Biology subjects

Vankova, P.

Publications and source records attributed to Vankova, P..

7 recordsLinked to original sources

bioRxiv2025

Structural mechanisms of allosteric regulation in the human cis-prenyltransferase complex

Human cis-prenyltransferase (hcis-PT) synthesizes long-chain isoprenoids essential for N-linked protein glycosylation. This heteromeric complex comprises the catalytic subunit DHDDS and the regulatory Nogo-B receptor (NgBR). Although NgBR dramatically enhances DHDDS activity, the molecular basis for this allosteric regulation remains unclear. Here, we combined crystallography, hydrogen-deuterium exchange mass spectrometry (HDX-MS), molecular dynamics simulations, and network analysis to uncover the structural dynamics and communication pathways within hcis-PT. By solving the apo structure of hcis-PT, we reveal only a localized flexibility at the active site and the NgBR C-terminus. However, HDX-MS demonstrated widespread substrate-induced stabilization, particularly at the NgBR {beta}D-{beta}E loop, highlighting it as an allosteric hub. Functional mutagenesis scanning identified NgBRS249 as critical for enzymatic activity, independent of structural perturbations. Network analysis of MD simulations pinpointed this residue as a central node in inter-subunit communication, with perturbations disrupting downstream allosteric pathways, altering enzymatic activity. Our findings reveal a dynamic regulatory network centered at the inter-subunit interface, wherein specific NgBR residues modulate DHDDS activity through allosteric signaling. This work elucidates a conserved mechanism of subunit coordination in long-chain cis-prenyltransferases and suggests novel avenues for therapeutic targeting of hcis-PT-related disorders.

biochemistry↗

bioRxiv2025

Nanobodies as therapies for loss-of-function misfolding diseases.

Misfolding diseases that result in loss of function represent a considerable burden for both individuals and society. Primary hyperoxaluria type 1 (PH1) is a rare genetic disorder caused by mutations in the alanine:glyoxylate aminotransferase 1 (AGT) enzyme. The underlying molecular mechanisms causing PH1 are associated with protein misfolding (enhanced aggregation and mitochondrial mistargeting). The main therapeutic approach to increase patients lifespan and quality of life is a double kidney and liver transplantation. Alternative treatments such as gene and enzyme replacement and pharmacological chaperones are currently being introduced, but other alternatives are necessary. In this work, we developed and characterized a novel biotechnological approach using six single-domain nanobodies (NB-AGT-1 to -6) as potential therapeutics for PH1 misfolding. We show that NB-AGTs are very stable proteins and bind to pathogenic and non-pathogenic variants of AGT with extreme affinities (with Kd values from low nM to low pM). Structural studies showed that NB-AGTs bind to different epitopes of AGT with selectivity for different AGT variants. Experiments in cellular PH1 models showed that internalization of engineered NB-AGT-3 enhanced the specific activity of disease-associated variants. Overall, we show that NBs are a novel and promising approach to treat PH1 and other loss-of-function misfolding diseases.

biophysics↗

bioRxiv2025

New structural insights into the control of the retinoic acid receptors RAR/RXR by DNA, ligands and transcriptional coregulators

Retinoic acid receptors (RARs) are ligand-dependent transcription factors essential for various biological processes, including embryogenesis, differentiation, and apoptosis. RARs function as heterodimers with retinoid X receptors (RXRs) and regulate gene expression via retinoic acid response elements (RAREs). Their transcriptional activity is modulated by coregulators, with corepressors maintaining repression in the absence of ligand and coactivators enabling transcription upon ligand binding. Structural studies reveal that DNA binding induces conformational changes affecting coregulator interactions. However, the precise structural organization of RAR/RXR-coregulator complexes and the allosteric influence of DNA on receptor function remain incompletely understood. Our study presents an integrative analysis of the RAR/RXR heterodimer bound to four distinct and relevant RAREs (DR0, DR1, DR5, and IR0) in complex with either a corepressor (NCoR) or a coactivator (TIF-2) nuclear receptor interaction domain. By combining small-angle X-ray scattering, hydrogen/deuterium exchange mass spectrometry and molecular dynamics simulations, we revealed that the heterodimer adopts distinct conformations depending on the DNA sequence, influencing interdomain distances and receptor interactions. Additionally, we uncovered the dynamic interplay between ligand, DNA, and coregulator binding. This study provides new insights into the structural features of coregulator proteins and highlights the allosteric influence of RAREs on receptor function.

biochemistry↗

bioRxiv2025

Active site tyrosine residues in human NQO1 homodimer are critical for non-synchronous enzyme catalysis at the two active sites.

AbstractHuman NQO1 is a flavoenzyme essential for the redox metabolization of many substances and associated with wide-impacting diseases such as cancer and Alzheime[r]s. Recent X-ray crystallographic studies have proposed that a few residues at the active site of NQO1 (including Tyr126 and Tyr128) may control enzyme catalysis and functional negative cooperativity. In this work, we use rapid mixing pre-steady state kinetics and hydrogen-deuterium exchange followed by mass spectrometry (HDX-MS) to evaluate experimentally the role of Tyr126 and Tyr128 in NQO1 functionality by generating mutants to Phe, Ala and Glu. Mutations to Phe caused mild effects, whereas those to Ala significantly decreased hydride transfer efficiency and those to Glu virtually abolished NQO1 activity. Interestingly, structural stability studies by HDX-MS showed significant perturbations particularly affecting the binding site of NADH/NAD+ in the less conservative mutations (particularly to Glu). Mutations of Tyr126 and Tyr128 seem to also modulate the non-synchronous catalysis in the two active sites (negative cooperativity) as well as the selectivity for NADH/NADPH as coenzymes. Our work experimentally demonstrates the critical role of Tyr126 and Tyr128 in the flavin reductive half-reaction of the catalytic cycle of NQO1 in the negative cooperativity, and also suggests that phosphorylation of these two Tyr residues might shut down NQO1 activity reversibly.

biophysics↗

bioRxiv2023

Chloride intracellular channel (CLIC) proteins function as fusogens

Chloride Intracellular Channel (CLIC) family members uniquely transition between soluble and membrane-associated conformations. Despite decades of extensive functional and structural studies, CLICs function as ion channels remains debated, rendering our understanding of their physiological role incomplete. Here, we expose a novel function of CLIC5 as a fusogen. We demonstrate that purified CLIC5 directly interacts with the membrane and induces fusion, as reflected by increased liposomal diameter and lipid and content mixing between liposomes. Moreover, we show that this activity is facilitated by acidic pH, a known trigger for CLICs transition to a membrane-associated conformation, and that increased exposure of the hydrophobic inter-domain interface is crucial for this process. Finally, mutation of a conserved hydrophobic interfacial residue diminishes the fusogenic activity of CLIC5 in vitro and impairs excretory canal extension in C. elegans in vivo. Together, our results unravel the long-sought physiological role of these enigmatic proteins.

biochemistry↗

bioRxiv2021

Structural basis for long-chain isoprenoids synthesis by cis-prenyltransferases

Isoprenoids are the largest group of natural products, found in all living organisms and play an essential role in numerous cellular processes. These compounds are synthesized by prenyltransferases, catalyzing the condensation reaction between an allylic diphosphate primer and a variable number of isopentenyl diphosphate (C5) units. This superfamily of enzymes can be subdivided into trans- or cis-prenyltransferases according to the stereoisomerism of the product. The cis branch can be further classified according to product length. While the active site volume was suggested to determine the final length in enzymes synthesizing short- and medium-chain products (up to C60), long-chain enzymes (up to C120) and rubber synthases (>C10,000) fail to conform to this paradigm. Here, to resolve the structural basis for long-chain isoprenoid synthesis, we focused on the human cis-prenyltransferase complex (hcis-PT). This enzyme, peripheral to the endoplasmic reticulum membrane, produces the precursor for dolichol phosphate, a membrane residing glycosyl carrier. In line with its crucial role in the cellular protein glycosylation machinery, disease-causing mutations in hcis-PT were shown to result in a wide spectrum of clinical phenotypes. The crystallographic structures of hcis-PT in four different substrate/product-bound conformations revealed an outlet enabling product elongation into the bulk solvent. Moreover, hydrogen-deuterium exchange mass spectrometry analysis in solution showed that the hydrophobic active site core is flanked by dynamic regions consistent with separate inlet and outlet orifices. Finally, using a fluorescent substrate analog and a fluorescently-labeled lipid nanodiscs, we show that product elongation and membrane association are closely correlated. Together, our results support directional product synthesis in long-chain enzymes and rubber synthases, with a distinct substrate inlet and product outlet, allowing direct membrane insertion of the elongating isoprenoid during catalysis. This mechanism uncouples active site volume from product length and circumvents the need to expulse hydrophobic product into a polar environment prior to membrane insertion.

biochemistry↗

bioRxiv2020

Structure of the human heterotetrameric cis-prenyltransferase complex

The human cis-prenyltransferase (hcis-PT) is an enzymatic complex essential for protein N-glycosylation. Synthesizing the precursor of the glycosyl carrier dolichol-phosphate, we reveal here that hcis-PT exhibits a novel heterotetrameric assembly in solution, composed of two catalytic dehydrodolichyl diphosphate synthase (DHDDS) and two inactive Nogo-B receptor (NgBR) subunits. The 2.3 [A] crystal structure of the complex exposes a dimer-of-heterodimers arrangement, with DHDDS C-termini serving as homotypic assembly domains. Furthermore, the structure elucidates the molecular details associated with substrate binding, catalysis, and product length determination. Importantly, the distal C-terminus of NgBR transverses across the heterodimeric interface, directly participating in substrate binding and underlying the allosteric communication between the subunits. Finally, mapping disease-associated hcis-PT mutations involved in blindness, neurological and glycosylation disorders onto the structure reveals their clustering around the active site. Together, our structure of the hcis-PT complex unveils the dolichol synthesis mechanism and its perturbation in disease.

biochemistry↗