Biology subjects

Serysheva, I. I.

Publications and source records attributed to Serysheva, I. I..

5 recordsLinked to original sources

bioRxiv2024

Functional investigation of a putative calcium-binding site involved in the inhibition of inositol 1,4,5-trisphosphate receptor activity

A wide variety of factors influence inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) activity resulting in modulation of intracellular Ca2+ release. This regulation is thought to define the spatio-temporal patterns of Ca2+ signals necessary for the appropriate activation of downstream effectors. The binding of both IP3 and Ca2+ are obligatory for IP3R channel opening, however, Ca2+ regulates IP3R activity in a biphasic manner. Mutational studies have revealed that Ca2+ binding to a high-affinity pocket formed by the ARM3 domain and linker domain promotes IP3R channel opening without altering the Ca2+ dependency for channel inactivation. These data suggest a distinct low-affinity Ca2+ binding site is responsible for the reduction in IP3R activity at higher [Ca2+]. We determined the consequences of mutating a cluster of acidic residues in the ARM2 and central linker domain reported to coordinate Ca2+ in cryo-EM structures of the IP3R type 3. This site is termed the "CD Ca2+ binding site" and is well-conserved in all IP3R sub-types. We show that the CD site Ca2+ binding mutants where the negatively charged glutamic acid residues are mutated to alanine exhibited enhanced sensitivity to IP3-generating agonists. Ca2+ binding mutants displayed spontaneous elemental Ca2+ events (Ca2+ puffs) and the number of IP3-induced Ca2+ puffs was significantly augmented in cells stably expressing Ca2+ binding site mutants. When measured with "on-nucleus" patch clamp, the inhibitory effect of high [Ca2+] on single channel-open probability (Po) was reduced in mutant channels and this effect was dependent on [ATP]. These results indicate that Ca2+ binding to the putative CD Ca2+ inhibitory site facilitates the reduction in IP3R channel activation when cytosolic [ATP] is reduced and suggest that at higher [ATP], additional Ca2+ binding motifs may contribute to the biphasic regulation of IP3-induced Ca2+ release.

biochemistry↗

bioRxiv2022

Structural dynamics underlying gating and regulation in IP3R channel

Inositol-1,4,5-trisphosphate receptors (IP3Rs) are activated by IP3 and Ca2+ and their gating is regulated by various intracellular messengers that finely tune the channel activity. Here, using single particle cryo-EM analysis we determined 3D structures of the nanodisc-reconstituted IP3R1 channel in two ligand-bound states. These structures provide unprecedented details governing binding of IP3, Ca2+ and ATP in IP3R1, revealing conformational changes that couple ligand-binding to channel opening. Using a GMM based deep learning approach and 3D variability analysis, we extracted dynamic properties of the key protein domains. From this, we find that IP3 binding relies upon intrinsic flexibility of the cytoplasmic ARM2 domain. Our results highlight a key role of dynamic side chains surrounding the ion conduction path in regulating gating behavior of IP3R channels. Altogether, this work defines a structural platform for mechanistic understanding of the molecular dynamics underlying ligand-binding, activation and regulation of the IP3R activity.

biophysics↗

bioRxiv2022

Functional Determination of Calcium Binding Sites Required for the Activation of Inositol 1,4,5-trisphosphate receptors.

Inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs) initiate a diverse array of physiological responses by carefully orchestrating intracellular calcium (Ca2+) signals in response to various external cues. Notably, IP3R channel activity is determined by several obligatory factors including IP3, Ca2+ and ATP. The critical basic amino acid residues in the N-terminal IP3-binding core (IBC) region that facilitate IP3 binding are well characterized. In contrast, the residues conferring the biphasic regulation by Ca2+ are yet to be ascertained. Using comparative structural analysis of Ca2+ binding sites identified in two main families of intracellular Ca2+-release channels, ryanodine receptors (RyRs) and IP3Rs, we identified putative acidic residues coordinating Ca2+ in the cytosolic calcium sensor region in IP3Rs. We determined the consequences of substituting putative Ca2+ binding, acidic residues in IP3R family members. We show that the agonist-induced Ca2+ release, single channel open probability (P0) and Ca2+ sensitivities are markedly altered when the negative charge on the conserved acidic side chain residues are neutralized. Remarkably, neutralizing the negatively charged side chain on two of the residues individually in the putative Ca2+ binding pocket shifted the Ca2+ required to activate IP3R to higher concentrations, indicating that these residues likely are a component of the Ca2+ activation site in IP3R. Taken together, our findings indicate that Ca2+ binding to a well conserved activation site is a common underlying mechanism resulted in increased channel activity shared by IP3Rs and RyRs.

physiology↗

bioRxiv2020

In situ structure of the AcrAB-TolC efflux pump at subnanometer resolution

In Gram-negative bacteria, tripartite efflux pump AcrAB-TolC plays a prominent role in antibiotic resistance. We have used high resolution cryo-ET to visualize the structure of Escherichia coli AcrAB-TolC at a 7 [A] resolution in intact cells. The resulting structures show the detailed architecture of the assembled complex embedded into cell envelope. Interactions with the inner membrane enable crosstalk between AcrB and TolC through AcrA, suggesting that assembly in the native cellular environment is critical for the pump activation mechanism, where the allosteric activating signal is triggered by the alternate binding of AcrA to the lipid membrane and AcrB porter domain. We establish a platform for high resolution in situ structural studies of bacteria efflux pump, which can yield critical information in understanding complex assemblies function. One sentence summaryA 7 [A] in situ structure of the AcrAB-TolC pump from bacteria by cryo-ET reveals mechanisms for active efflux.

biophysics↗

bioRxiv2018

Cryo-EM Reveals Ligand Induced Allostery Underlying InsP3R Channel Gating

Inositol-1,4,5-trisphosphate receptors (InsP3Rs) are cation channels that mobilize Ca2+ from intracellular stores in response to a wide range of cellular stimuli. The paradigm of InsP3R activation is the coupled interplay between binding of InsP3 and Ca2+ that switches the ion conduction pathway between closed and open states to enable the passage of Ca2+ through the channel. However, the molecular mechanism of how the receptor senses and decodes ligand-binding signals into gating motion remains unknown. Here we present the electron cryo-microscopy structure of InsP3R1 from rat cerebellum determined to 4.1 [A] resolution in the presence of activating concentrations of Ca2+ and adenophostin A (AdA), a structural mimetic of InsP3 and the most potent known agonist of the channel. Comparison with the 3.9 [A]-resolution structure of InsP3R1 in the Apo-state, also reported herein, reveals the binding arrangement of AdA in the tetrameric channel assembly and striking ligand-induced conformational rearrangements within cytoplasmic domains coupled to the dilation of a hydrophobic constriction at the gate. Together, our results provide critical insights into the mechanistic principles by which ligand-binding allosterically gates InsP3R channel.

biophysics↗