural similarity between two structures . A TM-score of more than 0.5 indicates a model of correct topology. Figure 1:Resonance forms of retinal. The arrows indicate the equilibrium between the two forms and the corresponding blue/red shift of the maximum absorption wavelength. blue region of the spectrum (below 500 nm) are not well understood. The substitutions alanine to serine at position 180, phenylalanine to tyrosine at position 277, and alanine to threonine at position 285 have been shown to induce a red shift of the absorption peak, but no information on the tuning mechanism at the atomic level is available [2, 6, 12]. In the literature, it is known that amino acid positions 122 and 189 are important to determine the differences between opsins and rhodopsin . From the sequence alignment shown in Figure 2, it can be noted that, in human and bovine rhodopsins, an isoleucine is present in position 189, whereas this position is occupied by a proline residue in human opsins (Pro185 in S opsin and Pro205 in M and L opsins). The mutagenesis studies of Ile189 to Pro showed an increase in the rate of decay of the Meta II state . Thus, the absolute conservation of proline in position 189 affects the kinetics of decay of the activated state and the recycling of retinal . Also, Glu122 is conserved only in rhodopsin. In this regard, it is well known that in rhodopsin a glutamate in position 122 and an isoleucine in position 189 determine the typical characteristics of the visual pigments of rods: higher thermal stability, slower decay of the photointermediate, and slower regeneration of pigment in the dark . An additional factor that modulates the sensitivity of pigments and the absorption wavelength is the modulation of the interaction between the protonated Schiff base and counterions present in the retinal molecular environment . Given the absence of experimental structures for human rhodopsin and S, M, and L opsins, in this work, molecular modeling techniques have been used to obtain Centola and Polticelli: Modeling of human visual pigments143 Figure 2:Amino acid sequence alignment of bovine rhodopsin, human rhodopsin, and S, M, and L opsins. The alignment positions are colored according to BLOSUM62 score; a higher score corresponds to a darker blue color. Residues building up the retinal environment in bovine rhodopsin (using as a selection criterion a distance lower than 5 Å from the retinal moiety) are indicated by red stars below the alignment. The chromophore has been positioned within the constructed structural models by best fitting each model onto the bovine rhodopsin crystal structure. This procedure correctly positioned the retinal moiety within bonding distance from Lys296 (numbering refers to the bovine enzyme). In fact, the length of the bond connecting the N atom of Lys296 to the C15 atom of retinal ranges between 1.45 and 1.46 Å in all models, matching the distance measured on the bovine rhodopsin crystal structure (1.46 Å). Regarding the retinal environment, in each model, the conformation of the nonconserved residues surrounding the chromophore has been optimized using the SwissPdbViewer  rotamer library to avoid steric clashes and maximize favorable interactions such as hydrogen bonds. The molecular models of the proteins of interest were analyzed using the molecular graphics programs SwissPdbViewer  and UCSF Chimera . The schematic representation of the molecular environment of retinal in rhodopsins and opsins was obtained with LigPlot+ . Results and discussion Figure 2 shows the amino acid sequence alignment of bovine and human rhodopsins and human S, M, and L opsins. As seen in Figure 2, there is a high degree of conservation among all the sequences. In fact, most residues known to be involved in rhodopsin/opsin function are conserved. The primary Schiff base counterion Glu113 is conserved in both human rhodopsin and opsins. Pro267 is responsible for a distortion in helix VI, and Pro291 and Pro303, which are located in the vicinity of Lys296 (numbering of human rhodopsin), covalently bound to retinal to form the Schiff base . Glutamic acid and arginine residues in positions 134 and 135 are conserved in all proteins, whereas Tyr136 that is present in human and bovine rhodopsin and S opsin is replaced conservatively by tryptophan in M and L opsins. Glu147, which plays an important role in connecting the end of helices III and IV , is conserved in all proteins. The Glu181 residue, the second counterion of the Schiff base in the photocycle 144Centola and Polticelli: Modeling of human visual pigments , is conserved in human rhodopsin and S opsin, but is replaced by a His residue in M and L opsins. Given the high degree of sequence identity observed, the three-dimensional structure of bovine rhodopsin (PDB code: 1F88 ) is used as a template for the prediction of the structural models of human rhodopsin and S, M, and L opsins. The superposition of the three-dimensional structure of bovine rhodopsin on the structural models of human rhodopsin and opsins is shown in Figure 3. All four structural models display very good overall quality as assessed by their C-score  and TM-score  values calculated by I-TASSER and reported in Supplemental Material Table S1 (see Methods for details). As expected, the overall fold of human proteins is very similar to that of bovine rhodopsin, although some differences are observed at the level of the surface loops. The structural models of human proteins have been used to analyze the retinal environment to study the molecular basis of the different spectroscopic and functional properties that characterize human rhodopsin and opsins. The schematic diagrams of the retinal environment in bovine rhodopsin, human rhodopsin, and S, M, and L opsins, obtained using LigPlot+ , are shown in Supplemental Material Figures S1S5. As expected, the retinal environment of bovine and human rhodopsin is almost identical. A very similar retinal environment is also observed in the S opsin model (Supplemental Material Figure S3), with the conservation of fundamental residues, such as Lys293 (ortholog of Lys296 of rhodopsins), the primary Schiff base counterion Glu110 (ortholog of Glu113 of rhodopsins), and the second counterion Glu178 (Glu181 in bovine rhodopsin). In M and L opsins (Supplemental Material Figures S4 and S5, respectively), the primary counterion is conserved as Glu129, whereas, as stated above, the Glu181 ortholog position is occupied by a His residue (His197). The location of the above-cited counterions is shown in Figure 4. As seen in Figure 4, the second counterion (Glu181 in bovine rhodopsin) is conserved only in human rhodopsin (Glu181) and S opsin (Glu178). However, the analysis of the molecular models of M and L opsins evidenced the presence of a glutamic acid residue (Glu102) whose distance from the Schiff base (5.7 Å) is compatible with the role Figure 3:Superimposition of the three-dimensional structure of bovine rhodopsin (tan) on the structural models of (A) human rhodopsin (light blue), (B) S opsin (blue), (C) M opsin (green), and (D) L opsin (orange). The retinal moiety is shown in ball-and-stick representation and colored in red. Figure 4:Relative position of the acidic groups located in the retinal environment in bovine and human rhodopsins and human S, M, and L opsins. Residues and the corresponding labels are colored according to the color scheme of Figure 3. Centola and Polticelli: Modeling of human visual pigments145 as second counterion in stabilizing the Schiff base in the Meta I state (Figure 4) . Indeed, although Glu181 in rhodopsin (and Glu178 in S opsin) is located on the opposite side of the retinal moiety with respect to the position of Glu102 in M and L opsins, its distance from the Schiff base (6.9 Å) is even higher than that of Glu102. The molecular models of M and L opsins confirm the prediction that these proteins display the presence of His197 and Lys200 in the region hosting Glu181 in bovine rhodopsin . These residues would form a binding side for a chloride ion, whose negative charge has been hypothesized to play a role as second counterion of the Schiff base . However, in rhodopsins, the second counterion, in its protonated (neutral) form, has been hypothesized to participate directly in the mechanism of hydrolysis of the Schiff base and release of the retinal . This is obviously not possible for a chloride ion, whereas Glu102, being able to reversibly protonate, could play a similar role during M and L opsin photocycle. As mentioned above, the changes in the retinal environment between rhodopsins and opsins are responsible for the spectral tuning of these visual pigments. The molecular models presented in this work help rationalize the role of amino acid substitutions in the tuning mechanism. In fact, as seen in Figure 5, in M and L opsins, there are amino acid substitutions that increase the polarity of the retinal environment. In particular, the Ala164 residue present in bovine and human rhodopsins is replaced by Ser180 in M and L opsins. Interestingly, Ser180 is located in the vicinity of Trp183, a residue found in that position only in M and L opsins, being substituted by valine in S opsin and by cysteine in bovine and human rhodopsins (Figure 2). The distance between Ser180 and Trp183 is compatible with the formation of a side chain-to-side chain hydrogen bond. Thus, Ser180, in addition to directly increasing the polarity of the retinal environment, could do it indirectly by relocating the side chain of Trp183. In L opsin, additional substitutions are of particular interest. In fact, in this latter protein, Ala285 present in M opsin (Ala296 in bovine rhodopsin) is replaced by Thr285, and Phe277 (Phe261 in bovine rhodopsin) is replaced by Tyr277. The substitution of Phe277 (conserved in rhodopsin and S and M opsins) is one of the main factors responsible for the selective modification of the absorption spectrum of L opsin. In fact, the analysis of the structural model of this protein evidences that the hydroxyl group of Tyr277 is at only 3.6 Å distance from the C5 atom of retinal, which, in one of the "red-shifted" resonance forms of the cofactor, carries a net positive charge (Figures 1 and 5). Thus, Tyr277, by selectively stabilizing the electronic delocalization of retinal, would be one of the factors responsible for the red shift of the absorption spectrum of retinal in L opsin. A similar consideration applies to residue 285 (alanine in rhodopsin and S and M opsins and threonine in L opsin). These data confirm that the shift of the maximum absorption of retinal to higher wavelengths in M opsin and, to a higher degree, in L opsin is caused not just by an increase in the polarity of the retinal microenvironment with respect to rhodopsin and S opsin. In detail, the analysis of the molecular model of L opsin indicates that the hydroxyl groups of Tyr277 and Thr285 are in the correct position to establish strong electrostatic interactions with the delocalized positive charge of retinal. These interactions likely promote the delocalization of retinal electrons, which in turn determines the red shift of the maximum absorption wavelength of retinal [6, 7]. Conclusions The main result of this work consists of the availability, for the first time, of molecular models of human rhodopsin and opsins S, M, and L, which allows to analyze at an atomic level the relationship between the structure of these proteins and their functional properties. In this regard, one of the most important and original results is the identification of Glu102 in M and L opsins as the putative second counterion, which is thought to be missing in these opsins. The analysis of the models allows also to pinpoint the main reason for the different maximum absorption of M and L opsins with respect to rhodopsin and S opsin. As far as M opsin is concerned, Figure 5:Relative position of the amino acid residues responsible for the spectral tuning of retinal in bovine rhodopsin and human M and L opsins. See text for details. Residues and the corresponding labels are colored according to the color scheme of Figure 3. 146Centola and Polticelli: Modeling of human visual pigments this characteristic seems to be due to an increase of the polarity of the retinal environment (due to the presence of Ser180 and Trp183). In L opsin, this increase in polarity is accompanied by a stabilization of the positive charge delocalized on the retinal C5 atom due to specific interactions with residues Thr285 and Tyr277. These interactions would induce a reorganization of the electronic distribution of the chromophore and then red shift its maximum absorption wavelength. Author contributions: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission. Research funding: None declared. Employment or leadership: None declared. Honorarium: None declared. Competing interests: The funding organization(s) played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication. 10. Nathans J. Determinants of visual pigment absorbance: identification of the retinylidene Schiff's base counterion in bovine rhodopsin. Biochemistry 1990;29:974652. 11. Nathans J. Determinants of visual pigment absorbance: role of charged amino acids in the putative transmembrane segments. Biochemistry 1990;29:93742. 12. Chan T, Lee M, Sakmar TP. Introduction of hydroxyl-bearing amino acids causes bathochromic spectral shifts in rhodopsin: amino acid substitutions responsible for red- green color pigment spectral tuning. J Biol Chem 1992;267:947880. 13. Lamb TD, Collin SP, Pugh EN Jr. Evolution of the vertebrate eye: opsins, photoreceptors, retina and eye cup. Nat Rev Neurosci 2007;8:96076. 14. Kuwayama S, Imai H, Hirano T, Terakita A, Shichida Y. Conserved proline residue at position 189 in cone visual pigments as a determinant of molecular properties different from rhodopsins. Biochemistry 2002;41:1524552. 15. Hunt DM, Carvalho LS, Cowing JA, Parry JW, Wilkie SE, Davies WL, et al. Spectral tuning of shortwave-sensitive visual pigments in vertebrates. Photochem Photobiol 2007;83:30310. 16. Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, et al. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 1997;25:3389402. 17. Berman HM, Westbrook J, Feng Z, Gilliland G, Bhat TN, Weissig H, et al. The Protein Data Bank. Nucleic Acids Res 2000;28:23542. 18. Palczewski K, Kumasaka T, Hori T, Behnke CA, Motoshima H, Fox BA, et al. Crystal structure of rhodopsin: a G protein-coupled receptor. Science 2000;289:73945. 19. Zhang Y. I-TASSER server for protein 3D structure prediction. BMC Bioinform 2008;9:40. 20. Roy A, Kucukural A, Zhang Y. I-TASSER: a unified platform for automated protein structure and function prediction. Nat Prot 2010;5:72538. 21. Yang J, Yan R, Roy A, Xu D, Poisson J, Zhang Y. The I-TASSER Suite: protein structure and function prediction. Nat Methods 2015;12:78. 22. Zhang Y, Skolnick J. Scoring function for automated assessment of protein structure template quality. Proteins 2004;57:70210. 23. Guex N, Peitsch MC. SWISS-Model and the Swiss-PdbViewer: an environment for comparative protein modeling. Electrophoresis 1997;18:271423. 24. Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, et al. UCSF Chimera a visualization system for exploratory research and analysis. J Comput Chem 2004;25:160512. 25. Laskowski RA, Swindells MB. LigPlot+: multiple ligand-protein interaction diagrams for drug discovery. J Chem Inf Model 2011;51:277886.
Bio-Algorithms and Med-Systems – de Gruyter
Published: Sep 1, 2016
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