Download Mechanisms of the Spectral Shifts for Retinitis Pigmentosa Mutants

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: [email protected] and [email protected]
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 6th
and 3rd 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 11cis 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 (λmax) 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 λmax‘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 proteinmembrane 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 tightbinding (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.