Crystal structure ofHalobacterium salinarumhalorhodopsin with a partially depopulated primary chloride-binding site

Abstract

The transmembrane pump halorhodopsin in halophilic archaea translocates chloride ions from the extracellular to the cytoplasmic side upon illumination. In the ground state a tightly bound chloride ion occupies the primary chloride-binding site (CBS I) close to the protonated Schiff base that links the retinal chromophore to the protein. The light-triggered trans-cis isomerization of retinal causes structural changes in the protein associated with movement of the chloride ion. In reverse, chemical depletion of CBS I in Natronomonas pharaonis halorhodopsin (NpHR) through deprotonation of the Schiff base results in conformational changes of the protein: a state thought to mimic late stages of the photocycle. Here, crystals of Halobacterium salinarum halorhodopsin (HsHR) were soaked at high pH to provoke deprotonation of the Schiff base and loss of chloride. The crystals changed colour from purple to yellow and the occupancy of CBS I was reduced from 1 to about 0.5. In contrast to NpHR, this chloride depletion did not cause substantial conformational changes in the protein. Nevertheless, two observations indicate that chloride depletion could eventually result in structural changes similar to those found in NpHR. Firstly, the partially chloride-depleted form of HsHR has increased normalized B factors in the region of helix C that is close to CBS I and changes its conformation in NpHR. Secondly, prolonged soaking of HsHR crystals at high pH resulted in loss of diffraction. In conclusion, the conformation of the chloride-free protein may not be compatible with this crystal form of HsHR despite a packing arrangement that hardly restrains helices E and F that presumably move during ion transport.

Utility of B-Factors in Protein Science: Interpreting Rigidity, Flexibility, and Internal Motion and Engineering Thermostability

“…Using carefully performed B-factor analyses on the basis of normalized values, inter alia, it was found that large movements of two helices (E and F) during the photocycle are essentially unrestrained by packing effects and that the crystal lattice is not disrupted . Later this research was extended …”

“…102 Later this research was extended. 103 In a different recent contribution, J. Li and co-workers utilized B-factors in a very different way. 68 They stressed the importance of distinguishing between true protein interactions and crystal packing contacts, which is of obvious significance when developing reliable structural bioinformatics procedures.…”

Pumping mechanism of NM-R3, a light-driven bacterial chloride importer in the rhodopsin family

“…Two crystal structures of NM-R3 in the dark state have been published, and two chloride ions are found; Cl-1 lies close to the Schiff base, and Cl-2 lies in an extracellular surface pocket (11,12). The network of bonds between chloride ions and highly ordered water molecules in NM-R3 is different from that of other chloride-pumping rhodopsins (4,14,15). Overlaying NM-R3 and NpHR with secondary-structure matching (SSM) (16) shows that the proteins share 17% sequence identity, and a core region of 202 C atoms gives a root mean square deviation (RMSD) of 1.79 Å.…”

Two Consecutive Polar Amino Acids at the End of Helix E are Important for Fast Turnover of the Archaerhodopsin Photocycle

“…We considered that the observed changes could be related to the “bent structure” at the C‐terminus of helix E, where T164 and S165 are located. Despite their low sequence similarities, the “bent structure” is conserved in most microbial rhodopsins, including proton pumps , sensory rhodopsins and halorhodopsins , suggesting that the structure may be functionally important in different types of microbial rhodopsins (Figure ). On closer inspection of amino acid sequences in this region, these microbial rhodopsins seem to apply different strategies to attain a “bent structure”.…”