Structural Basis for Calcium-induced Inhibition of Rhodopsin Kinase by Recoverin

Abstract

Recoverin, a member of the neuronal calcium sensor branch of the EF-hand superfamily, serves as a calcium sensor that regulates rhodopsin kinase (RK) activity in retinal rod cells. We report here the NMR structure of Ca2+-bound recoverin bound to a functional N-terminal fragment of rhodopsin kinase (residues 1-25, called RK25). The overall main-chain structure of recoverin in the complex is similar to structures of Ca2+-bound recoverin in the absence of target (<1.8Å root-mean-square deviation). The first eight residues of recoverin at the N terminus are solvent-exposed, enabling the N-terminal myristoyl group to interact with target membranes, and Ca2+ is bound at the second and third EF-hands of the protein. RK25 in the complex forms an amphipathic helix (residues 4-16). The hydrophobic face of the RK25 helix (Val-9, Val-10, Ala-11, Ala-14, and Phe-15) interacts with an exposed hydrophobic groove on the surface of recoverin lined by side-chain atoms of Trp-31, Phe-35, Phe-49, Ile-52, Tyr-53, Phe-56, Phe-57, Tyr-86, and Leu-90. Residues of recoverin that contact RK25 are highly conserved, suggesting a similar target binding site structure in all neuronal calcium sensor proteins. Site-specific mutagenesis and deletion analysis confirm that the hydrophobic residues at the interface are necessary and sufficient for binding. The recoverin-RK25 complex exhibits Ca2+-induced binding to rhodopsin immobilized on concanavalin-A resin. We propose that Ca2+-bound recoverin is bound between rhodopsin and RK in a ternary complex on rod outer segment disk membranes, thereby blocking RK interaction with rhodopsin at high Ca2+. Recoverin, a member of the neuronal calcium sensor branch of the EF-hand superfamily, serves as a calcium sensor that regulates rhodopsin kinase (RK) activity in retinal rod cells. We report here the NMR structure of Ca2+-bound recoverin bound to a functional N-terminal fragment of rhodopsin kinase (residues 1-25, called RK25). The overall main-chain structure of recoverin in the complex is similar to structures of Ca2+-bound recoverin in the absence of target (<1.8Å root-mean-square deviation). The first eight residues of recoverin at the N terminus are solvent-exposed, enabling the N-terminal myristoyl group to interact with target membranes, and Ca2+ is bound at the second and third EF-hands of the protein. RK25 in the complex forms an amphipathic helix (residues 4-16). The hydrophobic face of the RK25 helix (Val-9, Val-10, Ala-11, Ala-14, and Phe-15) interacts with an exposed hydrophobic groove on the surface of recoverin lined by side-chain atoms of Trp-31, Phe-35, Phe-49, Ile-52, Tyr-53, Phe-56, Phe-57, Tyr-86, and Leu-90. Residues of recoverin that contact RK25 are highly conserved, suggesting a similar target binding site structure in all neuronal calcium sensor proteins. Site-specific mutagenesis and deletion analysis confirm that the hydrophobic residues at the interface are necessary and sufficient for binding. The recoverin-RK25 complex exhibits Ca2+-induced binding to rhodopsin immobilized on concanavalin-A resin. We propose that Ca2+-bound recoverin is bound between rhodopsin and RK in a ternary complex on rod outer segment disk membranes, thereby blocking RK interaction with rhodopsin at high Ca2+. Calcium ion (Ca2+) in retinal rod cells plays a critical role in regulating the phototransduction cascade in vision (1Polans A. Baehr W. Palczewski K. Trends Neurosci. 1996; 19: 547-554Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar, 3Yarfitz S. Hurley J.B. J. Biol. Chem. 1994; 269: 14329-14332Abstract Full Text PDF PubMed Google Scholar). Recoverin, a 23 kDa Ca2+-binding protein and member of the EF-hand superfamily, serves as a Ca2+ sensor in retinal rods (4Dizhoor A.M. Ray S. Kumar S. Niemi G. Spencer M. Rrolley D. Walsh K.A. Philipov P.P. Hurley J.B. Stryer L. Science. 1991; 251: 915-918Crossref PubMed Scopus (469) Google Scholar, 5Makino C.L. Dodd R.L. Chen J. Burn M.E. Roca A. Simon M.I. Baylor D.A. J. Gen. Physiol. 2004; 123: 729-741Crossref PubMed Scopus (137) Google Scholar). Recoverin prolongs the lifetime of photoexcited rhodopsin by inhibiting rhodopsin kinase only at high Ca2+ levels (5Makino C.L. Dodd R.L. Chen J. Burn M.E. Roca A. Simon M.I. Baylor D.A. J. Gen. Physiol. 2004; 123: 729-741Crossref PubMed Scopus (137) Google Scholar, 8Erickson M.A. Lagnado L. Zozulya S. Neubert T.A. Stryer L. Baylor D.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6474-6479Crossref PubMed Scopus (62) Google Scholar). Hence, recoverin makes the desensitization of rhodopsin responsive to Ca2+, and the shortened lifetime of photoexcited rhodopsin at low Ca2+ levels may promote visual recovery and contribute to the adaptation to background light. More recently, recoverin was shown to have a different role in synaptic termini and was found localized in the rod inner segment (9Sampath A.P. Strissel K.J. Elias R. Arshavsky V.Y. McGinnis J.F. Chen J. Kawamura S. Rieke F. Hurley J.B. Neuron. 2005; 46: 413-420Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Upon light activation, 98% of recoverin is detected in the rod inner segment, whereas in the dark more than 10% of recoverin returns to the outer segment (10Strissel K.J. Lishko P.V. Trieu L.H. Kennedy M.J. Hurley J.B. Arshavsky V.Y. J. Biol. Chem. 2005; 280: 29250-29255Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar), consistent with the conventional role of recoverin in the inhibition of RK. Recoverin contains four EF-hand Ca2+ binding motifs and a myristoyl or related fatty acyl group covalently attached at the N terminus (11Dizhoor A.M. Ericsson L.H. Johnson R.S. Kumar S. Olshevskaya E. Zozulya S. Neubert T.A. Stryer L. Hurley J.B. Walsh K.A. J. Biol. Chem. 1992; 267: 16033-16036Abstract Full Text PDF PubMed Google Scholar). The cooperative binding of two Ca2+ to the second and third EF-hands (EF-2 and EF-3) induces the binding of myristoylated, but not unmyristoylated recoverin to rod outer segment disc membranes (12Zozulya S. Stryer L. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11569-11573Crossref PubMed Scopus (294) Google Scholar, 13Dizhoor A.M. Chen C.K. Olshevskaya E. Sinelnikova V.V. Phillipov P. Hurley J.B. Science. 1993; 259: 829-832Crossref PubMed Scopus (169) Google Scholar). The three-dimensional structures of myristoylated recoverin with 0, 1, and 2 Ca2+ bound have been determined by NMR spectroscopy (14Tanaka T. Ames J.B. Harvey T.S. Stryer L. Ikura M. Nature. 1995; 376: 444-447Crossref PubMed Scopus (285) Google Scholar, 16Ames J.B. Ishima R. Tanaka T. Gordon J.I. Stryer L. Ikura M. Nature. 1997; 389: 198-202Crossref PubMed Scopus (429) Google Scholar). In the Ca2+-free state, the myristoyl group is sequestered in a deep hydrophobic cavity inside the protein. The binding of two Ca2+ leads to the extrusion of the myristoyl group and exposure of hydrophobic residues. The Ca2+-induced exposure of the myristoyl group, termed Ca2+-myristoyl switch, enables recoverin to bind to membranes only at high Ca2+.Ca2+-bound recoverin on the membrane surface also binds to rhodopsin kinase (RK) 2The abbreviations used are: RK, rhodopsin kinase; HSQC, heteronuclear single quantum coherence; ITC, isothermal titration calorimetry; NCS, neuronal calcium sensor; NOE, nuclear Overhauser effect; NOESY, two-dimensional NOE spectroscopy; R*, light-excited rhodopsin; Ni-NTA, nickelnitrilotriacetic acid. (17Chen C.K. Inglese J. Lefkowitz R.J. Hurley J.B. J. Biol. Chem. 1995; 270: 18060-18066Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar) and inhibits the kinase activity only at high Ca2+ levels (6Kawamura S. Nature. 1993; 362: 855-857Crossref PubMed Scopus (323) Google Scholar, 18Klenchin V.A. Calvert P.D. Bownds M.D. J. Biol. Chem. 1995; 270: 16147-16152Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). By contrast, the Ca2+-free form of recoverin does not bind and inhibit RK. Ca2+-bound recoverin interacts with RK exclusively at residues 1-25 (19Levay K. Satpaev D.K. Pronin A.N. Benovic J.L. Slepak V.Z. Biochemistry. 1998; 37: 13650-13659Crossref PubMed Scopus (68) Google Scholar), and deletion of these residues also abolishes the kinase interaction with light-excited rhodopsin (20Higgins M.K. Oprian D.D. Schertler G.F. J. Biol. Chem. 2006; 281: 19426-19432Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). According to surface plasmon resonance measurements, the N-terminal RK peptide binds to recoverin with micromolar affinity and only in the presence of Ca2+ (19Levay K. Satpaev D.K. Pronin A.N. Benovic J.L. Slepak V.Z. Biochemistry. 1998; 37: 13650-13659Crossref PubMed Scopus (68) Google Scholar, 20Higgins M.K. Oprian D.D. Schertler G.F. J. Biol. Chem. 2006; 281: 19426-19432Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Recoverin is structurally similar to other NCS proteins (Fig. 1A): Frq1 (21Ames J.B. Hendricks K.B. Strahl T. Huttner I.G. Hamasaki N. Thorner J. Biochemistry. 2000; 39: 12149-12161Crossref PubMed Scopus (107) Google Scholar), frequenin (22Bourne Y. Dannenberg J. Pollmann V.V. Marchot P. Pongs O. J. Biol. Chem. 2001; 276: 11949-11955Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar), neurocalcin (23Vijay-Kumar S. Kumar V.D. Nat. Struct. Biol. 1999; 6: 80-88Crossref PubMed Scopus (105) Google Scholar), J.B. A.M. Ikura M. Palczewski K. Stryer L. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus (137) Google Scholar), R. Palczewski K. J. Biol. 2006; PubMed Scopus Google Scholar), and W. Y. K. S. Neuron. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). The of NCS proteins are an four EF-hand the in the first EF-hand that to bind Ca2+, and an N-terminal In all NCS the four EF-hands form two in a that with the of EF-hand in J. Biol. PubMed Scopus Google Scholar) and O. J. Biol. PubMed Scopus Google Scholar). of the NCS structures is a groove lined by hydrophobic Trp-31, Phe-35, Phe-49, Ile-52, Tyr-53, Phe-56, Tyr-86, and in the N-terminal of recoverin that in all other NCS proteins (Fig. 1, in hydrophobic residues in and have been in target W. Y. K. S. Neuron. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, A.N. Olshevskaya A.M. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar, S. K. K. K. Kawamura S. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). We report here the structure of Ca2+-bound recoverin in bound to a functional fragment of rhodopsin kinase (residues 1-25, to as RK25). is the first structure of a Ca2+-myristoyl protein bound to a functional target protein. The structure that RK25 forms a amphipathic hydrophobic surface interacts with the hydrophobic groove of recoverin We propose that recoverin binding to N-terminal helix rhodopsin binding to the kinase deletion of the N-terminal RK residues abolishes interaction with light-excited rhodopsin (20Higgins M.K. Oprian D.D. Schertler G.F. J. Biol. Chem. 2006; 281: 19426-19432Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). The interaction of RK25 with recoverin here is different structures of target with other EF-hand proteins M. A.M. G. A. Science. 1992; PubMed Scopus Google Scholar), W. Y. K. S. Neuron. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar), J.L. M.D. M.J. M.A. K. M.A. 1995; Full Text PDF PubMed Scopus Google Scholar), and S. A. K. Y. Nature. PubMed Scopus Google Scholar) and may the of target unmyristoylated recoverin with the protein was in the the recoverin in and to PubMed Scopus Google Scholar, J.B. Tanaka T. Stryer L. Ikura M. Biochemistry. 1994; PubMed Scopus Google Scholar). recoverin was the of hydrophobic interaction as (12Zozulya S. Stryer L. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11569-11573Crossref PubMed Scopus (294) Google Scholar, Y. S. D. Hurley J.B. J. A. 2000; PubMed Scopus Google Scholar). to an in and with a at a of 2 the of to and was than as by functional fragment of RK (residues 1-25, with and with a was in E. the the RK25 in and PubMed Scopus Google Scholar, J.B. Tanaka T. Stryer L. Ikura M. Biochemistry. 1994; PubMed Scopus Google Scholar). RK25 with was the of in and affinity on a to the of to the protein was by and by to the The peptide was in and affinity The RK25 peptide was to and to a of used in NMR The recoverin-RK25 complex used in NMR was by a of Ca2+-bound recoverin 2 and at to of RK25 RK25 in 2 and at The complex was to a used in NMR of recoverin-RK25 at of was in with a of and recoverin in the presence and absence of Ca2+ and N-terminal The to The with of and (Fig. rhodopsin immobilized on A.M. P.P. PubMed Scopus Google Scholar) to the binding of recoverin and to a as J. F. Palczewski K. Ikura M. Ames J.B. J. Biol. Chem. 2005; 280: Full Text Full Text PDF PubMed Scopus Google Scholar). of RK25 and Ca2+-bound recoverin high Ca2+ 2 and of the was in the and RK25 was in the at with an RK25 of of Ca2+-bound recoverin in of was to the to titration was the to for of of the as J. F. Palczewski K. Ikura M. Ames J.B. J. Biol. Chem. 2005; 280: Full Text Full Text PDF PubMed Scopus Google Scholar, A. J. Biol. Chem. 1993; Full Text PDF PubMed Google Scholar). NMR for NMR analysis of or recoverin bound to of RK25 in of a and 2 or RK25 bound to of also for the NMR NMR at on a or with a interface and with and a with an a and with The on a of recoverin bound to RK25 (Fig. and RK25 bound to recoverin (Fig. The of complex and The quantum on a of recoverin protein bound to RK25 J.B. Tanaka T. Ikura M. Stryer L. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar) as as recoverin bound to RK25 not as W. M.J. 1994; PubMed Scopus Google Scholar). of of and by on a that 10% in recoverin or RK25 D. T. G. K. Biochemistry. PubMed Scopus Google Scholar). and as A.M. Nat. Struct. Biol. 1997; Google Scholar, T. Ames J.B. M. Stryer L. Ikura M. J. 1998; PubMed Scopus Google Scholar) on a of recoverin-RK25 with the of complex and The resonance and and to and structure in the recoverin-RK25 The Biochemistry. 1992; PubMed Scopus Google Scholar) for a and NOE for and a of the overall NOE and and are of residues in an and NOE and a are of residues in a The of the structure analysis of recoverin-RK25 complex are in and side-chain NMR of recoverin-RK25 complex as T. Ames J.B. M. Stryer L. Ikura M. J. 1998; PubMed Scopus Google Scholar) and the NMR the for and Scholar), as S. Harvey T.S. S. Ikura M. 1994; Full Text Full Text PDF PubMed Scopus Google Scholar). of as T. Ames J.B. M. Stryer L. Ikura M. J. 1998; PubMed Scopus Google Scholar) by analysis of and of recoverin bound to RK25 and RK25 bound to In to the the in the structure Ca2+ bound to residues 1, and in EF-hand (EF-2 and for structures and the structures of The and are and The an for and are and of the and was by more than or Recoverin to RK25 and have shown that recoverin interacts with the N-terminal residues of RK (19Levay K. Satpaev D.K. Pronin A.N. Benovic J.L. Slepak V.Z. Biochemistry. 1998; 37: 13650-13659Crossref PubMed Scopus (68) Google Scholar, 20Higgins M.K. Oprian D.D. Schertler G.F. J. Biol. Chem. 2006; 281: 19426-19432Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). the interaction of recoverin bound to RK25 by first a RK25 in E. that of RK25 and or other that RK25 is a of (Fig. that interacts with myristoylated and unmyristoylated forms of Ca2+-bound recoverin and does not interact with Ca2+-free myristoylated recoverin (Fig. Ca2+-bound recoverin not the Ca2+-free also interacts with rhodopsin to A.M. P.P. PubMed Scopus Google Scholar, M.A. 1991; PubMed Scopus Google Scholar) (Fig. suggesting that Ca2+ induces recoverin to form a ternary complex with RK25 and RK25 does not bind to immobilized rhodopsin not but the recoverin-RK25 complex binds to the rhodopsin consistent with a ternary The of recoverin binding to RK25 was (Fig. The titration was at as of Ca2+-bound recoverin RK25 in a of the a that RK25 binds to Ca2+-bound recoverin with a a of and a binding of NMR of NMR of recoverin in that the of of RK25 (Fig. and that recoverin binds to RK25 in a complex NMR The of recoverin in the complex the of with that recoverin in the complex is structurally and NMR J. Chem. 1995; Scopus Google Scholar) and light analysis that the complex is and NMR The NMR of RK25 bound to of recoverin highly with suggesting that RK25 structure and is bound to recoverin in the complex (Fig. By contrast, the of RK25 in the absence of recoverin of a structure not of the RK25 residues in the complex and that these residues are The RK25 residues in the complex that NMR structure (Fig. as determined by of and NOE More than of the NMR and NOE for the recoverin-RK25 complex as as the for the in The and have been the The structures the NMR in are in The overall structure of recoverin-RK25 is a complex with overall by by The structures structures of a of and the to the structure was to for main-chain atoms and for all atoms in of The main-chain structure of the complex in is as a in and for the recoverin-RK25 to to to bound at and of on the Recoverin, residues residues atoms of of all atoms of all of Residues on the Recoverin, residues residues in a The structure of recoverin in the complex (Fig. is similar to that of Ca2+-bound recoverin in J.B. Ishima R. Tanaka T. Gordon J.I. Stryer L. Ikura M. Nature. 1997; 389: 198-202Crossref PubMed Scopus (429) Google Scholar) and the Ca2+-bound forms of other NCS proteins (22Bourne Y. Dannenberg J. Pollmann V.V. Marchot P. Pongs O. J. Biol. Chem. 2001; 276: 11949-11955Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar, W. Y. K. S. Neuron. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, K. F. J. W. W. S. P. K.J. Neuron. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, Zozulya S. Stryer L. 1993; Full Text PDF PubMed Scopus Google Scholar). The unmyristoylated N-terminal of recoverin (residues is and structurally as is in all other Ca2+-bound NCS proteins and in to the highly sequestered N-terminal of Ca2+-free myristoylated recoverin (14Tanaka T. Ames J.B. Harvey T.S. Stryer L. Ikura M. Nature. 1995; 376: 444-447Crossref PubMed Scopus (285) Google Scholar). of and are in (residues (residues (residues (residues (residues (residues (residues (residues (residues (residues (residues (residues (residues and (residues (Fig. Recoverin contains two of four and are and form the N-terminal and form the and interact and form a at the interface between the two EF-hand of a structure similar to the structure of EF-hands in structures of J. Biol. PubMed Scopus Google Scholar) and O. J. Biol. PubMed Scopus Google Scholar). The four EF-hands of recoverin are in a and form a structure with a hydrophobic groove on the surface of the N-terminal in in and The structure of RK25 in the complex of an amphipathic (residues The hydrophobic surface of the RK25 helix Val-10, Ala-11, Phe-15) interacts with the exposed hydrophobic groove on recoverin Phe-35, Phe-49, Ile-52, Tyr-53, Phe-56, Phe-57, Tyr-86, and residues on the face of the RK25 helix are The structure of RK25 in the complex is by hydrophobic with as RK25 in is of NMR residues of recoverin than residues of RK25 at the binding interface (Fig. The hydrophobic face of the RK25 helix in interacts with residues in the exposed hydrophobic groove of recoverin in in are RK25 residues Val-10, Ala-11, Ala-14, and The group of the of and the of and The side-chain of are on by of and The side-chain of contact the group of and of and The group of the and of and and of The group of makes contact with the and of and of The side-chain group of is to the of and The of makes contact with the of and The of hydrophobic in for the high binding affinity and (Fig. a mutagenesis analysis of RK25 that Val-10, and are for the binding interaction with recoverin (20Higgins M.K. Oprian D.D. Schertler G.F. J. Biol. Chem. 2006; 281: 19426-19432Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). In determined the structure of recoverin bound to a functional N-terminal fragment of rhodopsin kinase The overall main-chain of recoverin in the complex (Fig. is similar to that of recoverin in in EF-hand and other NCS proteins (22Bourne Y. Dannenberg J. Pollmann V.V. Marchot P. Pongs O. J. Biol. Chem. 2001; 276: 11949-11955Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar, S. Kumar V.D. Nat. Struct. Biol. 1999; 6: 80-88Crossref PubMed Scopus (105) Google Scholar, W. Y. K. S. Neuron. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, K. F. J. W. W. S. P. K.J. Neuron. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, Zozulya S. Stryer L. 1993; Full Text PDF PubMed Scopus Google Scholar). The first residues at the N terminus of recoverin are structurally in the exposure of the N-terminal myristoyl Ca2+ is bound to recoverin at and The four EF-hands form two by a the protein an overall The N-terminal of recoverin contains an exposed hydrophobic groove (Fig. and that interacts with an amphipathic of RK25 (Fig. The between recoverin and RK25 are (Fig. and the and high affinity binding (Fig. mutagenesis on recoverin S. K. K. K. Kawamura S. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar) and RK (20Higgins M.K. Oprian D.D. Schertler G.F. J. Biol. Chem. 2006; 281: 19426-19432Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar) have shown that of the hydrophobic residues at the binding interface (Fig. are for the high affinity The interaction between recoverin and rhodopsin kinase in may the of kinase The Ca2+-myristoyl of recoverin Ca2+-induced extrusion of the N-terminal myristoyl group (12Zozulya S. Stryer L. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11569-11573Crossref PubMed Scopus (294) Google Scholar, 16Ames J.B. Ishima R. Tanaka T. Gordon J.I. Stryer L. Ikura M. Nature. 1997; 389: 198-202Crossref PubMed Scopus (429) Google is structurally to Ca2+-induced inhibition of RK (17Chen C.K. Inglese J. Lefkowitz R.J. Hurley J.B. J. Biol. Chem. 1995; 270: 18060-18066Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar, 18Klenchin V.A. Calvert P.D. Bownds M.D. J. Biol. Chem. 1995; 270: 16147-16152Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar, P.D. V.A. Bownds M.D. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). hydrophobic residues of recoverin that interact with rhodopsin kinase and to residues that contact the N-terminal myristoyl group in the structure of Ca2+-free recoverin (14Tanaka T. Ames J.B. Harvey T.S. Stryer L. Ikura M. Nature. 1995; 376: 444-447Crossref PubMed Scopus (285) Google Scholar). Ca2+-induced extrusion of the myristoyl group in recoverin J.B. Ishima R. Tanaka T. Gordon J.I. Stryer L. Ikura M. Nature. 1997; 389: 198-202Crossref PubMed Scopus (429) Google Scholar) a hydrophobic groove in the N-terminal that serves as a target binding site for rhodopsin kinase (Fig. The Ca2+-induced exposure of the N-terminal hydrophobic recoverin binds to rhodopsin kinase only at high Ca2+ In the Ca2+-free state, the covalently attached myristoyl group the N-terminal hydrophobic groove and the target binding In Ca2+-induced extrusion of the myristoyl group of recoverin exposure of residues that bind and inhibit RK. Ca2+-induced inhibition of RK by recoverin a that light In the dark or Ca2+ levels in retinal rods are high 2000; PubMed Google Scholar, Y. Neurosci. 1996; 19: Full Text PDF PubMed Scopus Google Scholar) and Ca2+-bound recoverin inhibits RK, prolongs the lifetime of light-excited rhodopsin and of the rod (5Makino C.L. Dodd R.L. Chen J. Burn M.E. Roca A. Simon M.I. Baylor D.A. J. Gen. Physiol. 2004; 123: 729-741Crossref PubMed Scopus (137) Google Scholar). Ca2+ levels L. Baylor D.A. Nature. 1994; PubMed Scopus Google Scholar), in the of Ca2+-free recoverin RK (17Chen C.K. Inglese J. Lefkowitz R.J. Hurley J.B. J. Biol. Chem. 1995; 270: 18060-18066Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar) and of RK V.A. Calvert P.D. Bownds M.D. J. Biol. Chem. 1995; 270: 16147-16152Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar), of (6Kawamura S. Nature. 1993; 362: 855-857Crossref PubMed Scopus (323) Google Scholar). The binds to of and desensitization of the rod kinase binding to recoverin is structurally related to target binding in other EF-hand proteins and (Fig. EF-hand proteins form a of two EF-hands that an exposed to interact with a segment of target proteins in In the two N-terminal EF-hands interact with of a target helix peptide kinase (Fig. M. A.M. G. A. Science. 1992; PubMed Scopus Google Scholar). N-terminal EF-hands of interact in a related with an the of (Fig. W. Y. K. S. Neuron. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). The of the RK25 helix bound to recoverin (Fig. is different that of and The RK25 helix to the of whereas the helix is to the of The of the target helix with to in the complex to on and that interact with In the helix contains a of residues and that form with in to the RK25 helix that forms hydrophobic with the RK25 helix Ala-11, and Phe-15) makes with in recoverin that are not in or different with the of the target helix with to a for target The structure of the recoverin-RK25 complex with a deletion and mutagenesis analysis of RK (20Higgins M.K. Oprian D.D. Schertler G.F. J. Biol. Chem. 2006; 281: 19426-19432Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar) the of inhibition of rhodopsin kinase by recoverin (Fig. structure of the recoverin-RK25 complex an exposed N-terminal myristoyl group that propose may the complex to the membrane surface in to In in suggesting that Ca2+-bound recoverin may form a ternary complex with RK and rhodopsin (Fig. the of these propose the for kinase In the Ca2+ levels are Ca2+-bound recoverin is at the membrane interacts on with rhodopsin and on the other with the N-terminal helix of RK. In recoverin is between rhodopsin and RK and thereby may in the kinase of the ternary interaction is that recoverin is to rhodopsin and RK in that the inhibition is of retinal rods leads to a of Ca2+ 2000; PubMed Google Scholar, Stryer L. Nature. PubMed Scopus Google Scholar) Ca2+ to that the myristoyl group to sequestered inside the protein. The of the myristoyl group two recoverin to the membrane surface the sequestered myristoyl group is to as a membrane the sequestered myristoyl group the hydrophobic residues Phe-35, Phe-49, Ile-52, Tyr-53, Phe-56, and that interact with the RK N-terminal RK to Ca2+-free Ca2+-free recoverin also recoverin immobilized rhodopsin in the presence of M.A. 1991; PubMed Scopus Google Scholar). The of Ca2+-free recoverin rhodopsin and RK a that RK The N-terminal RK helix been in deletion of the N-terminal helix abolishes RK binding to (20Higgins M.K. Oprian D.D. Schertler G.F. J. Biol. Chem. 2006; 281: 19426-19432Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). The of recoverin the N-terminal binding to RK and thereby promote on the RK are at the to and We for with NMR for with for NMR and and for for NMR and with