RLBP1; crystal structure; vitamin A; retinitis pigmentosa; 11-cis-retinal; visual cycle; 11 vitamin A; X-ray structure
Bolze Christin S, Helbling Rachel E, Owen Robin L, Pearson Arwen R, Pompidor Guillaume, Dworkowski Florian, Fuchs Martin R, Furrer Julien, Golczak Marcin, Palczewski Krzysztof, Cascella Michele, Stocker Achim (2014), Human cellular retinaldehyde-binding protein has secondary thermal 9-cis-retinal isomerase activity., in Journal of the American Chemical Society
, 136(1), 137-46.
Helbling Rachel E, Bolze Christin S, Golczak Marcin, Palczewski Krzysztof, Stocker Achim, Cascella Michele (2013), Cellular retinaldehyde binding protein-different binding modes and micro-solvation patterns for high-affinity 9-cis- and 11-cis-retinal substrates., in The journal of physical chemistry. B
, 117(37), 10719-29.
Cascella Michele, Bärfuss Simon, Stocker Achim (2013), Cis-retinoids and the chemistry of vision., in Archives of biochemistry and biophysics
, 539(2), 187-95.
Helbling Rachel E, Aeschimann Walter, Simona Fabio, Stocker Achim, Cascella Michele (2012), Engineering tocopherol selectivity in α-TTP: a combined in vitro/in silico study., in PloS one
, 7(11), 49195-49195.
He Xiaoqin, Lobsiger Joel, Stocker Achim (2012), Molecular clues to Bothnia-type retinal dystrophy., in Anderson R.E., Ash J., Grimm C., LaVail M.M., Hollyfield J.G., Rickman C.B. (ed.), Springer US, US, 589-94.
1. SUMMARYBackground: Retinal is the photon coupling cofactor of rhodopsin in the photoreceptors of the vertebrate eye retina. The photon driven cis-trans isomerization of the 11-cis-retinal triggers a cascade of signaltransduction reactions which eventually result in the visual response. Once isomerized 11-trans-retinal is no longer active and must be exchanged for fresh 11-cis-retinal. Vision in vertebrates therefore depends on the continuous regene¬ration of 11-cis-retinal from all-trans-retinal. This regeneration is a multistep reaction called the visual cycle. The enzymes of this cycle are distributed between photo¬re¬ceptor cells and adjacent retinal pigment epithelium (RPE) cells. CRALBP, the retinaldehyde-binding protein, chaperones the reaction intermediates between the regeneration enzymes and prevents premature cis-trans isomerization. It possesses a high affinity 11-cis-retinoid binding pocket and forms protein-protein complexes with the membrane protein 11-cis-retinol dehydrogenase (RDH5), the plasma membrane adaptor protein EBP50 and possibly other components of the cycle. Dysfunction of CRALBP has been associated with a number of severe retinopathies.Working Hypothesis: CRALBP plays a pivotal role in the visual cycle. CRALBP gene defects can tighten or diminish interactions with retinoid substrates, with visual cycle enzymes and/or with the retinoid transporters in the plasma membrane. Understanding the 3D structure of CRALPB and the structural alterations in clinically relevant mutants is prerequisite for a molecular explanation of the visual cycle’s function, certain of its pathologies and for progress towards their therapy.Specific Aims: We have cloned human CRALBP in E. coli and solved its X-ray structure in complex with 11-cis-retinal. We have constructed six clinically relevant CRALBP missense mutants and so far solved the crystal structure of Bothnia dystrophy causing CRALBP mutant R234W. The next steps are: (i) X-ray structure of clinically relevant CRALBP mutants and quantitative in vitro characterization of retinoid ligand binding. (ii) Characterization of the interactions between CRALBP and regenerating enzymes. (iii) Design of a light-sensing in vitro visual cycle. (iv) Characterization of retinoid-processing membrane protein complexes.Experimental Design: (i) Clinically relevant mutants of CRALBP will be cloned, purified, crystallized and their X-ray structures will be solved. Interactions with retinoid substrates will be quantitatively characterized by photoisomerization experiments and by fluorescence spectroscopy (collaboration with Prof. Krzysztof Palczewski, Department of Pharmacology School of Medicine Case Western Reserve University, Cleveland, USA.). (ii) For this, protein interaction domains of human RDH5 and EBP50 will be cloned, purified and crystallized, alone and in complex with CRALBP. (iii) The robust human suspension cell culture system 293-F will be transiently co-transfected with combinations of visual cycle proteins including retinal G protein-coupled receptor (RGR) opsin and, CRALBP or alternatively one of its mutants. The in vitro regeneration of 11-cis-retinoids and retinoid intermediates will be traced by HPLC monitoring. (iv) Retinoid-processing membrane protein complexes will be isolated from transfected 293-F cells and characterized by blue-native electrophoresis, transmission electron microscopy and single particle analysis (collaboration with Prof. Dimitrios Fotiadis, Institute of Biochemistry and Molecular Medicine, Bern, Switzerland).Expected Value of Proposed Project: CRALBP is a highly abundant 11-cis-retinoid carrier in the human retina. Probing the structure-function relationships of CRALBP with its membrane bound interaction partners may provide molecular clues to the understanding of the vertebrate visual cycle and to retinal pathologies in man. This study may aid to address the issue of visual cycle regulation in the human eye.