Erix Wiliam Hernández-Rodríguez

Mechanisms of the Spectral Shifts for Retinitis Pigmentosa Mutants Explored by Quantum Mechanical/Molecular Mechanical Calculations.

Erix Wiliam Hernández-Rodríguez,^†,* Elsa Sánchez-García,^‡ Ana Lilian Montero-Alejo,^¶ Luis Alberto Montero-Cabrera,^¶ Walter Thiel^‡,*

^†Departamento de Bioquímica, Instituto de Ciencias Básicas y Preclínicas“Victoria de Girón”, 11600 Havana City, Cuba and Charité Centrum für Innere Medizin und Dermatologie, Biomedizinishes Forschungszentrum, Campus Virchow, Charité-Universitätsmedizin, 13353 Berlin, Germany.

^¶Laboratorio de Química Computacional y Teórica. Departamento de Química Física, Universidad de La Habana, 10400 Havana City, Cuba.

^‡Max-Planck-Institut für Kohlenforschung Mülheim an der Ruhr, 45470 Germany.

^*Corresponding author. Email: [e-mail] and [e-mail]

ABSTRACT: Retinitis Pigmentosa (RP) is a pathological condition associated to blindness due to a progressive retinal degeneration. RP-linked mutations leading to a less stable rhodopsin and changes at the retinal binding site (RBS) can also cause deviations of absorption spectra, affecting crucial functions; even when the stability could be reached natural or artificially. Mutation effects are largely unknown beyond stability; its solution sheds light onto the molecular mechanisms related to optical spectra and dark-state geometry. To evaluate the stability, geometries, electric influence and vertical excitation energies in the dark state of mutated human rhodopsins carrying the abnormal substitutions M207R or S186W at RBS, we mainly calculated rhodopsins within or out a lipidic bilayer using Molecular Dynamics, combined Quantum Mechanical/Molecular Mechanical approach, for ground state properties and the ab initio DFT/MRCI and TDDFT methods for excited state calculations. Both mutations appear diminishing diminished the rhodopsin stability, even when the folding and retinal binding could take place in these mutants. Substantial blue-shifted absorption spectra were observed as well as geometrical deviations and electric changes causing an unoptimal geometry for the photoisomerization and the possibility of an increased energy in the dark state of mutated rhodopsins. The energy excess could lead to harmful reactions. The calculations explored in large regions of the conformational chromophore space were accurate, in very good agreement with available experimental data, providing a reliable atomistic insight on the mechanisms of these mutations near chromophore, which can open new therapeutic strategies with the purpose of minimizing the surplus energy and its consequences or/and using stabilizing molecules suggested in recent advances.

Introduction

Vision is one of the most valued senses for humans. Ophthalmological pathologies as Retinitis Pigmentosa (RP), a condition associated to blindness, have been increasing since years ago as an important public health problem (1). RP refers to a heterogeneous group of mostly inherited diseases characterized by progressive retinal degeneration due to death of the rod photoreceptor cells, the vertebrate photoreceptors dedicated to dim light vision (2, 3), and the outer field of vision is lost first when mutations affects the rhodopsin, the visual pigment in rods; central vision may be impaired at the beginning if the primary injury is in the visual pigments of cone photoreceptor cells, causing difficulty in tasks such as identifying colors and reading (4), although in some cases, other proteins and cells in retina some affected primarily (2). Even when a high genetic heterogeneity has been found, over 120 point mutations have been discovered identified in the rhodopsin gene. The large majority of these mutations cause autosomal dominant form (ADRP) of the pathology (3, 5). Patients suffering ADRP caused by rhodopsin mutations display night blindness, progressive loss of peripheral and, eventually, central vision and the characteristic accumulation of the intraretinal pigment deposits named lipofuscin, from which these dystrophies get its name (3-7), its pathogenesis and some mutation mechanisms are unclear; no a definitive treatment is available today.

Rhodopsin, the prototype of G-protein coupled receptor (GPCR) superfamily (8, 9), is a heptahelical transmembrane (TM) receptor protein expressed in retina, composed by four building blocks, (a) an apoprotein opsin with 348 amino acid residues, (b) a covalently bound chromophore (retinal), (c) two palmitoyl residues linked to Cys322 and Cys323 and (d) two sugars linked to Asn2 and Asn15 (10). In the dark state, the chromophore is the 11-cis-retinal, forming a retinal protonated Schiff´s base (RPSB) linkage with Lys296 at the rhodopsin binding pocket (RBP). Visible light absorption at ~500 nm (11) by the pigment triggers the isomerization of the 11-cis of the RPSB to the nonprotonated all-trans form of the retinal Schiff´s base (RBS) linked to the opsin upon a single photon absorption (10, 12), this reaction occurs with high efficiency characterized by a quantum yield in the range 0.65-0.67 (12, 13); providing rhodopsin with the energy to form the active state (14, 15); the primary photoproduct, photorhodopsin, is formed within a very short time (200 fs) and thermally relaxes within a few picoseconds to a distorted all-trans configuration, bathorhodopsin (10), which establishes the equilibrium with a blue-shifted intermediate (BSI) before forming lumirhodopsin. Metarhodopsin I intermediate is the next one followed by metarhodopsin II, the active conformation for G-protein coupling, Meta II state is formed by translocation of the Schiff’s base proton to residue Glu113, which plays the role of counterion for the RPSB in the dark state (12, 13). This sequence of events results in excitation of the visual nerve and perception of light in the brain (16).

RP-associated mutations can cause an impairment of protein folding or expression or retinal binding (17), affecting the rhodopsin function; but in other cases these processes can take place and/or could be favoured by drugs (18, 19), being the mutation influence on absorption and photoisomerization a more relevant aspect.

Extensive mutagenesis analyses on rhodopsin has shown that key residues as Pro267 are important, structurally; substitutions at that position can impair the transducin activation by affecting the structure of the G-protein interaction site and with implication for opsin folding, membrane insertion, assembly, and/or function (20). On the other hand, researches have been focused on RP-associated mutations for cysteines in rhodopsin, showing impossibility for regeneration with 11-cis retinal and transportation to plasma membrane (21). It is well documented by these studies that the disulfide bond forming Cys110 and Cys187 is necessary for an appropriate folding and receptor function (22).

Trp265 mutations have a large effect on the spectral properties of rhodopsin, changing the retinal binding site (20) as well as substitutions at Phe261 and Gly121, positions from the 6^th and 3^rd transmembrane helices, respectively, influences in the early steps of the photoisomerization, causing blue-shifted intermediates (23). In vitro, studies using recombinant rhodopsin for carrying amino acid substitutions associated with RP in different positions of the rhodopsin structure, including the RBP, show a modified spectral behavior (24, 25). Several other mutants studied related to, or not, with RP have been E113Q, E181Q, G90D, A292S, A269T, H211C and D83N displaying a diverse light-absorption pattern (16). A plentiful biochemical knowledge, molecular biology data on retinal proteins and high-resolution structural data by X-ray of bovine rhodopsin are available nowadays, for understanding the structure-function relationships at a pertinent molecular level for this prototypical GPCR (13, 26).

These advances with other experimental studies are a wealth used by theoretical studies to obtain new knowledge, besides to reproduce and to explain experimental data. The vast majority of researches on normal or wild-type (WT) and mutants structures have been performed for bovine rhodopsin and other retinal proteins with high-resolution structure data resolved experimentally; despite large experimental and computational studies in the last years as described and the plentiful knowledge reached on mechanism regulating the absorbance of 11-cis retinal chromophore in bovine rhodopsin (27), little data have been reported for human rhodopsin and its mutants, a not available crystal structure for this protein has been an important handicap.

Analyzing the mechanisms for RP-associated mutations needs structural information and hence it is crucial the human rhodopsin structure; although, its crystal structure is not solved until now, it is possible to achieve a reasonable three-dimensional (3D) structure, by means of Homology Modelling (28) combined with optimizations based on Molecular Mechanics (MM) and Molecular Dynamics (MD) methods (29), taking into account the high sequence similarity with bovine rhodopsin and enough biochemical data available. Subsequently, a reliable structure to calculate spectral properties and retinal geometry from human rhodopsin can be obtained, using QM/MM MD and QM/MM optimizations in different mediums (29, 30). On the other hand, robust methods for calculating the electronic spectra (31) can be used on an applicative optimized structure of human rhodopsin.

Hence, mechanisms of RP-associated mutations implicated with absorption shifts and photoisomerization, affecting the RBP in human rhodopsin, could be resolved appropriately, by theoretical methods at a very molecular level nowadays.

Obviously, rhodopsin mutations associated with RP act in many ways, from strong misfolding to severe impairments of folding/expression and/or binding retinal (32). However, other mutations can cause RP, changing amino acid residues near chromophore; even when the folding, binding retinal and light absorption take place somehow, its mechanisms are yet largely unknown.

M207R and S186W mutations cause ADRP (17) and affecting the RBP. A recent experimental study reports that the mutation M207R allows the rhodopsin folding, rhodopsin regeneration with 11-cis-retinal, increasing the experimental chromophore concentration and light absorption was detected in the dark state, outlining possible protonation-state changes for the SBL (33). A low misfolding at the second extracellular loop has been reported for the mutation S186W (34) and that thermally destabilize rhodopsin and to increase the rate of thermal isomerization, indirectly it is shown light absorption and folding in some grade (35); experimental studies about this mutation are not well documented.

Some authors of this paper previously published calculations of the wavelength of absorption maxima (λ_max) for these RP-associated mutations on a simpler and very reduced RBP model. Therefore, it constitutes the natural precedent for the present work. However, no estimation of the protonation states was then performed and vertical excitation energies were calculated with a simpler method, only allowing important hypotheses on light absorption patterns, although only reproducing λ_max‘s near to experimental values (36). Such previous paper left opened a wide field for the present work.

On the other hand, a very recent computational study using FoldX analysis on disease-linked rhodopsin mutations included to M207R and S186W. The research found a change in protein stability, in a mutation more than in other, classifying these mutations within Class IIa; hence, no interference with retinal binding area can be possible for these mutants, since the mutations was not within Class IIb. Both substitutions could destabilize in some extent the protein according to FoldX predictions (the higher ∆∆G, the more severe folding effects). The study also reported that the disease mechanism could be especially valuable for the misfolding Class II mutants, taking into account the advances made using small molecules to stabilize a protein that otherwise tends to misfold (37). It is needful to deepen into the mechanisms of these mutations beyond the protein stability; since for these mutations the folding and retinal binding are possible naturally, or through drugs; no evaluation at a very molecular level on the mutation (M207R or S186W) effect concerning optical spectra and/or farther processes has been reported yet; studies in that sense are required for improving a deeper and necessary understanding for prospective therapies.

It is reported that the optical properties of the chromophores in retinal proteins are modulate by the protein moiety. Roughly speaking, three mechanisms are described for the spectral tuning: first, distortion of the retinal geometry as a result of steric interactions with the protein binding pocket; second, interaction of the RPBS with the counterion Glu113 balancing its positive charge; and third, interaction of the retinal with the rest polar amino acid residues lining the binding pocket (38).

Since 207 and 186 positions drop into the RBP and near chromophore in the 3D rhodopsin structure, M207R and S186W mutations could induce structural changes, affecting the dark state geometry of the RBP, including chromophore, and/or electric perturbations, impairing the appropriate mechanisms of photoisomerization and/or spectral tuning.

Calculating the spectra and absorption shifts with accuracy for human rhodopsin and retinal proteins as a very challenging problem, even using combined Quantum Mechanical/Molecular Mechanical (QM/MM) approaches; where the entire protein into account and thus are viable in understanding the visual event (16, 39). Several models, from the more simple to the more complexes or realistic and diverse methods have been employed for calculating the optical spectra (27, 36). At present, spectral properties of rhodopsin can be studied with reasonable exactness using combined QM/MM approaches (36).

Specific conditions are important for an accurate calculation of the absorption spectra: (a) highly accurate methods must be used, (b) geometrical parameters of the chromophore must be properly described because the spectrum is highly sensitive to the chromophore geometry, (c) chromophore polarization by the environment as well as environment polarization must be considered, (d) dispersion effects should be taken into account, (e) a sampling of the conformations is necessary to calculate absorption energies and spectra that are directly comparable to those from the experiment. Most approaches rely on geometry optimization, evaluating the spectrum at a single point in configuration space. However, this may not necessarily lead to representative structures (40).

On the grounds of the previous considerations, here we apply combined QM/MM methods (30, 41) for calculating the protonated states of all titratable amino acid residues of rhodopsins and including a large RBP as active region. We are modelling all WT- and- mutant systems, either isolated or within a lipid membrane, as obtained from a previous homology modelling combined with MM optimization and MD. Then we evaluate the effect of M207R and S186W mutations on the absorption spectra, retinal molecular geometry, overall electronic changes in the involved sites and its interaction with the protein environment in the dark state from mutants of human rhodopsin. We also analyze possible mechanisms on how these mutations can be related to energy balancing, photoisomerization and some potential side or harmful reactions.

Structural and stability characteristics are followed either through classical MD’s of protein-membrane complexes or by QM/MM MD and QM/MM geometry optimization techniques. In this case we used as QM component both a self-consistent charge density functional tight-binding (SCC-DFTB) method and the B3LYP functional for DFT. Vertical excitation energies were determined by time-dependent DFT (TDDFT) and a DFT-based multi-reference configuration interaction treatment (DFT/MRCI). We found not previously reported studies on these mutations with such methodologies for establishing our further conclusions.

Anticipating our results, we found a decreased stability due to these mutations. We will see that a substantial blue-shifted absorption spectra appears in amino acid substitution carrying mutants. They appear mustly caused by the RP-associated mutations M207R and S186W, with regard to WT rhodopsins. The new found light-absorption patterns were consistent with geometric and electronic changes leading to abnormal geometries that influence the photoisomerization and energy balances in the dark state of the RBP.