Conformational dynamics of recoverin's Ca<sup>2+</sup>‐myristoyl switch probed by <sup>15</sup>N NMR relaxation dispersion and chemical shift analysis

Xu, Xia; Ishima, Rieko; Ames, James B.

doi:10.1002/prot.23014

Cited by 20 publications

(24 citation statements)

References 48 publications

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“…Detectable chemical shift differences are seen for residues in EF1 (residues 26, 27, and 33) that are implicated in RetGC1 binding (36). Somewhat larger chemical shift changes are seen in EF2 (residues 55, 62, and 68), which represent residues at the domain interface and suggest Ca 2ϩ -dependent structural contacts between EF2 and EF3 like that seen previously for recoverin (38,45). The largest chemical shift differences (highlighted in red in Fig.…”

Section: Casupporting

confidence: 51%

“…These Ca 2ϩ -dependent contacts to EF3 cause a change in packing angle between EF2 and EF3 at the domain interface (see the dotted line in Fig. 6C) that is somewhat reminiscent of the Ca 2ϩ -dependent domain swiveling observed for recoverin (38,45).…”

Section: Discussionmentioning

confidence: 84%

“…The corresponding residues in recoverin ( Fig. 1) also exhibited broad NMR resonances, and 15 N NMR relaxation dispersion studies revealed that these residues at the domain interface exhibit backbone dynamics on the millisecond timescale (45). Ca 2ϩ -induced rearrangement of residues at the domain interface in recoverin gives rise to a 45-degree swiveling of the two domains (38 -induced rearrangement at the domain interface (Fig.…”

Section: Discussionmentioning

confidence: 95%

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Structure of Guanylyl Cyclase Activator Protein 1 (GCAP1) Mutant V77E in a Ca2+-free/Mg2+-bound Activator State

Lim

Peshenko

Olshevskaya

et al. 2016

Journal of Biological Chemistry

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2؉switch helix changes solvent accessibility of Thr-171 and Leu-174 that affects the domain interface. Although the Ca 2؉ switch helix is not part of the RetGC1 binding site, insertion of an extra Gly residue between Ser-173 and Leu-174 as well as deletion of Arg-172, Ser-173, or Leu-174 all caused a decrease in Ca 2؉ binding affinity and abolished RetGC1 activation. We conclude that Ca 2؉ -dependent conformational changes in the Ca 2؉ switch helix are important for activating RetGC1 and provide further support for a Ca 2؉ -myristoyl tug mechanism.Guanylyl cyclase activating proteins (GCAPs) 2 belong to the neuronal calcium sensor (NCS) branch of the calmodulin superfamily (1-3) and regulate Ca 2ϩ -sensitive activity of retinal guanylyl cyclase (RetGC) in rod and cone cells (4 -6). Phototransduction in retinal rods and cones is modulated by intracellular Ca 2ϩ sensed by GCAPs (7,8), and defects in Ca 2ϩ signaling by GCAPs are linked to retinal diseases (9). GCAP proteins in the Ca 2ϩ -free/Mg 2ϩ -bound state activate RetGC (10), whereas Ca 2ϩ -bound GCAPs inhibit RetGC at high Ca 2ϩ levels maintained in the dark (11-13).The GCAPs (GCAP1 (6), GCAP2 (14), GCAP3 (15), and GCAP4 -8 (16) are all ϳ200-amino acid residue proteins containing a covalently attached N-terminal myristoyl group and four EF-hand motifs (EF1 through EF4; Fig. 1 RetGC (10,18,19). The x-ray crystal structure of Ca 2ϩ -bound GCAP1 (20) and NMR structure of GCAP2 (21) showed that the four EF-hands form two semiglobular domains (EF1 and EF2 in the N-domain and EF3 and EF4 in the C-domain); Ca 2ϩ is bound at EF2, EF3, and EF4, and the N-terminal myristoyl group in GCAP1 is buried inside the Ca 2ϩ -bound protein flanked by hydrophobic residues at the N and C termini (see the red residues in Fig. 1 Experimental ProceduresExpression and Purification of GCAP1 and Mutants-Mutations were introduced in a bovine GCAP1 coding plasmid using a "splice by overlap extension" approach as previously described (24). Myristoylated GCAP1 and its mutants were produced in Escherichia coli strain harboring yeast N-myristoyl

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Section: Casupporting

confidence: 51%

Section: Discussionmentioning

confidence: 84%

Section: Discussionmentioning

confidence: 95%

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Structure of Guanylyl Cyclase Activator Protein 1 (GCAP1) Mutant V77E in a Ca2+-free/Mg2+-bound Activator State

Lim

Peshenko

Olshevskaya

et al. 2016

Journal of Biological Chemistry

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“…In addition, NMR pulsed-field gradient diffusion studies (49) on Ca 2+ -bound recoverin determined a translational diffusion coefficient (D = 1 × 10 −10 m 2 /s) consistent with a molar mass of a protein dimer (~44 kDa). By contrast, Ca 2+ -free recoverin was shown previously to be monomeric under the same conditions (47). …”

Section: Resultsmentioning

confidence: 81%

Double Electron–Electron Resonance Probes Ca²⁺-Induced Conformational Changes and Dimerization of Recoverin

Myers

et al. 2013

Biochemistry

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Recoverin, a member of the neuronal calcium sensor (NCS) branch of the calmodulin superfamily, is expressed in retinal photoreceptor cells and serves as a calcium sensor in vision. Ca 2+ -induced conformational changes in recoverin cause extrusion of its covalently attached myristate (termed Ca 2+ -myristoyl switch) that promote translocation of recoverin to disk membranes during phototransduction in retinal rod cells. Here we report double electron-electron resonance (DEER) experiments on recoverin that probe Ca 2+ -induced changes in distance as measured by the dipolar coupling between spin labels strategically positioned at engineered cysteine residues on the protein surface. The DEER distance between nitroxide spin-labels attached at C39 and N120C is 2.5 ±0.1 nm for Ca 2+ -free recoverin and 3.7 ±0.1 nm for Ca 2+ -bound recoverin. An additional DEER distance (5 -6 nm) observed for Ca 2+ -bound recoverin may represent an intermolecular distance between C39 and N120. 15 N NMR relaxation analysis and CW-EPR experiments both confirm that Ca 2+ -bound recoverin forms a dimer at protein concentrations above 100 M, whereas Ca 2+ -free recoverin is monomeric. We propose that Ca 2+ -induced dimerization of recoverin at the disk membrane surface may play a role in regulating Ca 2+ -dependent phosphorylation of dimeric rhodopsin. The DEER approach will be useful for elucidating dimeric structures of NCS proteins in general for which Ca 2+ -induced dimerization is functionally important but not well understood. KeywordsRecoverin; NMR; EPR; calcium; Ca 2+ -myristoyl switch; DEER; vision; phototransduction; EFhand † This work was supported by NIH grants EY012347 (J.B.A.) and RR11973 (UC Davis NMR), and DOE grant DE-FG02-09ER16117 (R.D.B.). * To whom correspondence should be addressed. Tel: (530) 752-6358. Fax: (530) 752-8995. jbames@ucdavis.edu or rdbritt@ucdavis.edu. 1 Abbreviations: DEER, double electron-electron resonance; CW-EPR, continuous wave electron paramagnetic resonance; HSQC, heteronuclear single quantum coherence; IPTG, isopropyl -D-1-thiogalactoside; NMR, nuclear magnetic resonance; SDS PAGE, sodium dodecylsulfate polyacrylamide gel electrophoresis; R 1 , longitudinal relaxation rate constant; R 2 , transverse relaxation rate constant; RMSD, root-mean-squared deviation; TM, phase memory time. SUPPORTING INFORMATION AVAILABLESupporting information includes 15 N-1 H HSQC spectra of Ca 2+ -free recoverin N120C (Fig. S1); Ca 2+ -binding data for recoverin N120C (Fig. S2); DEER data and analysis for Ca 2+ -free and Ca 2+ -bound recoverin N120C (Fig. S3); and Q-band DEER data recorded at longer 2 times (Fig. S4). This material will be available free of charge online at http://pubs.acs.org. [20][21][22]. In the Ca 2+ -free state, the myristoyl group is sequestered in a deep hydrophobic cavity inside the protein, forming a compact structure (Fig. 1A). The binding of two Ca 2+ causes a large protein conformational change that leads to extrusion of the N-terminal myristoyl group (Fig. 1B). The Ca 2+ -induced ...

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“…Instead, it seems that inhibition of the actions of recoverin, which requires sequestration of its myristoyl tail and extrusion from the disc membrane, requires larger and longer changes in intracellular free Ca 2ϩ , like those occurring during steady moderate light. The dynamics of the Ca 2ϩ -myristoyl switch has been recently described (43) and presumably confers the light dependence of recoverin translocation to the inner segment upon steady illumination (see below).…”

Section: Light Adaptation: Role Of Calciummentioning

confidence: 99%

Photoreceptor Signaling: Supporting Vision across a Wide Range of Light Intensities

Arshavsky

Burns

2012

Journal of Biological Chemistry

178

169

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For decades, photoreceptors have been an outstanding model system for elucidating basic principles in sensory transduction and biochemistry and for understanding many facets of neuronal cell biology. In recent years, new knowledge of the kinetics of signaling and the large-scale movements of proteins underlying signaling has led to a deeper appreciation of the photoreceptor's unique challenge in mediating the first steps in vision over a wide range of light intensities. First Steps in Vision Occur in Photoreceptor Outer SegmentsRetinal photoreceptors transduce information obtained in the form of absorbed photons into an electrical response that can be relayed across synapses to other neurons in the retina. In vertebrate photoreceptors, photon absorption and visual signaling take place in the outer segment (Fig. 1), a sensory cilium tightly packed with stacks of membranous discs containing extremely high densities of visual pigments and other signaling proteins. This morphological arrangement allows photons to be efficiently absorbed as they pass through the outer segment. The signal from activated visual pigment (rhodopsin in rods or cone opsins in cones) must then be sufficiently amplified to generate an electrical response that overcomes intrinsic noise. Transduction from the light-absorbing visual pigment into an electrical response utilizes a G protein signaling pathway termed the phototransduction cascade, which leads to a decrease in the second messenger cGMP and the closure of cGMP-sensitive cation channels. The resulting hyperpolarization transiently decreases the release of glutamate from the photoreceptor synaptic terminals, signaling the number of absorbed photons to the rest of the visual system. Remarkably, photoreceptors both detect low light levels (single photons in the case of rods) and continue to rapidly and reliably signal changes in light intensity as illuminance increases over 10 orders of magnitude during the course of a typical day. Phototransduction: Rhodopsin Activation, Amplification, and DeactivationPhototransduction has been the subject of many comprehensive reviews (1-4). Here, we provide a framework introduction and briefly summarize the latest findings that are shaping our understanding of this first step in vision.Phototransduction begins when a photon causes cis-transisomerization of the chromophore 11-cis-retinal, which induces a rapid conformational change to the protein's fully active form, R*. R* activates molecules of the G protein transducin by catalyzing GDP/GTP exchange on the transducin ␣-subunit, G␣ t (Fig. 1). G␣ t ⅐GTP separates from the transducin ␤␥-subunits and binds to the ␥-subunit of its effector, cGMP phosphodiesterase (PDE), 3 which releases this subunit's inhibitory constraint on the catalytic ␣-and ␤-subunits of PDE. Activated PDE rapidly hydrolyzes cGMP, thereby reducing its concentration in the cytoplasm and causing cGMP-sensitive cation channels in the plasma membrane to close. The closure of channels reduces the inward cation current, resulting in a tra...

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Conformational dynamics of recoverin's Ca²⁺‐myristoyl switch probed by ¹⁵N NMR relaxation dispersion and chemical shift analysis

Cited by 20 publications

References 48 publications

Structure of Guanylyl Cyclase Activator Protein 1 (GCAP1) Mutant V77E in a Ca2+-free/Mg2+-bound Activator State

Structure of Guanylyl Cyclase Activator Protein 1 (GCAP1) Mutant V77E in a Ca2+-free/Mg2+-bound Activator State

Double Electron–Electron Resonance Probes Ca²⁺-Induced Conformational Changes and Dimerization of Recoverin

Photoreceptor Signaling: Supporting Vision across a Wide Range of Light Intensities

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Conformational dynamics of recoverin's Ca2+‐myristoyl switch probed by 15N NMR relaxation dispersion and chemical shift analysis

Cited by 20 publications

References 48 publications

Structure of Guanylyl Cyclase Activator Protein 1 (GCAP1) Mutant V77E in a Ca2+-free/Mg2+-bound Activator State

Structure of Guanylyl Cyclase Activator Protein 1 (GCAP1) Mutant V77E in a Ca2+-free/Mg2+-bound Activator State

Double Electron–Electron Resonance Probes Ca2+-Induced Conformational Changes and Dimerization of Recoverin

Photoreceptor Signaling: Supporting Vision across a Wide Range of Light Intensities

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Product

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Conformational dynamics of recoverin's Ca²⁺‐myristoyl switch probed by ¹⁵N NMR relaxation dispersion and chemical shift analysis

Double Electron–Electron Resonance Probes Ca²⁺-Induced Conformational Changes and Dimerization of Recoverin