Relocating the Active-Site Lysine in Rhodopsin and Implications for Evolution of Retinylidene Proteins

Relocating the active-site lysine in rhodopsin and implications for evolution of retinylidene proteins

Erin L. Devine, Daniel D. Oprian1, and Douglas L. Theobald1

Department of Biochemistry, Brandeis University, Waltham, MA 02454

Edited by Steven O. Smith, Stony Brook University, Stony Brook, NY, and accepted by the Editorial Board July 8, 2013 (received for review April 10, 2013) Type I and type II rhodopsins share several structural features retinal chromophore is attached to the protein covalently by including a G protein-coupled receptor fold and a highly conserved means of a protonated Schiff base to the e-amino group of a Lys active-site Lys residue in the seventh transmembrane segment of residue, Lys216, in the seventh transmembrane α-helix. Upon the protein. However, the two families lack significant sequence absorption of light, a key intermediate, M, in the proton pumping similarity that would indicate common ancestry. Consequently, cycle forms in which the Schiff base nitrogen is no longer pro- the rhodopsin fold and conserved Lys are widely thought to have tonated. With the exception of a few fungal proteins of unknown arisen from functional constraints during convergent evolution. To function, the GPCR fold and the active-site Lys are also conserved test for the existence of such a constraint, we asked whether it among all type I rhodopsin homologs. were possible to relocate the highly conserved Lys296 in the visual Despite the striking structural and functional similarities of pigment bovine rhodopsin. We show here that the Lys can be the type I and type II rhodopsins, there is no significant sequence moved to three other locations in the protein while maintaining identity between these two families that would suggest a common the ability to form a pigment with 11-cis-retinal and activate the G ancestral origin (1). It is widely believed that the common fold protein transducin in a light-dependent manner. These results con- and active-site Lys are products of convergent evolution resulting tradict the convergent hypothesis and support the homology of from functional constraints on the proteins (1, 7–12). To test this type I and type II rhodopsins by divergent evolution from a com- hypothesis, we have focused on the visual pigment rhodopsin and mon ancestral protein. asked whether it is possible to move the active-site Lys296 to a different location in the protein. We attempted to move the he retinylidene proteins are integral membrane proteins that Lys to five different locations: two positions in transmembrane Tcovalently bind a retinal chromophore. Amino acid sequence helix (TM) 2, one in TM3, one to a different location in TM7, comparison divides these proteins into two families known as and one in the β-hairpin loop connecting TM4 and TM5 that type I and type II rhodopsins (1). Type I rhodopsins, such as forms part of the retinal binding pocket. Surprisingly, four of the bacteriorhodopsin from the archaeon Halobacterium salinarum, five mutants combine with 11-cis-retinal to form pigments with function as light-driven ion transporters, channels, and photo- near wild-type spectral properties, and three of these four taxis receptors. Type II rhodopsins, best known for the visual activate transducin in a light-dependent manner with specific pigment of mammalian rod photoreceptor cells, function pri- activities approaching that of wild-type rhodopsin. These marily as photosensitive receptor proteins in metazoan eyes and results demonstrate that an absolutely conserved, common — — in certain extraocular tissues. Henceforth, we will use the term structural feature theSchiffbaseLysinhelixseven is not ’ “rhodopsin” to refer to the visual pigment of bovine rod pho- required for rhodopsin s photosensitive function, contradicting a toreceptor cells and “bacteriorhodopsin” to refer to the light- key prediction of convergent evolution resulting from functional driven proton pump of H. salinarum. constraint. Rhodopsin is a prototypical member of the large family of G Results protein-coupled receptors (GPCRs; specifically class A GPCRs) The K296G and K296A mutant rhodopsins, in which the active- (2). It is composed of an apoprotein (called “opsin”) and an 11- site Lys has been removed, cannot covalently bind 11-cis-retinal cis-retinal chromophore, resulting in a pigment with λ = 500 max (13). However, both mutants can bind a Schiff base complex of nm. The GPCR fold comprises seven transmembrane α-helices 11-cis-retinal and an external alkyl amine, thereby producing oriented in a particular spatial arrangement with a specific a pigment with near wild-type properties (absorption maximum connectivity (SCOP classification scop.b.g.c.A; ref. 3). In rho- approximately 490 nm and restored ability to light-activate dopsin, the N terminus resides in the intradiscal (i.e., extracel- transducin) (13). The apoprotein forms of K296G and K296A lular) space and the C terminus in the cytoplasm. The 11-cis- activate transducin in the dark without retinal, an unusual ability retinal chromophore is covalently attached to the protein by known as constitutive activity (14). This behavior results from BIOCHEMISTRY means of a protonated Schiff base to the e-amino group of disruption of an internal salt bridge between the Lys296 e-amino Lys296 in the seventh helix. The GPCR fold and active-site Lys group and the Glu113 carboxylate, the so-called Schiff base are absolutely conserved among all visual pigments of higher “counterion” (15–17). eukaryotes. Because K296G and K296A cannot bind 11-cis-retinal co- Upon absorption of light, the 11-cis-retinal isomerizes to the valently, they have lost the ability to activate transducin in a all-trans form. The protein responds with a conformational light-dependent manner. We wondered, therefore, whether we change leading to an enzymatic cascade that begins with activation of the G protein transducin and ends with closure of cation channels in the plasma membrane and hyperpolarization of the Author contributions: E.L.D., D.D.O., and D.L.T. designed research; E.L.D. performed re- rod cell (4). A key intermediate in the photoactivation of rho- search; and E.L.D., D.D.O., and D.L.T. wrote the paper. dopsin is the species metarhodopsin II (MII) (5). MII is the only The authors declare no conflict of interest. intermediate capable of activating transducin and is characterized λ = This article is a PNAS Direct Submission. S.O.S. is a guest editor invited by the Editorial by an absorption maximum in the near UV ( max 380 nm), Board. resulting from deprotonation of the Schiff base. 1To whom correspondence may be addressed. E-mail: [email protected] or dtheobald@ Like rhodopsin, bacteriorhodopsin adopts the GPCR fold brandeis.edu. (1, 3, 6). Bacteriorhodopsin is oriented with the N terminus in This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. the extracellular space and the C terminus in the cytoplasm. The 1073/pnas.1306826110/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1306826110 PNAS | August 13, 2013 | vol. 110 | no. 33 | 13351–13355 Downloaded by guest on September 24, 2021 could introduce a Lys residue at a different site to rescue 11-cis- retinal binding and restore light-dependent activation of transducin. We selected five different sites for the newly introduced Lys residues (Fig. 1 and Fig. S1): Gly90, Thr94, Ala117, Ser186, and Phe293. All five positions are in the active site, located within 9 Å (range of 5–9 Å) of the retinal C15. The five sites are distributed among several different secondary structure elements, including TM2 (Gly90 and Thr94), TM3 (Ala117), the two-stranded β-sheet connecting TM4 and 5 (Ser186), and finally TM7 (Phe293), the same transmembrane segment as the original Lys296 of the WT protein. In each case, the target amino acid was changed to a Lys residue within the context of the N2C/ D282C thermally stable mutant rhodopsin that also contains a mutation of the active-site Lys296 (to either a Gly or an Ala). In total, we made 10 mutant proteins. We evaluated pigment function with three criteria: (i) ability to bind the 11-cis-retinal chromophore covalently via a Schiff base to the introduced Lys; (ii) ability to form a pigment with a long-wavelength absorption maximum; and (iii) ability to activate the G protein transducin in a light-dependent manner. With the exception of K296G/A117K and K296A/A117K, all of the mutant proteins bind retinal and form a pigment with a long-wavelength absorption maximum (Fig. 2 and Table 1). The mutants K296G/F293K, K296A/F293K, and K296A/G90K have a low yield of long-wavelength pigment and a significant peak at 380 nm, indicating a fraction of unprotonated Schiff base or free retinal. However, the remaining mutants were isolated in good yield and exhibit near wild-type absorption maxima (λmax = 476–484 nm). We chose one mutant for each new Lys position to test for ability to activate transducin: K296G/G90K, K296A/ T94K, K296A/S186K, and K296G/F293K. Three mutants activate transducin in a light-dependent manner (Fig. 3). The K296A/S186K and K296G/G90K mutants ap- proach the activity observed with the wild-type protein, whereas the K296A/T94K mutant exhibits significantly less activity, in Fig. 2. Ability of rhodopsin mutants to form pigments with 11-cis-retinal. extent of reaction and initial rate. The initial rates for K296A/ Normalized UV-visible absorption spectra for dark-adapted pigments in 0.02% (wt/vol) DDM at pH 7.5 at room temperature. Insets are an expanded S186K and K296G/G90K are similar. However, the extent of the λ fl view of the long-wavelength max peak. Scale bars in upper left corner of reaction is less for K296A/S186K, probably re ecting the greater each panel (upper right of Insets) represent 0.03 absolute absorbance.

stability of the MII intermediate in K296G/G90K as indicated by the yield of 440-nm pigment following acid trap of light-exposed samples (Fig. S2). In contrast, the K296G/F293K mutant displays decidedly nonwild-type behavior (Fig. 3). This mutant rhodopsin activates transducin, but in a light-independent manner. The K296G/F293K mutant is active in the dark, and exposure to light does not change the rate of transducin activation (although the spectrum is clearly converted to a peak with a λmax of 380 nm; Fig. S2). The K296G and K296A mutants are both constitutively active, whereas wild-type rhodopsin is not. Hence, it was also of interest to determine how the introduced Lys residues affect the constitutive activity of the K296G and K296A parents. Only the S186K mutant displays residual constitutive activity, which is signiﬁ- cantly reduced relative to the parents (Fig. 4). Thus, all of the Lys mutations (even at position 293) successfully rescue wild- type behavior of the apoprotein forms. Discussion Recently, the rational design of retinal binding proteins has emphasized the importance of active site geometry for forming a protonated Schiff base (18, 19). Successful reengineering of the Cellular Retinoic Acid Binding Protein II required proper orientation of the Lys e-amino group relative to the carbonyl cis Fig. 1. The retinal-binding pocket of bovine rhodopsin (PDB ID code 1U19). center of 11- -retinal for nucleophilic attack. However, we 11-cis-retinal, orange; Glu113 counterion, red; Lys296, blue; positions at which describe four different Lys positions that are capable of binding a Lys was introduced (Gly90, Thr94, Ala117, Ser186, and Phe293), green. retinal (G90K, T94K, S186K, and F293K). Each of these alternative

13352 | www.pnas.org/cgi/doi/10.1073/pnas.1306826110 Devine et al. Downloaded by guest on September 24, 2021 Table 1. UV-visible absorption maxima of rhodopsin mutants

Construct Dark λmax, nm Light λmax, nm Acid trapped after light λmax,nm

WT 500 384 428 K296G —— — K296G/G90K 483 381 438 K296G/T94K 475 384 396 K296G/A117K —— — K296G/S186K 469 384 402 K296G/F293K 485 368 393 K296A —— — K296A/G90K 478 381 436 K296A/T94K 483 382 402 K296A/A117K —— — K296A/S186K 478 385 401 K296A/F293K 493 370 423

positions forces the Lys to react with retinal from drastically constitutive activity is neutralization of charge on the Schiff base different angles, indicating that ideal Bürgi–Dunitz and Flippin– counterion residue, Glu113. Lodge angles are not required for Schiff base formation in The K296G/F293K mutant forms a pigment with 11-cis-retinal rhodopsin. This result is perhaps not entirely surprising, because and activates transducin, but the activity does not depend on the Bürgi–Dunitz and Flippin–Lodge angles were originally de- light. The apoprotein form, in contrast, does not activate trans- scribed for two molecules that collide in solution, not held in a ducin. Thus, the K296G/F293K mutation suppresses the consti- specific orientation by a protein scaffold. tutive activity of the K296G parent and allows the protein to The Lys mutations may also provide insight into the mecha- form a protonated Schiff base with retinal, but once retinal is nism of rhodopsin activation. In current models, movement of bound, the protein is locked in an active conformation. This TM5 and TM6 is related to a corresponding displacement of the behavior is significantly different from that of the previously β-sheet connecting TM4 and 5 (EL2) (5, 20). In the functional reported dark-active rhodopsin mutant E113Q/M257Y (21). K296A/S186K mutant, the Schiff base is located on EL2. If ac- Individually, the E113Q and M257Y mutations constitutively tivation involves the movement of EL2 away from retinal in the activate rhodopsin. The double mutant is also constitutively ac- binding pocket, there must be considerable plasticity in the active tive but, in addition, displays dark activity not seen with either site, such that minor structural rearrangements are easily ac- single mutation alone. The dark activity of this mutant likely commodated by the mechanism leading to the global confor- results from conformational uncoupling of the transducin acti- mational change for active rhodopsin. vation domain and retinal-binding site. In contrast, the K296G/ Surprisingly, several second-site Lys mutations suppress the F293K apoprotein is inactive but becomes trapped in an active constitutive activity of the K296G and K296A parents (Fig. 4). conformation after binding retinal. In this way, K296G/F293K is With the exception of K296A/S186K, the constitutive activity of reminiscent of the “steric doorstop” mutant T118W described by all of the Lys mutants is reduced to within experimental error of Kono and coworkers (22). Rhodopsin activates transiently in the the N2C/D282C host. Second-site mutations that reverse the process of binding retinal, as if the protein adopts an active con- constitutive activity of a functional rhodopsin are rare. These formation while opening to allow the ligand to enter the binding results are consistent with a model in which a key determinant of pocket (23–25). In the T118W mutant, changing Thr118 to a bulkier side chain results in a steric clash with the 9-methyl group of the retinal, preventing closure of the binding pocket and trapping the protein in an active conformation. Perhaps the dark activity of the K296G/F293K mutant similarly results from a steric clash due to a slight repositioning of the chromophore. The K296G/A117K and K296A/A117K mutants deserve com- ment. Previous mutagenesis studies indicated that an acidic residue (either Asp or Glu) at the 117 position can substitute for the Schiff base counterion (26, 27). Thus, even before the crystal BIOCHEMISTRY structure of rhodopsin confirmed that Ala117 is one helical turn from Glu113, indirect methods had shown that Ala117 is close to the Schiff base nitrogen. In addition, the E113A/A117E mutant forms a protonated MII intermediate upon exposure to light (28), demonstrating that the 117 side chain is close to the Schiff base nitrogen in both the excited state and the ground (or dark) state of the protein. In the present study, we attempted to move the Schiff base Lys from position 296 in TM7 to position 117 in TM3, but the mutant is incapable of forming a pigment with added 11-cis-retinal (Fig. 2). The inability to form a pigment 35 Fig. 3. [ S]-GTPγS binding to transducin following light activation of select could simply indicate that the mutant protein does not fold mutant pigments. Each reaction contained 5 nM rhodopsin, 1 μM transducin, μ γ properly. However, in the A117K mutant, the Glu113 counterion 3 M GTP S in 10 mM Tris buffer at pH 7.5, 100 mM NaCl, 5 mM MgCl2, e 0.1 mM EDTA, and 0.01% (wt/vol) DDM. Blue circles, WT; green squares, may be so close to the Lys -amino group that the nitrogen K296G/G90K; orange triangles, K296A/S186K; maroon diamonds, K296G/ remains protonated, unable to provide a lone pair of electrons F293K; black inverted triangles, K296A/T94K; shading, reactions run in the for nucleophilic attack on the carbonyl carbon of the retinal. dark. Error bars represent the SD (n = 4). This interpretation is supported by the high yield of protein

Devine et al. PNAS | August 13, 2013 | vol. 110 | no. 33 | 13353 Downloaded by guest on September 24, 2021 active-site Lys to a location in TM7. The mutants clearly demonstrate that rhodopsin can retain function when the Lys is moved to a different location. These results support a divergent evolutionary scenario in which a common ancestor gave rise to the modern retinylidene proteins. One might wonder why the active-site Lys has not migrated to other locations during divergent evolution. Relocating the Lys would likely require an intermediate either with no active-site Lys or with Lys residues at both locations. Perhaps the Lys has been retained in TM7 because neither of these intermediates is capable of forming a viable pigment. Materials and Methods Materials. Synthesis and purification of 11-cis-retinal was as described (30). Dodecyl β-D-maltoside (DDM) was from Calbiochem. DMEM was purchased from GIBCO. Bovine growth serum and Dulbecco’s PBS were from HyClone Laboratories. GTP was from Amersham Biosciences, GTPγS was from Sigma- Fig. 4. Constitutive activation of transducin by select mutant opsins, mon- 35 Aldrich, and [ S]-GTPγS (1,250 Ci/mmol) was from Perkin-Elmer. Oligos for itored by [35S]-GTPγS binding to activated transducin. Each reaction con- mutagenesis were purchased from Integrated DNA Technologies. tained 5 nM opsin, 1 μM transducin, 3 μM GTPγS in 10 mM Tris buffer at pH The antirhodopsin monoclonal antibody 1D4 was from the National Cell 7.5, 100 mM NaCl, 5 mM MgCl , 0.1 mM EDTA, and 0.01% (wt/vol) DDM. 2 Culture Center (Minneapolis, MN). The 1D4-Sepharose 4B immunoaffinity Purple open triangles, K296A; light blue open squares, K296G; orange tri- matrix used to purify rhodopsin was prepared as described (30). The 1D4- angles, K296A/S186K; light blue +, K296A/A117K; green squares, K296G/ peptide, corresponding to the carboxyl-terminal 8 amino acids of rhodopsin, G90K; black inverted triangles, K296A/T94K; maroon diamonds, K296G/ was used to elute the protein from the immunoaffinity matrix. F293K; light purple X, K296G/A117K; blue circles, WT. Error bars represent fi the SD (n = 4). Transducin was puri ed from frozen bovine retinae (Schenk Packing Co.), as reported (30, 31). Transducin concentration was determined spectro- − − photometrically by using an absorption coefficient of 93,570 M 1·cm 1 at 35 γ fi from the immunoaffinity column, because denatured rhodopsin 280 nm and by active-site titration using [ S]-GTP S of known speci cra- is usually retained on the column under our purification con- dioactivity (31). Transducin was monitored for contamination by rhodopsin using either a transducin activation assay or Western blot analysis with the ditions (29, 30). Close proximity of the Schiff base and Glu113 1D4 antibody. is also consistent with the loss of constitutive activity in this mutant (Fig. 4) (14). Mutagenesis, Expression, and Purification of the Proteins. All mutations were Is the highly conserved active site Lys residue in TM7 of type I made in the context of the thermally stable N2C/D282C mutant of bovine and type II rhodopsins a consequence of convergent evolution rhodopsin, which was considered to be WT in this study. The N2C/D282C due to a shared functional constraint on two unrelated protein mutant contains an engineered disulfide bond between two introduced families? If so, it should not be feasible to move the active-site cysteine residues, N2C and D282C (30). The engineered disulfide confers Lys to another location in the protein and retain the ability to enhanced thermal stability to the opsin form of the protein (30, 32). Crystal structures of the N2C/D282C mutant, in both the dark state (33) and active form a functional pigment with retinal. In fact, we were able to state (34, 35), show the protein to be identical to that of native rhodopsin construct multiple functional type II pigments in which the active (36–39), except for the missing oligosaccharyl chain at position 2 and the site Lys is moved to three different positions in different sec- presence of electron density corresponding to a disulfide bond connecting ondary structure elements. Therefore, photosensitive rhodopsin the two side-chain sulfur atoms at positions 2 and 282. In addition, func- function does not require a Lys-retinal Schiff base linkage in tional studies performed to date show that this mutant behaves as does WT TM7. These results support the homology of type I and type II in all ways except with respect to stability of the opsin form in detergent rhodopsins by demonstrating that the shared Schiff base position solution (30, 32). is not a product of convergence due to functional constraint. Mutations were introduced into the cDNA for rhodopsin by QuikChange mutagenesis (Stratagene). All rhodopsin mutants were expressed transiently Evolution has optimized modern type II rhodopsins over − in HEK293S-GnT1 cells by using calcium phosphate precipitation for trans- millions of years, regardless of whether type I and type II rho- fection (40). Proteins were purified and then reconstituted with 11-cis-retinal dopsins are convergent or divergent. Unlike with the natural essentially as described (30) except that mutants were eluted from the 1D4- rhodopsins, we have made little effort to optimize the function of Sepharose matrix following a 1-h incubation with 0.02% (wt/vol) DDM in

our mutant constructs, other than the N2C/D282C background 5 mM Hepes buffer at pH 7.5, 0.1 mM MgCl2,and3mMNaN3 containing mutations that confer some stability to the protein. It is plausible 80 μM 1D4-peptide at room temperature. Molar absorption coefficients e = −1· −1 e = that we could find other compensatory mutations that would were calculated relative to WT ( 500 40,600 M cm ; K296G/G90K, 483 −1 −1 −1 −1 42,200 M ·cm ; K296A/T94K, e483 = 36,300 M ·cm ; K296A/S186K, e478 = improve the function of our mutants. For example, other mu- −1 −1 −1 −1 36,100 M ·cm ; K296G/F293K, e485 = 24,200 M ·cm ) by acid-trapping tations could suppress the low constitutive activity of K296A/ −1 −1 the chromophore (e440 = 31,000 M ·cm ) in the dark with 0.5% (wt/vol) S186K or could red-shift the absorbance maximum of the SDS in 50 mM sodium phosphate buffer at pH 3.5 (final concentrations) as mutants by 10 nm. It is likewise possible that, say, the K296A/ has been described (41). S186K mutant would have no constitutive activity in a different protein background from another species; we have tried alter- Absorption Spectroscopy. UV-visible absorption spectra were recorded with nate Lys positions in only one protein from one species. In any a Hitachi model U-3210 that was specifically modified by the manufacturer case, convergent evolution can access all these potential variants for use in a darkroom. Data were collected with a microcomputer by using (different protein backgrounds and compensatory mutations). GraphPad Prism from GraphPad Software. All spectra were recorded with The fact that our crude mutagenesis found functional alternative samples at 25 °C and a path length of 1.0 cm. Pigments were bleached by fi Lys locations so easily—and that evidently nature has not—fur- exposure to light from a 300-W tungsten bulb ltered through a 435-nm cut- on filter for 30 s. ther underscores the implausibility of evolutionary convergence to the same Lys location in helix seven. Transducin Activation Assays. A filter-binding assay, described (30, 31, 41), was In summary, we have tested the hypothesis that the function of −1 −1 used to monitor the ability of mutant pigments or opsins (e280 = 65,000 M ·cm rhodopsin (in terms of binding retinal, formation of a long- used for all mutants) to catalyze the exchange of GDP for [35S]-GTPγS wavelength pigment, and activation of transducin) constrains the in transducin.

13354 | www.pnas.org/cgi/doi/10.1073/pnas.1306826110 Devine et al. Downloaded by guest on September 24, 2021 ACKNOWLEDGMENTS. We thank Stephanie M. McMorris for technical as- was supported by National Institutes of Health Grants EY007965 (to D.D.O.), sistance with cell culture and expression of rhodopsin mutants. This work 5T32GM007596 (to E.L.D.), and GM094468 and GM096053 (to D.L.T.).

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