Biology subjects

Kutti, V. R.

Publications and source records attributed to Kutti, V. R..

6 recordsLinked to original sources

bioRxiv2023

Factors affecting macromolecule orientations in thin films formed in cryo-EM

The formation of a vitrified thin film embedded with randomly oriented macromolecules is an essential prerequisite for cryogenic sample electron microscopy. Most commonly, this is achieved using the plunge freeze method described nearly 40 years ago. Although this is a robust method, the behaviour of different macromolecules shows great variation upon freezing and often needs to be optimized to obtain an isotropic, high-resolution reconstruction. For a macromolecule in such a film, the probability of encountering the air-water interface in the time between blotting and freezing and adopting preferred orientations is very high. 3D reconstruction using preferentially oriented particles often leads to anisotropic and uninterpretable maps. Currently, there are no general solutions to this prevalent issue, but several approaches largely focusing on sample preparation with the use of additives and novel grid modifications have been attempted. In this study, the effect of physical and chemical factors on the orientations of macromolecules was investigated through an analysis of select well-studied macromolecules and important parameters that determine the behaviour of proteins on cryo-EM grids were revealed. These insights highlight the nature of interactions that cause preferred orientations and can be utilized to systematically address orientation bias for any given macromolecule and provide a framework to design small-molecule additives to enhance sample stability and behaviour.

biochemistry↗

bioRxiv2023

Conformational diversity in class C GPCR positive allosteric modulation

The metabotropic glutamate receptors (mGlus) are class C G protein coupled receptors (GPCR) that form obligate dimers activated by the major excitatory neurotransmitter L-glutamate1,2. The architecture of mGlu receptor comprises an extracellular Venus-Fly Trap domain (VFT) connected to a transmembrane domain (7TM) through a Cysteine-Rich Domain (CRD). The binding of L-glutamate in the VFTs and subsequent conformational change results in the signal being transmitted to the 7TM inducing G-protein binding and activation 3-6. The mGlu receptors signal transduction can be allosterically potentiated by positive allosteric modulators (PAMs) binding to the 7TMs, which are of therapeutic interest in various neurological disorders7-9. Here, we report the cryoEM structures of metabotropic glutamate receptor 5 (mGlu5) purified with three chemically and pharmacologically distinct PAMs. We find that PAMs modulate the receptor equilibrium through their different binding modes, revealing how their interactions in the 7TMs impact the mGlu5 receptor conformational landscape and function. In addition, we identified a PAM-free but agonist-bound intermediate state that is stabilised by interactions mediated by intracellular loop 2. The activation of mGlu5 receptor is a multi-step sequential process in which the binding of the PAMs in the 7TM modulates the equilibrium towards the active state.

biochemistry↗

bioRxiv2023

Oryza genera-specific novel Histone H4 variant predisposes H4 Lysine5 Acetylation marks to modulate salt stress responses

Paralogous variants of canonical histones guide accessibility to DNA and function as additional layers of genome regulation. Across eukaryotes, the occurrence, mechanism of action and functional significance of several variants of core histones are well known except that of histone H4. Here we show that a novel variant of H4 (H4.V), expressing tissue-specifically among members of Oryza genera, mediates specific epigenetic changes contributing majorly to salt tolerance. H4.V was incorporated to specific chromosomal locations where it blocked deposition of active histone marks. Under salt stress, large scale re-distribution of H4.V enabled incorporation of stress dependent histone H4 Lysine5 Acetylation (H4K5Ac) marks. Mis-expression of H4.V led to defects at morphological level especially in reproductive tissues, and in mounting stress responses. H4.V mediated these alterations by condensing chromatin at specific genomic regions as seen with cryo-EM structure of reconstituted H4.V containing nucleosomes. These results not only uncovered the presence of a H4 variant in plants, but also a novel chromatin regulation of stress responses that might have contributed to success of semi-aquatic Oryza members under variable water-limiting conditions. One-line summaryHistone H4 variant predisposes chromatin for stress responses O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=199 SRC="FIGDIR/small/551207v1_ufig1.gif" ALT="Figure 1"> View larger version (53K): org.highwire.dtl.DTLVardef@101fa0forg.highwire.dtl.DTLVardef@c7fda9org.highwire.dtl.DTLVardef@1de9a0aorg.highwire.dtl.DTLVardef@1532641_HPS_FORMAT_FIGEXP M_FIG C_FIG

plant biology↗

bioRxiv2022

Structural insights into pore dynamics of human Pannexin isoforms

Pannexins are single-membrane large-pore ion channels that release ATP upon activation. Three isoforms of pannexins, 1, 2, and 3, perform diverse cellular roles, including inflammation, differentiation, neuropathic pain, and ATP release. In this study, we report the cryoEM structure of pannexin 3 at 3.9 [A] and characterize the structural differences with pannexin isoforms 1 and 2. We observe the organization of the Pannexin 3 vestibule into two distinct chambers with a wider pore radius in comparison to both PANX1 and 2 isoforms. We further report the structure of pannexin1 congenital mutant R217H in the resolution range of 3.9 [A]. The congenital mutant R217H in transmembrane helix3 (TM3), R217H induce structural changes that leads to a partially closed pore and altered ATP interaction propensities. The channel conductance of the congenital mutant displays weakened voltage sensitivity. The results showcase a complete comparison of the three pannexin isoform structures that along with the structure of Pannexin 1 congenital mutant highlight distinct structural features of pannexin isoforms and the allosteric role of distant substitutions in dictating channel behavior in Pannexin 1.

biochemistry↗

bioRxiv2021

Substrate dependent homeostatic control of c-di-AMP synthase (MsDisA) and hydrolase (MsPDE) from Mycobacterium smegmatis

Cyclic-di-nucleotide based secondary messengers regulate various physiological processes including the stress responses in bacteria. In the past decade, cyclic diadenosine monophosphate (c-di-AMP) has emerged as a crucial second messenger, implicated in fatty acid metabolism, antibiotic resistance, biofilm formation, virulence, DNA repair, ion homeostasis, sporulation etc. The level of c-di-AMP is maintained in the cell by the action of two opposing enzymes, namely diadenylate cyclase (DAC) and phosphodiesterase (PDE). In mycobacteria, this molecule is essential for its regulatory role in bacterial physiology and host-pathogen interactions. However, such modulation of c-di-AMP remains to be explored in Mycobacterium smegmatis. Here, we systematically characterised the c-di-AMP synthase (MsDisA) and a hydrolase (MsPDE) from M. smegmatis at different pH and osmolytic conditions in vitro. Our biochemical assays show that the MsDisA activity is enhanced during the alkaline stress and c-di-AMP is readily produced without any intermediates. At pH 9.4, the MsDisA promoter activity in vivo increases significantly, strengthening this observation. However, under physiological conditions, the activity of MsDisA was moderate with the formation of intermediates. To get further insights into the structural characteristics, we determined the cryo-EM structure of the MsDisA, revealing some interesting features. Biochemical analysis of individual domains shows that the N-terminal minimal region alone can form a functional octamer. Altogether, our results reveal the biochemical and structural regulation of mycobacterial c-di-AMP in response to various environmental stress.

biophysics↗

bioRxiv2021

Duox generated reactive oxygen species activate ATR/Chk1 to induce G2 arrest in Drosophila tracheoblasts

Progenitors of the thoracic tracheal system of adult Drosophila (tracheoblasts) arrest in G2 during larval life and rekindle a mitotic program subsequently. G2 arrest is dependent on ATR-dependent phosphorylation of Chk1 that is actuated in the absence of detectable DNA damage. We are interested in the mechanisms that activate ATR/Chk1 (Kizhedathu et al., 2018, 2020). Here we report that levels of reactive oxygen species (ROS) are high in arrested tracheoblasts and decrease upon mitotic re-entry. High ROS is dependent on expression of Duox, an H2O2 generating-Dual Oxidase. ROS quenching by overexpression of Superoxide Dismutase 1, or by knockdown of Duox, abolishes Chk1 phosphorylation and results in precocious proliferation. Tracheae deficient in Duox, or deficient in both Duox and regulators of DNA damage-dependent ATR/Chk1 activation (Claspin/ATRIP/TOPBP1), can induce phosphorylation of Chk1 in response to micromolar concentrations of H2O2 in minutes. The findings presented reveal that H2O2 activates ATR/Chk1 in tracheoblasts by a non-canonical, potentially direct, mechanism.

developmental biology↗