Mechanism of a molecular valve in the halorhodopsin chloride pump.

Andreea D. Gruia, Ana-Nicoleta Bondar, Jeremy C. Smith and Stefan Fischer*

Stucture  13, p. 617-627 (2005).  Full paper (PDF)



Halorhodopsin is an archaeal rhodopsin that uses light energy to pump chloride ions across the cellular plasma membrane during a photocycle triggered by retinal photoisomerization.
The mechanism by which chloride is transferred across the retinal chromophore after photoisomerization (the primary transfer step) is explored by computing all possible Minimum-Energy Pathways (MEP) for this process, using the Conjugate Peak Refinement (CPR) method.  This reveals that one pathway has an activation barrier (~9kcal/mol) significantly lower than the others.
Along this path, the chloride anion, driven by an interaction with the protonated Schiff base, transiently opens a passage between Ser115 and retinal, which is not present in the ground state structure. To allow this opening, flexible deformation of the protein more than 10Angstroem around retinal is necessary, mostly involving helix C.
This flexible deformation raises the chloride translocation barrier when retinal is in the all-trans ground-state.  Unlike macroscopic valve designs, chloride back-flow between photocycle is slowed down by the protein deformation, which serves as a valve spring.  This spring is tuned to allow differential ion flows in the pumping versus resting states that match the physiological times scales of the cell, thus creating a "kinetic" valve.
The all-trans retinal chromophore (orange) is covalently attached via a protonated Schiff base (blue) to Lys242 on helix G. 

Absorption of a photon trigers isomerization of the C13-C14 double bond of retinal into the 13-cis conformation.  This leads to a photocycle during which one chloride ion (green) is sequentially transfered from the extracellular to the cytoplasmic side of the membrane.

The arrows shows the chloride transfer in the order in which they occur. Step 1 is the 'primary' transfer step past the 13-cis retinal.

Chloride routes along the four different Minimum Energy Pathways found by the Conjugate Peak Refinement (CPR) method.

For each route, the chain of small spheres shows the position of the chloride, the bigger sphere indicating the position at the transition state.

Prefered path of the primary chloride transfer during pumping (retinal is 13-cis):
Path 1 has the lowest energy barrier (~9.2 kcal/mol).
To be seen: Chloride passes on the Ser115 side of retinal, in front of the Ser115 sidechain.  Chloride transiently enlarges a lumen by pushing away helix C and the palmitate.  H-bonds with water w24 and Ser115 are maintained until after the transition state is passed and the lumen starts reclosing.  They are replaced by a salt-bridge with the Schiff base and water w79.  Path 2 is very similar to path 1.
Download the movie , 1Mb

Chloride leaking in the resting state between photocycles (retinal in all-trans):
The energy barrier is 28kcal/mol, resulting in a very low rate of leaking, on the timescale of hours.  This prevents chloride backflow while the protein waits to be actived by the next photon. The route of the chloride is similar to that of path 1.
Download the movie , 1Mb

Energetically unfavorable paths for primary transfer (retinal is 13-cis):
Path 3 has an energy barrier of 17.2 kcal/mol, thus this path is unlikely.
To be seen: The chloride follows a route in the back of the sidechain of Ser115.
Download the movie , 1.6Mb

Path 4 has an energy barrier of 41.3 kcal/mol, thus this path is excluded.  There is no lower MEP with a chloride route on the Asp238 side of retinal.  The electrostatic repulsion between Asp238 and the chloride anion is responsible for the high barrier.
To be seen:  Chloride goes through the water cluster, Asp238 then bends away from the approaching anion and the chloride squeezes between retinal and Tyr210.
Download the movie , 1.6Mb

Collective motion of helices C and G allow the transient enlargement of a lumen near the Schiff base, on the Ser115 side of retinal, requiring flexible deformation of the protein up to 10 Angstroem around the Schiff base. This is facilitated by a built-in "breaking-point" in helix C, due to Pro117, located right next to the Schiff base.  For a description of the kinetic valve mechanism, see the paper.

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