Biology subjects

Rayaprolu, V.

Publications and source records attributed to Rayaprolu, V..

6 recordsLinked to original sources

bioRxiv2026

Long-range allosteric communication, double mutant cycles, and energetic coupling in SARS-CoV-2 spike protein

SARS-CoV-2 spike protein is continuously evolving, leading to new variants. While mutations in the receptor-binding domain (RBD) enhance binding to the ACE2 receptor and evade neutralizing antibodies, the function of mutations in the N-terminal domain (NTD) remains poorly understood. Using two independent methods, surface plasmon resonance (SPR) and cryo-EM, we show that NTD mutations increase the population of spike protein with the RBD in the "up" conformational state. SPR association and dissociation kinetics of spike binding to ACE2 and antibodies, analyzed using a coupled equilibrium model, determined the relative populations and indicated that the RBD-up-to-down transition is rate-limiting relative to the RBD-down-to-up transition. Advanced model fitting of cryo-EM Coulomb potential maps confirmed the populations. The combined effect of NTD and RBD mutations exceeds the sum of their individual effects, indicating long-range allosteric communication and energetic coupling within the spike protein.

biophysics↗

bioRxiv2024

A broadly-neutralizing antibody against Ebolavirus glycoprotein that potentiates the breadth and neutralization potency of other antibodies

Ebolavirus disease (EVD) is caused by multiple species of Ebolavirus. Monoclonal antibodies (mAbs) against the virus glycoprotein (GP) are the only class of therapeutic approved for treatment of EVD caused by Zaire ebolavirus (EBOV). Therefore, mAbs targeting multiple Ebolavirus species may represent the next generation of EVD therapeutics. Broadly reactive anti-GP mAbs were produced; among these, mAbs 11886 and 11883 were broadly neutralizing in vitro. A 3.0 [A] cryo-electron microscopy structure of EBOV GP bound to both mAbs shows that 11886 binds a novel epitope bridging the glycan cap (GC), 310 pocket and GP2 N-terminus, whereas 11883 binds the receptor binding region (RBR) and GC. In vitro, 11886 synergized with a range of mAbs with epitope specificities spanning the RBR/GC, including 11883. Notably, 11886 increased the breadth of neutralization by partner mAbs against different Ebolavirus species. These data provide a strategic route to design improved mAb-based next-generation EVD therapeutics.

immunology↗

bioRxiv2023

S1 hydrophobic residues alter the voltage sensing phosphatase enzymatic function and voltage sensing

The voltage sensing domain (VSD) is a four-helix modular protein domain that converts electrical signals into conformational changes, leading to open pores and active enzymes. In most voltage sensing proteins, the VSDs do not interact with one another and the S1-S3 helices are considered mainly as scaffolding. The two exceptions are the voltage sensing phosphatase (VSP) and the proton channel (Hv). VSP is a voltage-regulated enzyme and Hvs are channels that only have VSDs. To investigate the S1 contribution to VSP function, we individually mutated four hydrophobic amino acids in S1 to alanine (F127, I131, I134 and L137). We also combined these mutations to generate quadruple mutation designated S1-Q. Most of these mutations shifted the voltage dependence of activity to higher voltages though interestingly, not all substrate reactions were the same. The kinetics of enzymatic activity were also altered with some mutations significantly slowing down dephosphorylation. The voltage dependence of VSD motions were consistently shifted to lower voltages and indicated a second voltage dependent motion. Co-immunoprecipitation demonstrated that none of the mutations broke the VSP dimer indicating that the S1 impact could stem from intrasubunit and/or intersubunit interactions. Lastly, when the same alanine mutations were introduced into a genetically encoded voltage indicator, they dramatically altered the optical readings, making some of the kinetics faster and shifting the voltage dependence. These results indicate that the S1 helix in VSP plays a critical role in tuning the enzymes conformational response to membrane potential transients and influencing the function of the VSD.

biophysics↗

bioRxiv2022

Structure of the Inmazeb cocktail and resistance to escape against Ebola virus

Monoclonal antibodies can provide important pre- or post-exposure protection against disease for those not yet vaccinated or in individuals that fail to mount a protective immune response after vaccination. A key concern in use of monotherapy monoclonal antibody products lies in the high risk of mutagenic escape. Inmazeb (REGN-EB3), a three-antibody cocktail against Ebola virus, demonstrated efficacy in lessening disease course and improving survival in a randomized, controlled trial. Here we present the cryoEM structure at 3.1 [A] of the Ebola virus glycoprotein, determined without symmetry averaging, in a simultaneous complex with eight Fab fragments of antibodies in the Inmazeb cocktail. This structure allows modeling of previously disordered portions of the glycan cap, maps the non-overlapping epitopes of Inmazeb, and illuminates the basis for complementary activities, as well as residues that are critical for resistance to escape by each component of this cocktail and other clinically relevant antibodies. We also provide direct evidence that, unlike monotherapy treatments, including those targeting conserved epitopes, the Inmazeb protects against the rapid emergence of EBOV escape mutants and supports the benefit of the combination approach.

biophysics↗

bioRxiv2022

Glycan shield of the ebolavirus envelope glycoprotein GP

The envelope glycoprotein GP of the ebolaviruses is essential for host cell attachment and entry. It is also the primary target of the protective and neutralizing antibody response in both natural infection and vaccination. GP is heavily glycosylated with up to 17 predicted N-linked sites, numerous O-linked glycans in its disordered mucin-like domain (MLD), and three predicted C-linked mannosylation sites. Glycosylation of GP is important for host cell attachment to cell-surface lectins, as well as GP stability and fusion activity. Moreover, it has been shown to shield GP from neutralizing activity of serum antibodies. Here, we use mass spectrometry-based glycoproteomics to profile the site-specific glycosylation patterns of ebolavirus GP. We detect up to 16 unique O-linked glycosylation sites in the mucin-like domain, as well as two O-linked sites in the head and glycan cap domains of the receptor-binding GP1 subunit. Multiple O-linked glycans are observed at the S/T residues of N-linked glycosylation sequons, suggesting possible crosstalk between the two types of modifications. We also confirmed the presence of C-mannosylation at W288 in the context of trimeric GP. We find heterogenous, complex N-linked glycosylation at the majority of predicted sites as expected. By contrast, the two conserved sites N257 and N563 are enriched in unprocessed high-mannose and hybrid glycans, suggesting a role in host-cell attachment via DC-SIGN/L-SIGN. We discuss our findings in the context of antibody recognition to show how glycans contribute to and restrict neutralization epitopes. This information on how N-, O-, and C-linked glycans together build the heterogeneous glycan shield of GP can guide future immunological studies and functional interpretation of ebolavirus GP-antibody interactions.

biochemistry↗

bioRxiv2018

The voltage sensing phosphatase (VSP) localizes to the apical membrane of kidney tubule epithelial cells

Voltage-sensing phosphatases (VSPs) are transmembrane proteins that couple changes in membrane potential to hydrolysis of inositol signaling lipids. VSPs catalyze the dephosphorylation of phosphatidylinositol phosphates (PIPs) that regulate diverse aspects of cell membrane physiology including cell division, growth and migration. VSPs are highly conserved among chordates, and their RNA transcripts have been detected in the adult and embryonic stages of frogs, fish, chickens, mice and humans. However, the subcellular localization and biological function of VSP remains unknown. Using reverse transcriptase-PCR (RT-PCR), we show that both Xenopus laevis VSP (Xl-VSP1 and Xl-VSP2) mRNAs are expressed in early embryos, suggesting that both Xl-VSPs are involved in early tadpole development. To understand which embryonic tissues express Xl-VSP mRNA, we used in situ hybridization (ISH) and found Xl-VSP mRNA in both the brain and kidney of NF stage 32-36 embryos. By Western blot analysis with a VSP antibody, we show increasing levels of Xl-VSP protein in the developing embryo, and by immunohistochemistry (IHC), we demonstrate that Xl-VSP protein is specifically localized to the apical membrane of both embryonic and adult kidney tubules. We further characterized the catalytic activity of both Xl-VSP homologs and found that while Xl-VSP1 catalyzes 3- and 5-phosphate removal, Xl-VSP2 is a less efficient 3-phosphatase with different substrate specificity. Our results suggest that Xl-VSP1 and Xl-VSP2 serve different functional roles and that VSPs are an integral component of voltage-dependent PIP signaling pathways during vertebrate kidney tubule development and function.

cell biology↗