Structural waters define a functional channel mediating activation of the GPCR, rhodopsin

Abstract

Structural water molecules may act as prosthetic groups indispensable for proper protein function. In the case of allosteric activation of G protein-coupled receptors (GPCRs), water likely imparts structural plasticity required for agonist-induced signal transmission. Inspection of structures of GPCR superfamily members reveals the presence of conserved embedded water molecules likely important to GPCR function. Coupling radiolytic hydroxyl radical labeling with rapid H(2)O(18) solvent mixing, we observed no exchange of these structural waters with bulk solvent in either ground state or for the Meta II or opsin states. However, the radiolysis approach permitted labeling of selected side chain residues within the transmembrane helices and revealed activation-induced changes in local structural constraints likely mediated by dynamics of both water and protein. These results suggest both a possible general mechanism for water-dependent communication in family A GPCRs based on structural conservation, and a strategy for probing membrane protein structure.

Fig. 1.

High resolution LC-MS analysis of proteolytic fragments of rhodopsin results in ≈88% amino acid sequence coverage. (A) The primary sequence of rhodopsin with proteolytic peptides detected and confirmed by tandem MS analysis is indicated by bars below the amino acid sequence. Peptides detected following digestion with pepsin are underlined by dark gray bars, and those detected after cyanogen bromide digestion and LC-MS analysis are underlined by light gray bars. Residues that make up transmembrane domains of rhodopsin are colored red. (B) LC-MS/MS analysis reveals radiolytic modification of many residues (yellow sticks) found in the membrane embedded region of rhodopsin as shown in this crystallographic model. The shaded rectangle represents the membrane; regions detected by LC-MS/MS analysis are colored green and undetected regions are colored gray. The chromophore, 11-cis-retinylidene, is shown as orange sticks. Ordered waters are shown as red spheres in the transmembrane domain.

Fig. 2.

The extent of bulk water exchange into the membrane embedded domain of rhodopsin was measured by monitoring the incorporation of O¹⁸ from isotopically labeled hydroxyl radicals. Taking advantage of locations sensitive to hydroxyl radical oxidation, we monitored solvent uptake for several activated states of this receptor over time. Rapid mixing of H₂O¹⁸ containing buffer with rhodopsin, Meta II, and opsin was followed by equilibration (delay) periods of 50 ms, 500 ms, 5 s, and 30 s before X-ray exposure for 6 or 40 ms. No O¹⁸ hydroxyl radical incorporation occurred in any of these experiments except for the exposed amino terminal peptide. This indicates that radicals that modify residues within the helical bundle are formed in situ in intramembranous regions of rhodopsin, photoactivated receptor, and apo-protein. Only following dehydration and rehydration of rhodopsin with 97% H₂O¹⁸ water and subsequent X-ray exposure were low levels of O¹⁸ incorporation detected in resulting proteolytic fragments (A, blue ribbons) from the transmembrane domain (A, yellow spheres). For example, O¹⁸ labeling of M86 in peptide ⁸⁵FMVFGGF⁹¹ located in helix II was specifically detected by tandem mass spectrometry. The daughter ion spectrum of unmodified peptide ⁸⁵FMVFGGF⁹¹ is shown in panel B and the tandem MS spectrum of O¹⁶-modified M86 in panel C. Importantly, O¹⁸ hydroxyl labeling of M86 following X-ray exposure was only detected following full exchange of solvent after sample dehydration and rehydration with H₂O¹⁸ (D).

Fig. 3.

Pictorial summary of modification rate constants. Radiolytic modification rate constants were determined for many residues in rhodopsin (Left), Meta II (Center), and opsin (Right). Residues with rate constants >0.1 s⁻¹ are rendered as spheres colored by rate constant ranges: 0.5–1.2 s⁻¹, light blue; 1.3–3.9 s⁻¹, light green; 4.0–5.9 s⁻¹, green; 6.0–7.9 s⁻¹, light-yellow; 8.0–9.9 s⁻¹, yellow; 10–14.9 s⁻¹, light-orange; 15–25 s⁻¹, orange; >200 s⁻¹, red. Following photoactivation, modification rates increased for M86, C140, M143, the pair of residues in helix IV I154 and M155, M163, and M288. Residues exhibiting decreased modification rates were Y301, P303, and Y306 in helix VII. There also was a reduced modification rate of M86 and F116 in opsin as compared with the two other states. The mixed modification of peptide 137–146, comprising part of the C-II loop, showed a large increase in the rates of detectable modification for opsin relative to ground state and activated rhodopsin, whereas M183 in the E-II loop exhibited no change in modification rate as a function of receptor activation state. The carboxyl terminal peptide did not show a marked difference in modification rates between the three states of the receptor. Changes in rates of oxidation observed when comparing ground state and activated receptor reflect local structural changes upon formation of both Meta II and opsin.