Met125 is essential for maintaining the structural integrity of calmodulin\u2019s C-terminal domain

Sarah E D Nelson,Gianluigi Veglia,Robyn T Rebbeck,David D Thomas,Razvan L Cornea,Daniel K Weber

doi:10.1038/s41598-020-78270-w

Sarah E D Nelson, Gianluigi Veglia + Show 4 more

Open Access

https://doi.org/10.1038/s41598-020-78270-w

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Journal: Scientific Reports	Publication Date: Dec 1, 2020
Citations: 1	License type: open-access

Affiliation: University of Minnesota

Abstract

We have used NMR and circular dichroism spectroscopy to investigate the structural and dynamic effects of oxidation on calmodulin (CaM), using peroxide and the Met to Gln oximimetic mutations. CaM is a Ca2+-sensitive regulatory protein that interacts with numerous targets. Due to its high methionine content, CaM is highly susceptible to oxidation by reactive oxygen species under conditions of cell stress and age-related muscle degeneration. CaM oxidation alters regulation of a host of CaM’s protein targets, emphasizing the importance of understanding the mechanism of CaM oxidation in muscle degeneration and overall physiology. It has been shown that the M125Q CaM mutant can mimic the functional effects of methionine oxidation on CaM’s regulation of the calcium release channel, ryanodine receptor (RyR). We report here that the M125Q mutation causes a localized unfolding of the C-terminal lobe of CaM, preventing the formation of a hydrophobic cluster of residues near the EF-hand Ca2+ binding sites. NMR analysis of CaM oxidation by peroxide offers further insights into the susceptibility of CaM’s Met residues to oxidation and the resulting structural effects. These results further resolve oxidation-driven structural perturbation of CaM, with implications for RyR regulation and the decay of muscle function in aging.

Highlights

We have used NMR and circular dichroism spectroscopy to investigate the structural and dynamic effects of oxidation on calmodulin (CaM), using peroxide and the Met to Gln oximimetic mutations
It has been shown that the M125Q mutation weakens the interactions with ryanodine receptor (RyR) in a Ca2+-dependent manner, which is consistent with the altered response to Ca2+ observed in this study[29,43]
Recent EPR experiments focusing on the conformational equilibrium between closed-to-open conformations of WT-CaM and M125Q-CaM showed that the populations of these states are significantly affected by this single m utation[21]

Summary

Introduction

We have used NMR and circular dichroism spectroscopy to investigate the structural and dynamic effects of oxidation on calmodulin (CaM), using peroxide and the Met to Gln oximimetic mutations. CaM’s C a2+ sensitivity stems from the cooperatively paired EF-hand motifs in the N- and C-terminal domains, which are tethered by a flexible linker that facilitates CaM’s binding to a variety of cellular targets[2,5] For most targets, this binding interaction depends on the calcium occupancy state, either Ca2+-free (apo) or Ca2+-bound (holo). In addition to the hydrophobic environment around the Met residues, the EF-hand motifs contain paired aromatic residues in helices 1 and 4 that shift from nearly antiparallel orientation in the apo state to nearly perpendicular in the holo s tate[11,12,13,14,15,16] The stacking of these aromatic residues and stabilization of hydrophobic interactions throughout CaM’s domains play key roles in the cooperativity of C a2+ binding[13]. While some forms of oxidized CaM are selectively degraded by the 20S proteasome[38,41], CaM oxidation can be reversed by methionine sulfoxide reductase (Msr), and CaM’s oxidation level plays a role in cellular s ignaling[35]

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