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

Abstract

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.

Fig. 1. Comparison of the light-adapted (LA) AR3 (6S6C [http://doi.org/10.2210/pdb6S6C/pdb], white) and bR (5ZIM [http://doi.org/10.2210/pdb5ZIM/pdb] purple) crystal structures.

The approximate positions of the extracellular (EC) and intracellular (IC) membrane interfaces are shown as black dotted lines. The retinylidene chromophore (formed by the post-translational conjugation of retinal to a lysine sidechain) is shown in stick representation. The transmembrane helices (shown in ribbon representation) are labeled from A to G. The N termini of both proteins face the extracellular (EC) side of the membrane and the C-termini face the intracellular (IC) side.

Fig. 2. Comparison of the conformations of retinal in the DA (6GUX [http://doi.org/10.2210/pdb6GUX/pdb]) and LA (6S6C [http://doi.org/10.2210/pdb6S6C/pdb]) states of AR3.

In the DA state (left) the C13 = C14 retinal bond has been modeled with 70% cis and 30% trans isomers (colored in dark and light pink, respectively). In the LA state (right) retinal (colored in dark and light pink respectively) is modeled in the all-trans state only, but as two different conformers. Movement of the β-ionone ring is also observed in both structures. The 2F_obs–F_calc electron density maps (blue mesh) around the retinal and Lys226 are contoured at 1.2σ.

Fig. 3. Calculated potentials of mean force (PMF) for the isomerization of the C12–C13 = C14–C15 dihedral of retinal in DA (blue) and LA (red) AR3.

The PMF was computed by sampling the retinal isomerization from all-trans to 13-cis and vice versa. Each point on the curve is generated from two independent 0.5 ns QM(SCC-DFTB)/MM MD trajectories, initiated from two separated equilibrated starting structures. The protein backbone was fixed in place; however, all other atoms (including those in the chromophore and amino acid sidechains) were allowed to move.

Fig. 4. Structures of the pentagonal H-bond networks in AR3.

Predicted H-bonds are represented by yellow dashes (for distances see Supplementary Table 4) for DA (a) and LA AR3 (b). Selected amino acid sidechains are shown in stick representation with atoms colored using the CPK convention. Water molecules are shown as red spheres and retinal is colored pink. Wat401 is seen in two positions (A and B) at partial occupancy and the sidechain of Arg92 is seen in four conformations for both AR3 structures. Wat402 has single occupancy in AR3 (b) and bR (Wat602 in Supplementary Fig. 8b) in the LA state.

Fig. 5. Structures of the Proton Release Complex (PRC) of AR3.

Glu204, Glu214 and the associated network of H-bonded water molecules in DA (6GUX [http://doi.org/10.2210/pdb6GUX/pdb]) (a) and LA AR3 (6S6C [http://doi.org/10.2210/pdb6S6C/pdb]) (b). Selected amino acid sidechains are shown in stick representation with atoms colored using the CPK convention. Water molecules are shown as red spheres and retinal is colored pink. The 2F_obs–F_calc electron density maps (blue mesh) around the water molecules are contoured at 1.2σ.

Fig. 6. Comparison of the structures the N termini of AR3 and related microbial rhodopsins.

Overlay of structures of bR (5ZIM [http://doi.org/10.2210/pdb5ZIM/pdb] purple), AR1 (1UAZ [http://doi.org/10.2210/pdb1UAZ/pdb] green), AR2 (3WQJ [http://doi.org/10.2210/pdb3WQJ/pdb] blue), and LA AR3 (6S6C [http://doi.org/10.2210/pdb6S6C/pdb], white) showing the extracellular-facing omega loop, which is present in AR1, AR2, and AR3 but absent in bR. The inset shows details of the 6S6C [http://doi.org/10.2210/pdb6S6C/pdb] AR3 omega loop. Amino acids are shown in stick representation with atoms colored using the CPK convention. The 2F_obs–F_calc electron density map (blue mesh) is contoured at 2.3σ.