Biology subjects

Srinivasu, B. Y.

Publications and source records attributed to Srinivasu, B. Y..

5 recordsLinked to original sources

bioRxiv2026

Magnesium as a Conformational Gatekeeper of KRAS: Structural Dynamics and Therapeutic Implications

Magnesium serves as an essential cofactor for small GTPases, yet its structural role in regulating KRAS conformational dynamics and nucleotide exchange remains poorly understood. Here, we combine hydrogen-deuterium exchange mass spectrometry (HDX-MS), native mass spectrometry, and functional assays to elucidate how Mg2+ stabilizes the KRAS conformational ensemble and constrains transitions between GDP- and GTP-bound states. Depletion of Mg2+ triggers widespread increases in structural dynamics throughout KRAS--spanning the p-loop, 1-helix, switch I, nucleotide-binding region, and distal helices--revealing a global loosening of the protein fold that favors an open, nucleotide exchange-competent state. Mg2+ titration experiments demonstrate that individual structural elements exhibit distinct Mg2+ dependencies: the p-loop and 1-helix recover native dynamics at micromolar concentrations, whereas switch I requires millimolar levels, underscoring its exceptionally high sensitivity to Mg2+ for structural stabilization. KRAS bound to the catalytic domain of exchange factor SOS1 displays an HDX signature closely resembling the Mg2+-free state, indicating that SOS1 promotes nucleotide exchange by transiently perturbing Mg2+ coordination while simultaneously stabilizing switch I. Consistently, phosphomimetic KRAS S17E variant, which disrupts a critical Mg2+-coordinating residue, exhibits pronounced global destabilization--reinforcing the central importance of Mg2+ in maintaining structural integrity. Taken together our findings show that Mg2+ acts as a master regulator of KRAS structural dynamics and reveal Mg2+-sensitive hotspots that might represent promising targets for next-generation KRAS therapeutics.

biochemistry↗

bioRxiv2025

SARS CoV-2 spike adopts distinct conformational ensembles in situ

Engineered recombinant Spike (S) has been invaluable for determining S structure and dynamics and is the basis for the design of most prevalent vaccines. While these vaccines have been highly efficacious for short-term protection from infection, protection waned with the emergence of variants (alpha through omicron). Here we report differences in conformational dynamics between native, membrane-embedded full-length S and recombinant S. Our virus-like particle (VLP) model mimics the native SARS CoV-2 virion by displaying S assembled with auxiliary E, M, and N proteins in a native membrane environment that captures the entirety of quaternary interactions mediated by S. Display of S on VLP obviates the requirement for stabilizing modifications that have been engineered into recombinant S for enhanced expression and solubility. Amide hydrogen/deuterium exchange mass spectrometry (HDXMS) reveals altered interprotomer contacts in VLP S trimers attributable to the presence of auxiliary proteins, membrane anchoring, and lack of engineered modifications. Our results reveal decreased dynamics in the S2 subunit and at sites spanning interprotomer contacts in VLP S with minimal differences in the N-terminal domain (NTD) and receptor binding domain (RBD). This carries implications for display of epitopes beyond NTD and RBD. In summary, despite affording efficient structural characterization, recombinant S distorts the intrinsic conformational ensemble of native S displayed on the virus surface.

biophysics↗

bioRxiv2023

Oxygen-induced chromophore degradation in the photoswitchable red fluorescent protein rsCherry

Reversibly switchable monomeric Cherry (rsCherry) is a photoswitchable variant of the red fluorescent protein mCherry. We report that this protein gradually and irreversibly loses its red fluorescence in the dark over a period of months at 4{degrees}C and a few days at 37{degrees}C. We also find that its ancestor, mCherry, undergoes a similar fluorescence loss but at a slower rate. X-ray crystallography and mass spectrometry reveal that this is caused by the cleavage of the p-hydroxyphenyl ring from the chromophore and the formation of two novel types of cyclic structures at the remaining chromophore moiety. Overall, our work sheds light on a new process occurring within fluorescent proteins, further adding to the chemical diversity and versatility of these molecules.

biophysics↗

bioRxiv2020

Functional self-nonamerization of the Type III translocase chaperone/exported protein receptor

Type III protein secretion is widespread in Gram-negative pathogens. It comprises the injectisome with a surface-exposed needle and an inner membrane translocase. The translocase contains the SctRSTU export channel enveloped by the export gate subunit SctV that binds chaperone/exported clients and forms a putative ante- chamber. We probed the assembly, function, structure and dynamics of SctV from enteropathogenic E.coli (EPEC). In both EPEC and E.coli lab strains, SctV forms peripheral oligomeric clusters that are detergent-extracted as homo-nonamers. Membrane-embedded SctV9 is necessary and sufficient to act as a receptor for different chaperone/exported protein pairs with distinct C-domain binding sites that are essential for secretion. Negative staining electron microscopy revealed that peptidisc-reconstituted His-SctV9 forms a tripartite particle of [~]22 nm with a N- terminal domain connected by a short linker to a C-domain ring structure with a [~]5 nm-wide inner opening. The isolated C-domain ring was resolved with cryo-EM at 3.1 [A] and structurally compared to other SctV homologues. Its four sub-domains undergo a three-stage "pinching" motion. Hydrogen-deuterium exchange mass spectrometry revealed this to involve dynamic and rigid hinges and a hyper-flexible sub-domain that flips out of the ring periphery and binds chaperones on and between adjacent protomers. These motions are coincident with pore surface and ring entry mouth local conformational changes that are also modulated by the ATPase inner stalk. We propose a model that the intrinsic dynamics of the SctV protomer are modulated by chaperones and the ATPase and could affect allosterically the other subunits of the nonameric ring during secretion.

molecular biology↗

bioRxiv2020

PyHDX: Derivation and visualization of protection factors from Hydrogen-Deuterium Exchange Mass Spectrometry at near residue resolution

Hydrogen Deuterium Exchange Mass Spectrometry is a powerful monitor of protein intrinsic dynamics, yet the interpretation, visualization and cross-comparison of HDX-MS datasets is challenging. Here we present PyHDX, an open-source python package and web server, that batch-extracts the universal quantity Gibbs free energy at residue level over multiple protein conditions and homologues. {Delta}G values relate to protein normal modes and together provide a universal measure of protein flexibility. AvailabilityPyHDX source code is released under the MIT license and can be accessed on GitHub.

bioinformatics↗