Free backbone carbonyls mediate rhodopsin activation

Abstract

Conserved prolines in the transmembrane helices of G protein-coupled receptors (GPCRs) are often considered to function as hinges that divide the helix into two segments capable of independent motion. Depending on their potential to hydrogen-bond, the free C=O groups associated with these prolines can facilitate conformational flexibility, conformational switching or stabilize receptor structure. To address the role of conserved prolines in family A GPCRs, we focus on bovine rhodopsin, a GPCR in the visual receptor subfamily, using solid-state NMR spectroscopy. The free backbone C=O groups on helices H5 and H7 are found to stabilize the inactive rhodopsin structure through hydrogen-bonds to residues on adjacent helices. In response to light-induced isomerization of the retinal chromophore, hydrogen-bonding interactions involving these C=O groups are released facilitating H5 and H7 repacking onto the transmembrane core of the receptor. These results provide insights into the multiple structural and functional roles prolines play in membrane proteins.

Epistatic interactions influence terrestrial–marine functional shifts in cetacean rhodopsin

“…In rhodopsin, a central role of D83 appears to be stabilization of the dark state via H-bonds to N55 and a water molecule that is bonded to other residues [7,33,34]. Site N55 forms a H-bond with the carbonyl backbone of A299 in the dark state, which creates an indirect H-bond link between site 83 and site 299 (D83-N55-A299) [33,35]. During light activation, breakage and reformation of H-bonds in this network are key mechanisms by which the transmembrane helices rotate into the meta II conformation [34].…”

“…During light activation, breakage and reformation of H-bonds in this network are key mechanisms by which the transmembrane helices rotate into the meta II conformation [34]. In particular, recent work using solid-state NMR spectroscopic techniques have confirmed that the N55-A299 bond is broken during the transition to meta II [35]. This breakage may be due in part to reorientation of the N55 amine group away from A299 and towards D83 as seen in the bovine meta II crystal structure (figure 4a), which strengthens the D83-N55 bond [35,36].…”

G Protein-Coupled Receptors Contain Two Conserved Packing Clusters

“…At the other end of this network, the free C¼O at Ser7.46 hydrogen bonds with Asn1.50. A loss of the hydrogen bonding between Ser7.46 and Asn1.50 (51), and a corresponding increase in hydrogen bonding of the Asp2.50 carboxylic acid side chain (53) are observed upon rhodopsin activation. These changes may alter the local distortions of H7.…”

“…Although Pro5.50 forms important packing interactions, it also gives rise to a free C¼O group at position 5.46 that is able to switch hydrogen-bonding interactions upon activation. The free C¼O group associated with Pro7.50 also appears to function as a hydrogen-bonding switch in the second activation switch (see below) (51).…”

Complexity of Bovine Rhodopsin Activation Revealed at Low Temperature and Alkaline pH

“…41 Many structural studies reveal complex helix movements involving the rearrangements of H-bonding in the transmembrane region of the protein, and several states of the chromophore relaxation during activation. [21][22][23][24][25][26][27]42 Since the structural studies that reveal those motions are carried out by trapping intermediates at low temperatures and only some of the intermediates are likely to be trapped at low temperature, it is not possible to identify specific structures with specific intermediates observed here. Such correlations will require time-resolved structural studies under conditions that are comparable to photolysis conditions used in this study.…”