Structure and regulation of the BsYetJ calcium channel in lipid nanodiscs

Abstract

BsYetJ is a bacterial homolog of transmembrane BAX inhibitor-1 motif-containing 6 (TMBIM6) membrane protein that plays a key role in the control of calcium homeostasis. However, the BsYetJ (or TMBIM6) structure embedded in a lipid bilayer is uncharacterized, let alone the molecular mechanism of the calcium transport activity. Herein, we report structures of BsYetJ in lipid nanodiscs identified by double electron–electron resonance spectroscopy. Our results reveal that BsYetJ in lipid nanodiscs is structurally different from those crystallized in detergents. We show that BsYetJ conformation is pH-sensitive in apo state (lacking calcium), whereas in a calcium-containing solution it is stuck in an intermediate, inert to pH changes. Only when the transmembrane calcium gradient is established can the calcium-release activity of holo-BsYetJ occur and be mediated by pH-dependent conformational changes, suggesting a dual gating mechanism. Conformational substates involved in the process and a key residue D171 relevant to the gating of calcium are identified. Our study suggests that BsYetJ/TMBIM6 is a pH-dependent, voltage-gated calcium channel.

Regulation of mitochondrial proteostasis by the proton gradient

“…Thus, TMBIM5 appears to exert Ca 2+ /H + exchange activity, while other members of the TMBIM family act as pH-dependent Ca 2+ channels in membranes lacking a stable H + gradient (Bultynck et al, 2014;Guo et al, 2019;Li et al, 2020). Similar to the Na + /Ca 2+ exchanger NCLX (Katoshevski et al, 2021;Palty et al, 2010), TMBIM5 allows the efflux of Ca 2+ from mitochondria, raising the question why different Ca 2+ efflux routes have evolved.…”

“…The founding member of the TMBIM family, TMBIM6 (or BI-1) was suggested to be a pHsensitive Ca 2+ leak channel in the ER membrane, which can be activated by cytosolic protonation of a di-aspartyl moiety (Bultynck et al, 2014;Chang et al, 2014;Guo et al, 2019;Li et al, 2020). A Ca 2+ /H + antiporter activity of TMBIM6 has been discussed controversially, mainly because a Ca 2+ /H + exchanger can function only in the presence of a stable H + gradient that is not present between the ER lumen and the cytosol.…”

Location of the cross‐β structure in prion fibrils: A search by seeding and electron spin resonance spectroscopy

“…Error propagation in Tikhonov regularization cannot be predicted analytically. A common approach is to vary the starting time for the background fit and add 50% noise of the original noise level to estimate the uncertainty in the distance distributions 48,49 . In this study, we use the same method to obtain the uncertainties.…”

Simple Cryoprotectant-Free Method to Advance Pulsed Dipolar ESR Spectroscopy for Capturing Protein Conformational Ensembles

“…The determination of interspin distance distribution of the DEER measurement was performed using the DeerAnalysis program . Basically, the data were analyzed using the Tikhonov regularization based on the L-curve method, followed by a data refinement process using the maximum entropy method (MEM) to obtain the non-negative distance distributions as previously demonstrated. ,, The results were further validated using the Gaussian reconstruction method. , Error propagation in Tikhonov regularization cannot be predicted analytically. As previously demonstrated, a common approach is to vary the starting time for the background fit and also add 50% noise of the original noise level to estimate uncertainty in the distance distributions.…”

“…Double electron–electron resonance (DEER) , is a powerful pulsed dipolar electron spin resonance (ESR) spectroscopy tool for studying protein conformations, as it provides ensemble distributions of distances between pairs of engineered nitroxide spin labels in proteins. − In a combination with a cryogenic cold-plate (copper or aluminum)-based freeze-quench apparatus, ,− DEER can be further advanced to provide microsecond snapshots of protein conformational changes along the trajectory of a ligand binding or a folding event . DEER measurements are often performed at or below the liquid nitrogen temperature (∼80 K).…”