Role of Bulk Water in Hydrolysis of the Rhodopsin Chromophore

Abstract

Rhodopsin (Rho) is a prototypical G protein-coupled receptor that changes from an inactive conformational state to a G protein-activating state as a consequence of its retinal chromophore isomerization, 11-cis-retinal 3 all-trans-retinal. The photoisomerized chromophore covalently linked to Lys 296 by a Schiff base is subsequently hydrolyzed, but little is known about this reaction. Recent research indicates a significant role for tightly bound transmembrane water molecules in the Rho activation process. Atomic structures of Rho and hydroxyl radical footprinting reveal ordered waters within Rho transmembrane helices that are located close to highly conserved and functionally important receptor residues, forming a hydrogen bond network. Using 18 O-labeled H 2 O, we now report that water from bulk solvent, but not tightly bound water, is involved in the hydrolytic release of chromophore upon Rho activation by light. Moreover, small molecules (and presumably, water) enter the Rho structure from the cytoplasmic side of the membrane. Thus, this work indicates two distinct origins of water vital for Rho function.Rhodopsin (Rho), 3 the visual pigment in retinal rod photoreceptors, is a G protein-coupled receptor responsible for dim light vision (1, 2). Rho is composed of its apoprotein, opsin, and a retinylidene chromophore in an 11-cis-conformation covalently linked by a Schiff base to Lys 296 of the opsin (Fig. 1A). Photoactivation of Rho is initiated by isomerization of the retinylidene ligand to its all-trans-configuration (3), allowing the photoreceptor after deprotonation of the Schiff base to adopt the active Meta II state required for G protein (transducin) activation. The atomic structure of Rho led to the identification of crystallographically ordered water molecules adjacent to functionally important conserved protein residues (Fig. 1A) (4), supporting the conclusion that these waters are essential not just for structural stabilization of the receptor but also for the activation process. One of these tightly bound waters positioned close to the chromophore-binding pocket has been postulated to play a key role in the counterion switch between ground state (dark; Glu 113 ) and activated states (Glu 181 ) of Rho (5, 6). Moreover, Glu 113 and Glu 181 are also likely involved in the hydrolytic process via the carbinol ammonium ion and in a regeneration reaction between 11-cis-retinal and opsin. To form the Schiff base, Lys 296 must be deprotonated, the carbonyl group must be polarized, and water must be accommodated within the chromophore-binding site (1). Hydroxyl radical footprinting revealed local conformational changes in the Rho structure following its photoactivation, presumably mediated by the dynamics of both ordered water molecules and the protein (7,8). Moreover, protein footprinting combined with rapid H 2 18 O mixing methodology and deuterium-hydrogen exchange on C 2 His residues indicated that these tightly bound internal waters do not exchange with bulk solvent in ground state Rho, Meta II, or opsin...

Activation of G-protein-coupled receptors correlates with the formation of a continuous internal water pathway

“…For formyl peptide receptor 1 (FPR 1 ) and opioid receptors (ORs), using microsecond MD simulations, we showed that the influx of water molecules occurs towards the allosteric site, which is different from that in the human sphingosine-1-phosphate receptor 1 (S1PR 1 ) 30 . Jastrzebska et al 3 reported that water molecules can reach the opsin binding region from the intracellular side during the retinal isomerization in Rho, which is also seen in our MD simulations of Rho. These observations imply that there are two possible water penetration routes.…”

“…For G-protein-coupled receptors (GPCRs), which form the largest class of membrane proteins transmitting extracellular signals across the plasma membrane, internal water molecules have been proposed to play an important role for receptor activation but mechanistic models remain speculative 2 . Previous studies revealed that water molecules from the cytoplasmic region of rhodopsin (Rho) are involved in the hydrolytic release of retinal upon Rho activation by light 3,4 . Crystal structures of Rho and the beta-2 adrenergic receptor (b 2 AR) revealed further details.…”

Photocyclic behavior of rhodopsin induced by an atypical isomerization mechanism

“…Functional integrity of the preparation was examined spectrophotometrically by measuring the Rh6mr spectrum both in the dark and after a light stimulus. The orientation of Rh6mr or Rh in proteoliposomes was confirmed by Asp-N endoproteinase digestion (39,47), which cleaves opsin specifically between Gly329 and Asp330 in the C-terminal tail (48).…”

“…Briefly, a solution of commercially available soybean phospholipids (asolectin; Sigma-Aldrich) in buffer containing 10 mM MES (pH 5.9), 100 mM NaCl, and 1% n-nonyl-β-Dglucopyranoside (NG) was mixed with purified Rh6mr or Rh. The molar ratio of asolectin [assumed molecular mass, 760 (46)] to Rh6mr or Rh was 200:1 (39,47). The protein-lipid suspension was kept at 20°C for 2 h, followed by dialysis against buffer containing 10 mM MES (pH 5.9) and 100 mM NaCl (2,000-fold excess) at 4°C in the dark for 48 h with four buffer changes.…”

“…Rh6mr and Rh proteoliposomes were prepared as described previously (39). Briefly, a solution of commercially available soybean phospholipids (asolectin; Sigma-Aldrich) in buffer containing 10 mM MES (pH 5.9), 100 mM NaCl, and 1% n-nonyl-β-Dglucopyranoside (NG) was mixed with purified Rh6mr or Rh.…”