Biology subjects

Sloutsky, R.

Publications and source records attributed to Sloutsky, R..

3 recordsLinked to original sources

bioRxiv2020

CaMKII binds both substrates and effectors at the active site

Ca2+/calmodulin dependent protein kinase II (CaMKII) is a signaling protein required for long-term memory. Once activated by Ca2+/CaM, it sustains activity even after the Ca2+ dissipates. In addition to well-known autophosphorylation-mediated mechanism, interaction with specific binding partners also persistently activates CaMKII. A longstanding model invokes two distinct S- and T-sites. If an interactor binds at the T-site, it will preclude autoinhibition and allow substrates to be phosphorylated at the S-site. Here, we specifically test this model with X-ray crystallography, molecular dynamics simulations, and biochemistry. Our data are inconsistent with this model. Co-crystal structures of four different activators or substrates show that they all bind to a single continuous site across the kinase domain. We propose a mechanistic model that persistent CaMKII activity is facilitated by high affinity binding partners, which kinetically compete with autoinhibition by the regulatory segment to allow substrate phosphorylation.

biochemistry↗

bioRxiv2019

Heterogeneity in human hippocampal CaMKII transcripts reveals an allosteric role for the hub domain in activity regulation

Ca2+-calmodulin dependent protein kinase II (CaMKII) plays a central role in Ca2+ signaling throughout the body. Specifically in the hippocampus, CaMKII is required for learning and memory. CaMKII is encoded by four highly conserved genes in vertebrates: , {beta}, {gamma}, and {delta}. AllCaMKIIs are comprised of a kinase domain, regulatory segment, variable linker region, and hub domain responsible for oligomerization. The four genes differ primarily in linker length and composition due to extensive alternative splicing. Here, we unambiguously report the heterogeneity of CaMKII transcripts in 3 complex samples of human hippocampus using Illumina sequencing. Our results show that hippocampal cells contain a diverse collection of 70 CaMKII transcripts from all four CaMKII genes. We characterized the Ca2+/CaM sensitivity of hippocampal CaMKII variants spanning a broad range of linker lengths and compositions. We demonstrate that the effect of the variable linker on Ca2+/CaM sensitivity is conditional on kinase and hub domains. Moreover, we reveal a novel role for the hub domain as an allosteric regulator of kinase activity, which may provide a new pharmacological target for modulating CaMKII activity. Using small angle X-ray scattering and single-particle electron cryo-microscopy, we present evidence for extensive interaction between the kinase and the hub domain, even in the presence of a 30-residue linker. Taken together, we propose that Ca2+/CaM sensitivity in CaMKII is gene-dependent and includes significant contributions from the hub. Our sequencing approach combined with biochemistry provides new insights into understanding the complex pool of endogenous CaMKII. One Sentence SummaryCaMKII is a well-conserved protein that is essential for learning and memory. When CaMKII is mutated in a mouse, this mouse has difficulty learning and remembering how to get through a maze. The hippocampus is the part of the brain required for memory. Here, we used a specific experiment to determine every type of CaMKII that is in a human hippocampus. We found 70 different types and then asked how these differences affect CaMKII function. These data provide evidence that an assembly domain of CaMKII plays an unexpected role regulating its activity. This new finding helps us better understand endogenous CaMKII in the brain and provides a new mechanism for modulating CaMKII activity.

biochemistry↗

bioRxiv2017

Accuracy through Subsampling of Protein EvolutioN: Analyzing and reconstructing protein divergence using an ensemble approach

Mapping the history of ancestor divergence can yield critical insight into protein function. However, reconstruction of this evolutionary history is subject to a great deal of uncertainty, as selection and alignment of extant descendants to represent each ancestor dramatically affects phylogenetic inference of all ancestral nodes. Although historically problematic and somewhat ignored, here we capitalize on that variability by subsampling descendants of each ancestor, generating an ensemble of reconstructions, and integrating over the resulting topological diversity to improve the accuracy of ancestor divergence reconstruction, above the level of subsampling. First, we demonstrate that reproducibility of an all-sequence, single-alignment reconstruction, measured by comparing topologies inferred from 90% subsamples, can be used to assess the likely accuracy of single-alignment reconstructions. We believe this relationship results from modulation of the likelihood landscape around the true topology induced by alignments of resampled sequences. Second, we hypothesize that topological features observed consistently across an ensemble of subsampled reconstructions best reflect true evolutionary history, despite the inaccuracy of each individual reconstruction due to the failure by substitution models to infer the correct sequence of mutations that produced the input sequences. We present a scoring function that evaluates topologies on the basis of their consistency with features extracted from an ensemble, and a reconstruction algorithm (ASPEN) that produces and ranks topologies according to that function. We test our methodology on 600 simulated protein families and the LacI transcription factor family, and find that ASPEN topologies are significantly more accurate than single-alignment reconstructions.

molecular biology↗