Structures of the archaerhodopsin-3 transporter reveal that disordering of internal water networks underpins receptor sensitization

Many transmembrane receptors have a desensitized state, in which they are unable to respond to external stimuli. The family of microbial rhodopsin proteins includes one such group of receptors, whose inactive or dark-adapted (DA) state is established in the prolonged absence of light. Here, we present high-resolution crystal structures of the ground (light-adapted) and DA states of Archaerhodopsin-3 (AR3), solved to 1.1 Å and 1.3 Å resolution respectively. We observe significant differences between the two states in the dynamics of water molecules that are coupled via H-bonds to the retinal Schiff Base. Supporting QM/MM calculations reveal how the DA state permits a thermodynamic equilibrium between retinal isomers to be established, and how this same change is prevented in the ground state in the absence of light. We suggest that the different arrangement of internal water networks in AR3 is responsible for the faster photocycle kinetics compared to homologs.

Kinetic properties of chimeric class I aldehyde dehydrogenases for retinal isomers

Retinal dehydrogenase type 1 (RALDH1) catalyzes the oxidation of all-trans and 9-cis retinal to the respective retinoic acids (RAs), whereas another member of the aldehyde dehydrogenase (ALDH) family, the phenobarbital-induced aldehyde dehydrogenase (PB-ALDH), is very poorly active. We have previously generated chimeras between these 2 enzymes that displayed selectivity for retinal isomers in crude bacterial extracts. Here we have characterized the kinetic properties of the corresponding purified recombinant proteins. The all-trans selective chimera RALDH-131 converted all-trans retinal to all-trans RA with 2.9-fold lower efficiency than the wild-type RALDH1 and had only residual activity with 9-cis retinal. The converse chimera PB-131 was specific for 9-cis retinal, with no residual activity for all-trans retinal. MgCl2 inhibited the activities of RALDH1 and PB-131, but not of RALDH-131, suggesting that amino acids 132–510 in RALDH are necessary for inhibition by MgCl2. These data demonstrate that the chimeric enzymes act as retinal isomer-selective ALDHs, and suggest that these enzymes may be useful to study the roles of cis RA isomers in embryogenesis and differentiation in vivo.