Conformational dynamics of yeast calmodulin in the Ca<sup>2+</sup>‐bound state probed using NMR relaxation dispersion

Ogura, Kenji; Okamura, Hideyasu; Katahira, Masato; Katoh, Etsuko; Inagaki, Fuyuhiko

doi:10.1016/j.febslet.2012.06.031

Cited by 3 publications

(3 citation statements)

References 17 publications

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“…A key control involved the addition of 5% trifluoroethanol (TFE), which was shown to fully stabilize the native state and abolish any relaxation dispersion or evidence of exchange (Figure S4 of the Supporting Information). With the exception of CPMG studies of apoCaM, 48−50 nonvertebrate CaM, 51 or CaM mutants, 52 no prior observations of millisecond time scale exchange by CPMG measurements of CaM-4Ca 2+ , in the absence of other ligands, have been reported. Furthermore, 15 N CPMG relaxation dispersion experiments with 19 F-labeled CaM-4Ca 2+ did not reveal any dispersions.…”

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confidence: 99%

Conformational Selection and Functional Dynamics of Calmodulin: A ¹⁹F Nuclear Magnetic Resonance Study

Hoang

Prosser

2014

Biochemistry

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Calcium-bound calmodulin (CaM-4Ca(2+)) is innately promiscuous with regard to its protein interaction network within the cell. A key facet of the interaction process involves conformational selection. In the absence of a binding peptide, CaM-4Ca(2+) adopts an equilibrium between a native state (N) and a weakly populated near-native peptide-bound-like state (I), whose lifetime is on the order of 1.5 ms at 37 °C, based on (19)F nuclear magnetic resonance (NMR) Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion measurements. This peptide-bound-like state of CaM-4Ca(2+) is entropically stabilized (ΔS = 280 ± 35 J mol(-1) K(-1)) relative to the native state, water-depleted, and likely parental to specific bound states. Solvent depletion, conformational selection, and flexibility of the peptide-bound-like state may be important in priming the protein for binding. At higher temperatures, the exchange rate, kex, appears to markedly slow, suggesting the onset of misfolded or off-pathway states, which retards interconversion between N and I. (19)F NMR CPMG relaxation dispersion experiments with both CaM-4Ca(2+) and the separate N-terminal and C-terminal domains reveal the cooperative role of the two domains in the binding process and the flexibility of the N-terminal domain in facilitating binding. Thus, when calcium binds, calmodulin establishes its interaction with a multitude of protein binding partners, through a combination of conformational selection to a state that is parental to the peptide-bound state and, finally, induced fit.

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confidence: 99%

Conformational Selection and Functional Dynamics of Calmodulin: A ¹⁹F Nuclear Magnetic Resonance Study

Hoang

Prosser

2014

Biochemistry

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“…The transverse relaxation constant, R 2,eff , for each ν CPMG frequency was calculated as R 2,eff = −ln(I(ν CPMG )/I 0 )/T relax , where I(ν CPMG ) is the peak intensity obtained for a given value of ν CPMG and I 0 is the peak intensity obtained when the CPMG block is omitted. The CPMG dispersion curves were fitted numerically as a two-state exchange model using an in-house program described elsewhere2627. The fitting calculation extracted the following terms as the global parameters: the exchange rate, k ex ; the fractional population of the minor state, P b ; and the residue specific parameters, including the magnitude of the chemical shift difference between two states, Δω; the target function of fitting error, χ 2 ; and the nonexchangeable contribution of R 2,eff , R 2,0 .…”

Section: Methodsmentioning

confidence: 99%

Conformational change of Sos-derived proline-rich peptide upon binding Grb2 N-terminal SH3 domain probed by NMR

Ogura

Okamura

2013

Sci Rep

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Growth factor receptor-bound protein 2 (Grb2) is a small adapter protein composed of a single SH2 domain flanked by two SH3 domains. The N-terminal SH3 (nSH3) domain of Grb2 binds a proline-rich region present in the guanine nucleotide releasing factor, son of sevenless (Sos). Using NMR relaxation dispersion and chemical shift analysis methods, we investigated the conformational change of the Sos-derived proline-rich peptide during the transition between the free and Grb2 nSH3-bound states. The chemical shift analysis revealed that the peptide does not present a fully random conformation but has a relatively rigid structure. The relaxation dispersion analysis detected conformational exchange of several residues of the peptide upon binding to Grb2 nSH3. S rc homology 3 (SH3) domains are small protein modules consisting of approximately 60 amino acids that occur in cytoplasmic proteins, tyrosine kinases, and cytoskeletal proteins known to play important roles in signal transduction pathways 1 . SH3 domains mediate protein-protein interactions by recognizing prolinerich peptide sequences within target proteins 2 . Many structures of SH3 domains complexed with their specific ligand peptides have been solved by X-ray crystallography and NMR spectroscopy. These structural studies have revealed that proline-rich peptides adopt a polyproline type II (PPII) helix on the SH3 domain [3][4][5][6][7] . Growth factor receptor-bound protein 2 (Grb2) is a small adapter protein composed of a single SH2 domain flanked by two SH3 domains 8 . It has been shown that the N-terminal SH3 (nSH3) domain of Grb2 binds a proline-rich region present in the guanine nucleotide releasing factor, son of sevenless (Sos). The Grb2/Sos complex is an important component of a highly conserved pathway that transmits signals from the receptor to the nucleus and controls cell multiplication and differentiation 9 . Several structural studies for Grb2 nSH3 complexed with the Sos-derived peptide, which has a sequence of VPPPVPPRRR (denoted as VPP peptide), have been reported [10][11][12] . These structural studies have revealed the details of the interaction between the VPP peptide and the nSH3 domain. Grb2 nSH3 has three binding sites for the ligand peptide, denoted as S1, S2, and S3. The hydrophobic S1 and S2 sites bind Pro2-Pro3 and Val5-Pro6 of the VPP peptide, respectively. The negatively charged S3 site, which is crucial for the orientation of the bound peptide, binds the side chain of Arg8. Thus, the VPP peptide adopts a PPII helix and is bound to nSH3 in a class-II (minus) orientation with one face of the trigonal prism 13 . The structure of the VPP peptide in the free state has been also investigated using NMR spectroscopy and circular dichroism 14 ; the free VPP peptide shows a mixed conformer structure as a result of cis-trans isomerization around the Xaa-Pro bonds. Moreover, the VPP peptide is not a random coil; it takes on a ,60% PPII helix structure even in the free state.As mentioned above, the conformation of the VPP peptide has been well ...

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“…Calmodulin (CaM) is a highly conserved and ubiquitous Ca 2+ -binding protein that modulates the activity of many target enzymes, mainly in response to increasing intracellular Ca 2+ concentrations ( Cyert, 2001 ), which trigger distinct structural rearrangements, and modes of target activation ( Nakashima et al, 2012 ; Ogura et al, 2012b ; Ogura et al, 2012a ; Ishida et al, 2002 ). The number of identified target proteins for mammalian CaM is, according to the Calmodulin Target Database, nearly 300 ( Yap et al, 2000 ), whereas far fewer are known for the yeast CaM ( Cyert, 2001 ).…”

Section: Biological Backgroundmentioning

confidence: 99%

Modeling Calcium Signaling in S. cerevisiae Highlights the Role and Regulation of the Calmodulin-Calcineurin Pathway in Response to Hypotonic Shock

Spolaor

Rovetta

Nobile

et al. 2022

Front. Mol. Biosci.

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Calcium homeostasis and signaling processes in Saccharomyces cerevisiae, as well as in any eukaryotic organism, depend on various transporters and channels located on both the plasma and intracellular membranes. The activity of these proteins is regulated by a number of feedback mechanisms that act through the calmodulin-calcineurin pathway. When exposed to hypotonic shock (HTS), yeast cells respond with an increased cytosolic calcium transient, which seems to be conditioned by the opening of stretch-activated channels. To better understand the role of each channel and transporter involved in the generation and recovery of the calcium transient—and of their feedback regulations—we defined and analyzed a mathematical model of the calcium signaling response to HTS in yeast cells. The model was validated by comparing the simulation outcomes with calcium concentration variations before and during the HTS response, which were observed experimentally in both wild-type and mutant strains. Our results show that calcium normally enters the cell through the High Affinity Calcium influx System and mechanosensitive channels. The increase of the plasma membrane tension, caused by HTS, boosts the opening probability of mechanosensitive channels. This event causes a sudden calcium pulse that is rapidly dissipated by the activity of the vacuolar transporter Pmc1. According to model simulations, the role of another vacuolar transporter, Vcx1, is instead marginal, unless calcineurin is inhibited or removed. Our results also suggest that the mechanosensitive channels are subject to a calcium-dependent feedback inhibition, possibly involving calmodulin. Noteworthy, the model predictions are in accordance with literature results concerning some aspects of calcium homeostasis and signaling that were not specifically addressed within the model itself, suggesting that it actually depicts all the main cellular components and interactions that constitute the HTS calcium pathway, and thus can correctly reproduce the shaping of the calcium signature by calmodulin- and calcineurin-dependent complex regulations. The model predictions also allowed to provide an interpretation of different regulatory schemes involved in calcium handling in both wild-type and mutants yeast strains. The model could be easily extended to represent different calcium signals in other eukaryotic cells.

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Conformational dynamics of yeast calmodulin in the Ca²⁺‐bound state probed using NMR relaxation dispersion

Cited by 3 publications

References 17 publications

Conformational Selection and Functional Dynamics of Calmodulin: A ¹⁹F Nuclear Magnetic Resonance Study

Conformational Selection and Functional Dynamics of Calmodulin: A ¹⁹F Nuclear Magnetic Resonance Study

Conformational change of Sos-derived proline-rich peptide upon binding Grb2 N-terminal SH3 domain probed by NMR

Modeling Calcium Signaling in S. cerevisiae Highlights the Role and Regulation of the Calmodulin-Calcineurin Pathway in Response to Hypotonic Shock

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Conformational dynamics of yeast calmodulin in the Ca2+‐bound state probed using NMR relaxation dispersion

Cited by 3 publications

References 17 publications

Conformational Selection and Functional Dynamics of Calmodulin: A 19F Nuclear Magnetic Resonance Study

Conformational Selection and Functional Dynamics of Calmodulin: A 19F Nuclear Magnetic Resonance Study

Conformational change of Sos-derived proline-rich peptide upon binding Grb2 N-terminal SH3 domain probed by NMR

Modeling Calcium Signaling in S. cerevisiae Highlights the Role and Regulation of the Calmodulin-Calcineurin Pathway in Response to Hypotonic Shock

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Conformational dynamics of yeast calmodulin in the Ca²⁺‐bound state probed using NMR relaxation dispersion

Conformational Selection and Functional Dynamics of Calmodulin: A ¹⁹F Nuclear Magnetic Resonance Study

Conformational Selection and Functional Dynamics of Calmodulin: A ¹⁹F Nuclear Magnetic Resonance Study