NMR methods for analysis of CRALBP retinoid binding

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Retinoid interactions determine the function of the cellular retinaldehyde binding protein (CRALBP) in the rod visual cycle where it serves as an 11-cis-retinol acceptor for the enzymatic isomerization of all-trans-to 11-cis-retinol and as a substrate carrier for 11-cis-retinol dehydrogenase (RDH5). Based on preliminary NMR studies suggesting retinoid interactions with Met and Trp residues, human recombinant CRALBP (rCRALBP) with altered Met or Trp were produced and analyzed for ligand interactions. The primary structures of the purified proteins were verified for mutants M208A, M222A, M225A, W165F, and W244F, then retinoid binding properties and substrate carrier functions were evaluated. All the mutant proteins bound 11-cis-and 9-cis-retinal and therefore were not grossly misfolded. In vivo studies show that the cellular retinaldehyde binding protein (CRALBP) 1 functions in the retinal pigment epithelium (RPE) as an 11-cis-retinol acceptor in the isomerization step of the rod visual cycle (1). Following illumination, CRALBP knockout mice exhibit over a 10-fold delay in rhodopsin regeneration, 11-cis-retinal synthesis, and dark adaptation relative to WT animals (1). In vitro CRALBP can also serve as a substrate carrier (2, 3), stimulating the enzymatic oxidation of 11-cis-retinol by RPE 11-cis-retinol dehydrogenase (RDH5) and retarding its esterification by lecithin:retinol acyl transferase. In addition, CRALBP is required for in vitro hydrolysis of endogenous RPE 11-cis-reinyl ester (4) and is a component of an RPE visual cycle protein complex where it may serve other physiological roles (3). CRALBP is expressed in RPE, retinal Mü ller cells, ciliary epithelium, iris, cornea, pineal gland, and oligodendrocytes of the optic nerve and brain. The function of the protein in tissues other than RPE is not known, however, CRALBP has also been implicated as being important in an alternate visual cycle for cone visual pigment regeneration possibly involving Mü ller cells (1, 5). Mutations in the human CRALBP gene causing retinal pathology are addressed in the accompanying report (6). As part of ongoing efforts to define functional domains in this key visual cycle protein, we are characterizing CRALBP-ligand interactions and the structure of the retinoid binding pocket. Ligand interactions in this water-soluble, 316-residue protein are noncovalent and localized to C-terminal residues 120 -313 (7,8). Previous analyses of human recombinant CRALBP (rCRALBP) and site-directed mutants support residues Gln-210 and Lys-221 as components of the retinoid binding pocket (8,9). Preliminary NMR studies have suggested that rCRALBP undergoes limited structural changes upon photoisomerization of bound 11-cis-retinal and that Met and Trp residues may be associated with these ligand-dependent conformational changes (9, 10). As depicted in Fig. 1, four of the seven methionines in rCRALBP and both tryptophans are located in the C-terminal retinoid binding domain. To identify Met and Trp in the ligand binding cavity, we describe here the...

Section: Resultssupporting

confidence: 91%

Section: Resultsmentioning

confidence: 68%

mentioning

confidence: 94%

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Mapping the Ligand Binding Pocket in the Cellular Retinaldehyde Binding Protein

Yang

Shaw

et al. 2003

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“…The crystal structures for ␣TTP were determined with and without ␣-tocopherol in the binding pocket, yielding similar structures except for a solventexposed "lid" or "mobile lipid-exchange loop" (residues 198 -221) that limits access to the ligand binding cavity (14). Large conformational changes are required for entry or exit of ligand from the ␣TTP binding pocket (15), consistent with the liganddependent conformational changes observed in CRALBP by NMR (10,46). SPF was crystallized with an unidentified ligand in the binding pocket and the determined structure resembles the liganded conformation of ␣TTP with the lid in the closed position (17).…”

Section: The Rcralbp Ligand Binding Cavity Revealed By H/d Exchange-mentioning

confidence: 60%

Identification of CRALBP Ligand Interactions by Photoaffinity Labeling, Hydrogen/Deuterium Exchange, and Structural Modeling

Hasan

Liu

et al. 2004

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Cellular retinaldehyde-binding protein (CRALBP) 1 is a 36-kDa water soluble protein with a high affinity binding pocket for retinoids uniquely associated with vision, namely 11-cisretinal and 11-cis-retinol (1). Human CRALBP gene defects can either tighten or abolish retinoid interactions, which in turn can compromise substrate carrier interactions with 11-cis-retinol dehydrogenase and lead to several retinal pathologies (2). Targeted disruption of the CRALBP gene in mice impairs regeneration of both rod and cone visual pigments (3). These and other studies establish multiple functions for CRALBP in the retinal pigment epithelium (RPE), including roles as a major acceptor of 11-cis-retinol in the isomerization step of the rod visual cycle and as a facilitator of oxidation of 11-cis-retinol to 11-cis-retinal by 11-cis-retinol dehydrogenase (2-6). Ongoing proteomic studies support the existence of a RPE retinoid processing protein complex containing CRALBP (7). Direct CRALBP interactions have been demonstrated in vitro with 11-cis-retinol dehydrogenase (2) and with ERM (ezrin, radixin, moesin)-binding phosphoprotein 50 (EBP50), also known as sodium hydrogen exchanger regulator factor type 1 (NHERF-1) (8). Interactions with EBP50 have been suggested as a mechanism for localizing CRALBP to the apical RPE plasma membrane for export of 11-cis-retinal to the adjacent rod photoreceptor cells for visual pigment regeneration (8). The functions of CRALBP in tissues other than the RPE remain to be determined (i.e. in retinal Mü ller cells, ciliary epithelium, iris, cornea, pineal gland, and a subset of oligodendrocytes of the optic nerve and brain).To better understand CRALBP visual cycle functions, which require rapid association and dissociation of retinoid, we are characterizing the structure of the ligand binding pocket. Ligand interactions in CRALBP are non-covalent and previous structure-function studies (2, 9, 10) have implicated eight residues as possibly interacting with retinoid (Fig. 1). In this report, the interaction between human recombinant CRALBP and retinoid were characterized by mass spectrometry using hydrogen/deuterium exchange and photoaffinity labeling with 3-diazo-4-keto-11-cis-retinal (DK-11-cis-retinal). This photoaffinity analogue of 11-cis-retinal has previously been used to map the movement of ligand within rhodopsin following sensi-* This study was supported in part by National Institutes of Health Grants EY6603, EY14239, GM63020, a Research Center Grant from The Foundation Fighting Blindness, and funds from the Cleveland Clinic Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Tables I and II. ¶ This work was submitted in partial fulfillment of the requirements for a doctoral degree in chemistry from Cleveland State University. 1 The abbreviations used are: CRALBP, cellular retinaldehyde-binding protein; DK...

“…N uniformly labeled WT and mutant R233W rCRALBP were prepared by biosynthetic incorporation in E. coli strain BL21(DE3)LysS grown in defined minimal media (8,26). Purified mutant and WT rCRALBP with bound 11-cis-retinal (ϳ0.3 mM) were adjusted to 8% D 2 O (v/v) and transferred to 250 l of microcell NMR tubes (Shigemi Inc., Allison Park, PA) (8).…”

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confidence: 99%

Disease-causing Mutations in the Cellular Retinaldehyde Binding Protein Tighten and Abolish Ligand Interactions

Golovleva

Bhattacharya

et al. 2003

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Mutations in the human cellular retinaldehyde binding protein (CRALBP) gene cause retinal pathology. To understand the molecular basis of impaired CRALBP function, we have characterized human recombinant CRALBP containing the disease causing mutations R233W or M225K. Protein structures were verified by amino acid analysis and mass spectrometry, retinoid binding properties were evaluated by UV-visible and fluorescence spectroscopy and substrate carrier functions were assayed for recombinant 11-cis-retinol dehydrogenase (rRDH5). The M225K mutant was less soluble than the R233W mutant and lacked retinoid binding capability and substrate carrier function. In contrast, the R233W mutant exhibited solubility comparable to wild type rCRALBP and bound stoichiometric amounts of 11-cis-and 9-cis-retinal with at least 2-fold higher affinity than wild type rCRALBP. Holo-R233W significantly decreased the apparent affinity of rRDH5 for 11-cis-retinoid relative to wild type rCRALBP. Analyses by heteronuclear single quantum correlation NMR demonstrated that the R233W protein exhibits a different conformation than wild type rCRALBP, including a different retinoid-binding pocket conformation. The R233W mutant also undergoes less extensive structural changes upon photoisomerization of bound ligand, suggesting a more constrained structure than that of the wild type protein. Overall, the results show that the M225K mutation abolishes and the R233W mutation tightens retinoid binding and both impair CRALBP function in the visual cycle as an 11-cisretinol acceptor and as a substrate carrier.Mutations in the human gene RLBP1 encoding the cellular retinaldehyde-binding protein (CRALBP) 1 cause retinal pathology and have been associated with autosomal recessive retinitis pigmentosa (1), Bothnia dystrophy (2, 3), retinitis punctata albescens (4), fundus albipunctatus (5), and Newfoundland rod-cone dystrophy (6). These diseased phenotypes are all characterized by photoreceptor degeneration and night blindness (delayed dark adaptation) but differ in age of onset, rate of progression, and severity. The molecular basis for the clinical differences in these related retinal dystrophies is not well understood and no effective therapies exist for the pathology resulting from impaired CRALBP function.CRALBP is an abundant, 36-kDa protein in the cytosol of the retinal pigment epithelium (RPE) and Mü ller cells of the retina where it carries endogenous 11-cis-retinol and 11-cis-retinal (7). The CRALBP ligand binding cavity is mapped in the accompanying report (8). In vivo studies (9) show that CRALBP serves as a major 11-cis-retinol acceptor in the isomerization step of the visual cycle (7, 10, 11), stimulating the enzymatic isomerization of all-trans-to 11-cis-retinol in the rod visual cycle. However, CRALBP appears to function within an RPE protein complex (12) and to serve multiple functions. In vitro, CRALBP facilitates the oxidation of 11-cis-retinol to 11-cisretinal by 11-cis-retinol dehydrogenase (12, 13), retards 11-cisretinol esterification...