Biology subjects

Novikova, I. V.

Publications and source records attributed to Novikova, I. V..

7 recordsLinked to original sources

bioRxiv2022

Coupling to short linear motifs creates versatile PME-1 activities in PP2A holoenzyme demethylation and inhibition

Protein phosphatase 2A (PP2A) holoenzymes target broad substrates by recognizing short motifs via regulatory subunits. PP2A methylesterase 1 (PME-1) is a cancer-promoting enzyme and undergoes methylesterase activation upon binding to the PP2A core enzyme. Here we showed that PME-1 readily demethylates different families of PP2A holoenzymes and blocks substrate recognition in vitro. The high-resolution cryo-EM structure of a PP2A-B56 holoenzyme-PME-1 complex reveals that PME-1 disordered regions, including a substrate-mimicking motif, tether to the B56 regulatory subunit at remote sites. They occupy the holoenzyme substrate-binding groove and allow large structural shifts in both holoenzyme and PME-1 to enable multi-partite contacts at structured cores to activate the methylesterase. B56-interface mutations selectively block PME-1 activity toward PP2A-B56 holoenzymes and affect the methylation of a fraction of total cellular PP2A. The B56-interface mutations allow us to uncover B56-specific PME-1 functions in p53 signaling. Our studies reveal multiple mechanisms of PME-1 in suppressing holoenzyme functions and versatile PME-1 activities derived from coupling substrate-mimicking motifs to dynamic structured cores.

biochemistry↗

bioRxiv2022

Structure Of Eukaryotic Type III Glutamine Synthetase Lacking Ring-Ring Quaternary Contacts

There are three major classes of the enzyme Glutamine Synthetase (GS), denoted as GSI, GSII and GSIII in both prokaryotes and eukaryotes, that share widely conserved active-site residues but vary in average protein length, oligomeric assembly state and cofactor requirements. Key structures have been determined for all prokaryotic and eukaryotic GS classes except the GSIII class of eukaryotic origin, where even basic questions about its functional oligomeric state are unresolved. Here, we report the 3.2[A] cryo-EM structure of eukaryotic GSIII from the ancient organism of green algal lineage Ostreococcus tauri. Using a combined structural and biochemical approach, we demonstrate that O. tauri GSIII self-assembles and functions exclusively as a single hexameric ring. This is unlike most GSs in other classes, where they form double-stacked ring assemblies instead. Major structural differences in the ring-ring stacking across the classes suggest evolutionary pressure may have favored higher-order interactions that might be beneficial but are not necessarily required for the catalytic GS performance.

biochemistry↗

bioRxiv2022

Cryo-EM structure of the diapause chaperone artemin

The protein artemin constitutes over 10% of all protein in Artemia cysts during diapause and acts as both an RNA and protein chaperone. However, its mechanistic details remain elusive since no high-resolution structure of artemin exists. Here we report the full-length structure of artemin at 2.04 [A] resolution. The cryo-EM map contains density for an intramolecular disulfide bond between Cys22-Cys61 and resolves the entire C-terminus extending into the core of the assembled protein cage. We also provide data supporting the role of C-terminal helix F towards stabilizing the dimer form that is believed to be important for its chaperoning activity. We were able to destabilize this effect by placing a tag at the C-terminus to fully pack the internal cavity and cause limited steric hindrance.

biochemistry↗

bioRxiv2021

AutoMicroED: A semi-automated MicroED processing pipeline

Despite rapid adaptation of micro-electron diffraction (MicroED) for protein and small molecule structure determination to sub-angstrom resolution, the lack of automation tools for easy MicroED data processing remains a challenge for expanding to the broader scientific community. In particular, automation tools, which are novice user friendly, compatible with heterogenous datasets and can be run in unison with data collection to judge the quality of incoming data (similar to cryosparc LIVE for single particle cryoEM) do not exist. Here, we present AutoMicroED, a cohesive and semi-automatic MicroED data processing pipeline that runs through image conversion, indexing, integration and scaling of data, followed by merging of successful datasets that are pushed through phasing and final structure determination. AutoMicroED is compatible with both small molecule and protein datasets and creates a straightforward and reproducible method to solve single structures from pure samples, or multiple structures from mixed populations. The immediate feedback on data quality, data completeness and more parameters, aids users to identify whether they have collected enough data for their needs. Overall, AutoMicroED permits efficient structure elucidation for both novice and experienced users with comparable results to more laborious manual processing.

biophysics↗

bioRxiv2019

The tunable intrahexamer heteroassembly mechanism of pseudoenzyme/enzyme pair, PDX1.2/PDX1.3, with its molecular impact on vitamin B6 regulation

Pseudoenzymes have emerged as key regulatory elements in all kingdoms of life despite being catalytically non-active. Yet many factors defining why one protein is active while its homolog is inactive remain uncertain. For pseudoenzyme-enzyme pairs, the similarity of both subunits can often hinder conventional characterization approaches. In plants, a pseudoenzyme PDX1.2 positively regulates vitamin B6 production by association with its active catalytic homologs such as PDX1.3 through an unknown mechanism. Here we used an integrative experimental approach to learn that such pseudoenzyme-enzyme pair associations result in hetero-complexes of variable stoichiometry, which are unexpectedly tunable. We also present the atomic structure of the PDX1.2 pseudoenzyme as well as the population averaged PDX1.2-PDX1.3 pseudoenzyme-enzyme pair. Finally, we dissected hetero-dodecamers of each stoichiometry to understand the arrangement of monomers in the hetero-complexes and identified symmetry- imposed preferences in PDX1.2-PDX1.3 interactions. Our results provide a new model of pseudoenzyme-enzyme interactions and their native heterogeneity.

molecular biology↗

bioRxiv2018

Protein Structural Biology Using Cell-Free Platform from Wheat Germ

One of the biggest bottlenecks for structural analysis of proteins remains the creation of high yield and high purity samples of the target protein. Cell-free protein synthesis technologies are powerful and customizable platforms for obtaining functional proteins of interest in short timeframes while avoiding potential toxicity issues and permitting high-throughput screening. These methods have benefited many areas of genomic and proteomics research, therapeutics, vaccine development and protein chip constructions. In this work, we demonstrate a versatile and multistage eukaryotic wheat-germ cell-free protein expression pipeline to generate functional proteins of different sizes from multiple host organism and DNA source origins. We also developed a robust purification procedure, which can produce highly-pure (>98%) proteins with no specialized equipment required and minimal time invested. This pipeline successfully produced and analyzed proteins in all three major geometry formats used for structural biology including single particle analysis, and both two-dimensional and three-dimensional protein crystallography. The flexibility of the wheat germ system in combination with the multiscale pipeline described here provides a new workflow for rapid generation of samples for structural characterization that may not be amenable to other recombinant approaches.

molecular biology↗

bioRxiv2018

Cell-free production of full-length ice nucleating protein InaZ

Unlike inorganic or other synthetic alternatives, ice nucleating proteins (INPs) remain the most efficient ice nuclei today. Their potential applications in cryo-preservation, biomedicine, food industry and in the modulation of climate are widespread. Nevertheless, over several decades, cell-based recombinant methods have experienced multiple difficulties expressing these large proteins in full-length and in necessary yields while retaining functionality. As a result, our understanding of the structure and ice nucleation mechanism for this class of proteins is incomplete, and, most importantly, the full extent of possible applications unrealized. Using a wheat-germ cell-free expression pipeline, we successfully expressed and purified full-length ice nucleating protein InaZ from Pseudomonas syringae, known as a model INP. High protein yield and solubility has been achieved using this system. Ice nucleation experiments inside a dynamic environmental scanning electron microscope (ESEM) confirmed that the produced InaZ products remain functional. Preliminary structural assessments of these proteins using Transmission Electron Microscopy (TEM) showed experimental evidence for their structural organization as fibrils. We believe that the current platform will be suitable for expressing other INPs of interest and can be further employed as new engineering system either for industrial or scientific needs.

synthetic biology↗