Structure and Function in Rhodopsin. Single Cysteine Substitution Mutants in the Cytoplasmic Interhelical E−F Loop Region Show Position-Specific Effects in Transducin Activation

Yang, Ke; Dl, Farrens; Wl, Hubbell; Hg, Khorana

doi:10.1021/bi960848t

Cited by 99 publications

(96 citation statements)

References 24 publications

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“…These were all determined by the fluorescence assay (17) in 0.02% DM, the concentration of the detergent used in all the experiments now reported. It is noted that the times shown in Table 2 are shorter (about 70-80%) than those observed in 0.1% DM, in which the previous determinations were done (1,17).…”

Section: Resultsmentioning

confidence: 59%

Structure and function in rhodopsin: Peptide sequences in the cytoplasmic loops of rhodopsin are intimately involved in interaction with rhodopsin kinase

Creuzenet

et al. 1997

Proc. Natl. Acad. Sci. U.S.A.

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Phosphorylation of light-activated rhodopsin by the retina-specific enzyme, rhodopsin kinase (RK), is the primary event in the initiation of desensitization in the visual system. RK binds to the cytoplasmic face of rhodopsin, and the binding results in activation of the enzyme which then phosphorylates rhodopsin at several serine and threonine residues near the carboxyl terminus. To map the RK binding sites, we prepared two sets of rhodopsin mutants in the cytoplasmic CD and EF loops. In the first set, peptide sequences in both loops were either deleted or replaced by indifferent sequences. In the second set of mutants, the charged amino acids (E134, R135, R147, E239, K245, E247, K248, and E249) were replaced by neutral amino acids in groups of 1-3 per mutant. The deletion and replacement mutants in the CD loop showed essentially no phosphorylation, and they appeared to be defective in binding of RK. Of the mutants in the EF loop, that with a deletion of 13 amino acids, was also defective in binding to RK while the second mutant containing a replacement sequence bound RK but showed a reduction of about 70% in V max for phosphorylation. The mutants containing charged to neutral amino acid replacements in the CD and EF loops were all phosphorylated but to different levels. The charge reversal mutant E134R͞ R135E showed a 50% reduction in V max relative to wild-type rhodopsin. Replacements of charged residues in the EF loop decreased the K m by 5-fold for E239Q and E247Q͞K248L͞ E239Q. In summary, both the CD and EF cytoplasmic loops are intimately involved in binding and interaction of RK with light-activated rhodopsin.Light activation of rhodopsin initiates two biochemical cascades, one leading to visual sensitization and the other to desensitization. The primary event in the first cascade is the binding of transducin to light-activated rhodopsin (metarhodopsin II; Meta II), and that in the second is the binding of rhodopsin kinase (RK) to the same photointermediate resulting in its phosphorylation (2-4). While the structural requirements for interaction between Meta II and transducin have been investigated extensively, the study of RK-rhodopsin interaction has begun only recently. Akhtar and coworkers (5) dissected the action of RK into two distinct steps. The first involved binding to Meta II, which resulted in activation of the enzyme. Catalysis of phosphorylation then followed as a second step. Palczweski et al. (6) compared the activation of RK by a number of rhodopsin derivatives and concluded that the cytoplasmic EF loop was involved in binding to RK. More recently, Weiss and coworkers (7) studied the effects of certain amino acid replacements in cytoplasmic loops AB, CD, and EF and suggested the involvement of all the three loops in phosphorylation by RK.We now report on further characterization of the requirements for the binding of RK to Meta II and catalysis of the phosphorylation reaction. Two sets of mutants in the cytoplasmic loops CD and EF ( Fig. 1) (8) have been studied. In one set, pepti...

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Section: Resultsmentioning

confidence: 59%

Structure and function in rhodopsin: Peptide sequences in the cytoplasmic loops of rhodopsin are intimately involved in interaction with rhodopsin kinase

Creuzenet

et al. 1997

Proc. Natl. Acad. Sci. U.S.A.

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“…Although capable of forming both a spectral MII species and a PSB, following illumination a residual species with a max of ϳ480 nm persisted up to 10 h after illumination (data not shown). The cause of this is not known, although similar effects have been reported for other rhodopsin point mutations such as G90S and L226C (29,34,51).…”

Section: Rationale For Choice Of Loop E-2 Ion Pairmentioning

confidence: 55%

“…Site-directed mutagenesis was performed using a cassette-based strategy as described previously in the pMT4 plasmid (28,29), as well as overlap extension PCR (30) to generate EcoRI and NotI fragments containing either the R177C, R177K, R177Q or D190C, D190E, D190N mutations in the synthetic bovine rhodopsin gene (28). The sequences for the primers are as follows: R177C, 5ЈCGTCGGCTGGTCTTGCTAC-ATCCCGGAG3Ј; R177K, 5ЈGCTCGTCGGCTGGTCTAAGTACATCCC-GGAGGGCATGCAGTGC3Ј: R177Q, 5ЈGCTCGTCGGCTGGTCTCAGT-ACATCCCGGAGGGCATGCAGTGC3Ј; D190C, 5ЈCTCGTGCGGGATC-TGCTACTACACGCCG3Ј; D190E, 5ЈGGAGGGCATGCAGTGCTCGTG-CGGGATCGAGTACTACACGCCGC3Ј; and D190N, 5ЈGGAGGGCATG-CAGTGCTCGTGCGGGATCAACTACTACACGCCGC3Ј.…”

Section: Construction and Expression Of Rhodopsin Mutantsmentioning

confidence: 99%

Stability of Dark State Rhodopsin Is Mediated by a Conserved Ion Pair in Intradiscal Loop E-2

Janz

Fay

Farrens

2003

Journal of Biological Chemistry

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The rhodopsin crystal structure reveals that intradiscal loop E-2 covers the 11-cis-retinal, creating a "retinal plug." Recently, we noticed the ends of loop E-2 are linked by an ion pair between residues Arg-177 and Asp-190, near the highly conserved disulfide bond. This ion pair appears biologically significant; it is conserved in almost all vertebrate opsins and may occur in other G-protein-coupled receptors. We report here that the Arg-177/Asp-190 ion pair is critical for the folding and stability of dark state rhodopsin. We find ion pair mutants that regenerate with retinal are functionally and spectrally wild-type-like yet thermally unstable in their dark state because of rapid hydrolysis of the retinal Schiff base linkage. Surprisingly, Arrhenius analysis indicates that the activation energies for the hydrolysis process are similar between the ion pair mutants and wild-type rhodopsin. Furthermore, the ion pair mutants do not show increased reactivity toward hydroxylamine, suggesting that their instability is not caused by an increased exposure to bulk solvent. Our results indicate that the loop E-2 ion pair is important for rhodopsin stability and thus suggest that retinitis pigmentosa observed in patients with Asp-190 mutations may in part be the result of thermally unstable rhodopsin proteins.

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“…Reactivity of RLC Thiols-The conditions of the thiol reactivity experiments were similar to those used previously (33)(34)(35). UP-or P-HMM/S1 (0.75-2.5 mg/ml; 100 -200 l) in reaction buffer (50 mM MOPS (pH 7.0), 50 mM KCl, 1 mM EGTA, 2 mM MgCl 2 , and 50 M DTT with/without ATP or ADP) was incubated at 25°C with 300 M IAF.…”

Section: Methodsmentioning

confidence: 99%

Novel Sensors of the Regulatory Switch on the Regulatory Light Chain of Smooth Muscle Myosin

Mazhari

Selser

Cremo

2004

Journal of Biological Chemistry

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Here we used three different approaches to test this notion, which are reactivity of cysteine thiols, pyrene and acrylodan spectral analysis, and pyrene fluorescence quenching. All methods detected significant differences between the unphosphorylated and phosphorylated regulatory light chain N termini in heavy meromyosin, a double-headed subfragment with an intact regulatory switch. These differences were not observed for subfragment-1, a single-headed, unregulated subfragment. In the presence of either ATP or ADP, phosphorylation increased the solvent exposure and decreased the polarity of the environment about position 23 of the regulatory light chain of heavy meromyosin. These phosphorylation-induced structural changes were not as evident in the absence of nucleotides. Nucleotide binding to unphosphorylated heavy meromyosin caused a decrease in exposure and an increase in polarity of the N terminus, whereas the effects of nucleotide on phosphorylated heavy meromyosin were the opposite. We showed a direct correlation between the kinetics of nucleotide binding/turnover and the conformational change reported by acrylodan at position 23 of the regulatory light chain. Acrylodan-A23C also reports the heads up (extended) to flexed (folded) transition in unphosphorylated heavy meromyosin. This is the first demonstration of direct coupling of nucleotide binding to conformational changes in the N terminus of the regulatory light chain.Smooth muscle myosin (SMM) 1 and nonmuscle myosin are hexameric motor ATPases of ϳ500 kDa mass composed of pairs of HC, ELC, and RLC. A globular motor domain (N-terminal HC) and a light chain binding domain (HC ϩ ELC ϩ RLC) form a head (S1), and the two heads are dimerized by the HC C-terminal halves, which form a coiled-coil. The motor domain contains the catalytic site and the actin binding site (1). Only the head domain and its subfragments have been crystallized to date so there are no atomic resolution structures of a doubleheaded construct.The actin-activated ATPase activity and motor properties of SMM and nonmuscle myosin are regulated by phosphorylation of Ser-19 of the RLC, which is greater than 10 nm from the catalytic site (1-4). Phosphorylation enhances the ATPase activity by more than 1000-fold at saturating actin concentrations (5, 6). Domain requirements for regulation have been elucidated through studies of various proteolytic and expressed subfragments of SMM and nonmuscle myosin. HMM, which lacks the C-terminal two-thirds of the tail, is double-headed and regulated (5-7), but expressed HMM constructs with truncated tails too short to form stable double-headed structures are unregulated (8 -10) as is S1 (7,11,12) and single-headed myosin (13,14). Furthermore, a nonmuscle HMM construct without one of the motor domains but with both regulatory domains is not regulated (15). Therefore, two full heads connected together with enough coiled-coil to allow for dimerization are required for regulation.Removal of the RLC abolishes regulation (16), but the ELC can be removed with re...

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Structure and Function in Rhodopsin. Single Cysteine Substitution Mutants in the Cytoplasmic Interhelical E−F Loop Region Show Position-Specific Effects in Transducin Activation

Cited by 99 publications

References 24 publications

Structure and function in rhodopsin: Peptide sequences in the cytoplasmic loops of rhodopsin are intimately involved in interaction with rhodopsin kinase

Structure and function in rhodopsin: Peptide sequences in the cytoplasmic loops of rhodopsin are intimately involved in interaction with rhodopsin kinase

Stability of Dark State Rhodopsin Is Mediated by a Conserved Ion Pair in Intradiscal Loop E-2

Novel Sensors of the Regulatory Switch on the Regulatory Light Chain of Smooth Muscle Myosin

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