Biology subjects

Kastritis, P. L.

Publications and source records attributed to Kastritis, P. L..

At least 19 recordsLinked to original sources

bioRxiv2026

Structural basis for far-red light harvesting in a euglenophyte photosystem II supercomplex

Photosystem II (PSII) is in eukaryotic phototrophs is generally considered to operate within a more restricted spectral range than photosystem I (PSI), in which long-wavelength chlorophylls are a well-established feature of the peripheral antenna. Whether eukaryotic PSII can acquire comparable far-red-associated properties through lineage-specific antenna diversification has remained unclear. Here we present a 3.09 [A] cryo-electron microscopy structure of the C2S2M2L2 PSII supercomplex from Euglena gracilis, a euglenophyte species harbouring a secondary plastid and unusual light-harvesting system. We show that the euglenophyte-specific antenna protein LhcE9 occupies the position corresponding to canonical Lhcb5, but in a markedly different orientation that creates a distinct interface with the PSII core, particularly with CP43. Combined structural, spectroscopic, mutagenesis and proteomic analyses support LhcE9 as the stably bound PSII antenna subunit most closely associated with the far-red state in the supercomplex. Excitation-energy-transfer calculations further indicate two fast lineage-specific antenna-to-core routes mediated by LhcE9 and PsbX. Together, these findings reveal an unexpected mode of PSII antenna diversification and provide a structural framework for far-red-associated light harvesting in PSII.

plant biology↗

bioRxiv2026

SNPstar: A Web Server Linking Allelic Variation to Protein Structure and Function in Arabidopsis thaliana

Understanding how natural genetic variants affect protein structure and function is central to plant biology. The 1001 Genomes Project has catalogued millions of single nucleotide polymorphisms (SNPs) across more than a thousand Arabidopsis thaliana accessions, offering an unprecedented opportunity to relate sequence variation to three-dimensional protein structure and population context. Yet, realizing it requires tools that integrate these scales in one accessible framework. Here we present SNPstar, a web server that links allelic variation in A. thaliana to AlphaFold3-predicted structures through a gene-centric, interactive interface. SNPstar annotates each variant with descriptive features, thermodynamic stability estimates, protein domain context, and genome-wide association results, and computes haplotypes and proteotypes that group accessions by shared DNA or protein sequence. Researchers can characterize variants, visualize their structural context, map their geographic distribution, and prioritize accessions for experimental validation without local computational infrastructure. We demonstrate SNPstar with two case studies. The first recapitulates known loss-of-function variation in the cadmium transporter HMA3, validating that SNPstar prioritizes functionally consequential alleles. The second uses SNPstar-defined proteotypes to identify an N-terminal SNP combination in ARGONAUTE 2 that distinguishes accessions differing in in vitro siRNA-directed target cleavage, linking protein-coding variation to a measurable molecular phenotype. SNPstar thus helps translate natural variation into mechanistic insight.

plant biology↗

bioRxiv2026

A nested shell structure coordinates enzyme communication in pyruvate oxidation

The pyruvate dehydrogenase complex (PDHc)1 links glycolysis to the Krebs cycle by catalyzing the oxidative decarboxylation of pyruvate to acetyl-CoA and CO2, a process essential for life2,3. PDHc is formed by structural proteins (E3-binding protein, E3BP)4-7, enzymatic subunits (E1, E2, E3)4,6, and mobile lipoyl domains (LDs), the latter shuttling intermediates across active sites6,8. Although numerous details regarding pyruvate oxidation steps have been elucidated9, the precise organization of the entire PDHc remains unknown due to its large size and dynamic heterogeneity. Here, we employ in silico, in vitro, and in situ methods to propose a multi-scale model of PDHc that includes approximately one million atoms and to visualize multiple conformational states. This model reveals a [~]40-50 nm nested shell structure, formed by flexible linkers that spatially coordinate the E1 and E3 enzyme complexes around the E2-E3BP core scaffold. This structure acts as a molecular sieve, selectively guiding lipoyl arms while maintaining enzyme positioning with sub-nm precision. During catalysis, the nested shell structure expands and adopts a mechanically reinforced state comparable in magnitude to viral assemblies10. Our findings provide structural context for the textbook "link reaction"11, building on decades of biochemical knowledge, are transferable to functional aspects of other -ketoacid dehydrogenase complexes, and, ultimately, expand our understanding of primary metabolism as a whole. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=195 SRC="FIGDIR/small/724543v1_ufig1.gif" ALT="Figure 1"> View larger version (70K): org.highwire.dtl.DTLVardef@1615612org.highwire.dtl.DTLVardef@159bc8dorg.highwire.dtl.DTLVardef@69f8c1org.highwire.dtl.DTLVardef@14a83a3_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2026

A 37-million-particle dataset from over 250 experiments to accelerate data-driven cryo-EM analysis

Cryogenic Electron Microscopy (cryo-EM) has revolutionized structural biology by enabling near-atomic-resolution structure determination of biological macromolecules. Central to cryo-EM analysis are particles, namely 2D projections of biomolecules extracted from micrographs, which serve as the primary input for 3D reconstruction. While data-driven methods have transformed other scientific domains, their impact on cryo-EM remains limited because existing particle datasets are too small, too narrow in protein diversity, and lack rich per-particle annotations. We introduce cryoPANDA (cryo-EM Particles ANnotated DAtaset), comprising over 37 million annotated particles from 252 experiments spanning a wide range of protein types, more than 10-fold larger than prior collections. Each particle is accompanied by detailed annotations covering acquisition, classification, and re-construction metadata, alongside the corresponding 3D electrostatic potential map, the published EMDB map, and, where available, the PDB model. We validate cryoPANDA in two ways: first, by reconstructing hundreds of distinct high-resolution cryo-EM maps; and second, by training a DINOv2 foundation model and evaluating its learned representations on micrograph segmentation, particle picking, and particle clustering.

bioinformatics↗

bioRxiv2026

Structural basis of Arf1-driven membrane tubulation

Membrane tubules form at Golgi compartments to facilitate membrane and cargo flow in intracellular trafficking. Here we show that the small GTPase Arf1, an inducer of membrane curvature and key regulator of trafficking, is able to form strongly curved tubules in the presence of lipids and GTP{gamma}S in vitro without the need for further coat components. Using cryo-electron microscopy, we determined the structures of tubular Arf1-scaffolds with diameters of 195 and 215 [A] at 3.1 and 3.8 [A] resolutions, respectively. The nucleotide-bound globular domains of Arf1 form polar helical lattices (i.e. directional assemblies with distinct start/finish orientations), with conserved interfaces and a consistent back-to-face orientation along the filaments. The rigid coat is tethered to the membrane by a flexible linker and anchored by an amphipathic helix (AH) that is free to diffuse and make space within the leaflet, allowing for accommodation of transmembrane cargo. The diversity of tubular diameters observed would allow various cargo sizes to be accommodated in the lumen, while maintaining the local coat architecture. Apart from serving as tubular transport intermediates, Arf1-scaffolds may also play a role at the neck of COPI vesicle on the route to scission.

biophysics↗

bioRxiv2026

Revisiting p53:Sirt1 interaction in the light of controlling p53 acetylation levels

The NAD+-dependent deacetylase sirtuin 1 (Sirt1) is known to regulate the tumor suppressor p53 via deacetylation, but the structural basis of the protein-protein interaction between full-length p53 and Sirt1 has so far remained elusive. We apply an integrated approach, combining structural mass spectrometry (MS) with data-driven molecular docking, to study the interaction between human p53 and Sirt1. Sirt1 was found to bind exclusively to acetylated p53 forming complexes with a 1:1 stoichiometry, irrespectively of p53s oligomeric state. The lysine residue at position 582 (K582) in p53 was identified as predominant acetylation site showing a selective Sirt1-dependent deacetylation at this position. Cross-linking mass spectrometry (XL-MS) provided valuable distance constraints between p53 and Sirt1. Specifically, cross-links created between p53-K382 / Sirt1-K427 and p53-K120 / Sirt1-K622 give hints on a highly flexible interface. Molecular docking was conducted based on the distance constraints imposed by the cross-links, positioning Sirt1 at the DNA-binding and tetramerization domains of p53. This gives a rationale for a steric exclusion of additional Sirt1 molecules binding to p53. We present the first structural model of the full-length p53:Sirt1 (1:1) complex, establishing a mechanistic framework that links p53 activity to its Sirt1-controlled acetylation status.

biochemistry↗

bioRxiv2025

Direct evidence of acid-driven protein desolvation

Water and its ability to modulate the protonation states of biomolecules govern the physical chemistry of life, dictating their metabolic functions(1). However, how amino acid protonation alters protein hydration and solubility(2, 3) is an open question since Kuntz and Kauzmann proposed pH-driven protein desolvation in 1974(4). Here, in a series of high-resolution cryo-electron microscopy structures of a protein complex at different pH values (from pH 9.0 to 3.5) we examined thousands of observable hydration sites. Cryo-EM data, in agreement with constant-pH molecular dynamics simulations, show that nearly half of protein-bound waters exchanged with the bulk solvent upon acidification, with [~]100 waters lost per pH unit per molecule. The loss of waters was most significant around the side chains of glutamate and aspartate residues while specific polar residues, mostly asparagine, anchored persistent waters. A positionally conserved hydration layer was observed across all pH conditions, accounting for 40% of resolved waters. Those waters displayed denser packing than less persistent waters, forming a pH-independent solvation shell. Acid-induced water exchange also displaced bound iron, providing a mechanistic link between solvation and metal release. Our findings demonstrate the core principles of acid-driven protein desolvation, resolving a 50-year-old biochemical hypothesis(4).

biochemistry↗

bioRxiv2025

Structural basis of neurofibromin tetramerization and dimer-tetramer equilibrium

Human neurofibromin (NF1) is a tumor suppressor multidomain protein known to regulate cellular functions as a dimer. Using cryo-EM, we discovered that neurofibromin can form more highly organized structures and describe tetramerization and a dynamic dimer-to-tetramer equilibrium with extensive interlocked interfaces as well as structural flexibility. The new tetramer structure generates and modifies interaction surfaces and controls protein functions such as microtubule recruitment, thereby providing novel insights into cellular functions.

molecular biology↗

bioRxiv2025

Strategies for Mitigating Radiation Damage and Improving Data Completeness in 3D Electron Diffraction of Protein Crystals

While 3D electron diffraction (3D-ED or microcrystal electron diffraction, MicroED) has emerged as a promising method for protein structure determination, its applicability is hindered by high susceptibility to radiation damage, leading to decreasing signal-to-noise ratio in consecutive diffraction patterns that limits the quality (resolution and redundancy) of the data. In addition, data completeness may be restricted due to the geometrical limitations of current sample holders and stages. In this work, we introduce an approach that addresses these issues using a commonly available 200keV cryo-electron microscope. The multi-position acquisition technique we present here combines (a) multiple data acquisitions from a single crystal over several tilt ranges and (b) merging data from a small number of crystals, each tilted about a different axis. The robustness of this approach is demonstrated by the de novo elucidation of a protein-peptide complex structure from only two orthorhombic microcrystals.

biochemistry↗

bioRxiv2024

Mechanism of SHP2 activation by bis-Tyr-phosphorylated Gab1

The non-receptor tyrosine phosphatase SHP2 (PTPN11) is a regulator of diverse cellular functions including mitogenic activation and cell migration. SHP2 consists of two tandem SH2 domains followed by the catalytic domain, and is autoinhibited by the N-terminal SH2 domain that blocks access to the active site. Mutations that influence auto-inhibition have been implicated in cancer and other diseases, and allosteric inhibitors have been developed that stabilise the inactive state. The mechanism of SHP2 activation remains unclear, however. Here, we show that the intrinsically disordered bis-phosphorylated SHP2-activating peptide pY627pY659-Gab1 binds to both SH2 domains, undergoing a partial disorder-to-order transition in the process. In addition to eliciting changes in SH2 domain dynamics, the peptide reorganises their relative orientations to provide a new SH2-SH2 interface. Our data suggest an active conformation for SHP2 that is also applicable to the hematopoietic cell-specific SHP1 (PTPN6), shedding light on the activation mechanism of both enzymes and paving the way for the development of novel compounds that modulate SHP2 activity.

biochemistry↗

bioRxiv2024

IQD2 recruits KLCR1 to the membrane-microtubule nexus to promote cytoskeletal mechano-responsiveness in leaf epidermis pavement cells

Plant cells experience a variety of mechanical stresses from both internal and external sources, including turgor pressure, mechanical strains arising from heterogeneous growth between neighboring cells, and environmental factors like touch from soil, rain, or wind [1,2]. These stresses serve as signals at the cell-, tissue- and organismal level to coordinate plant growth during development and stress responses [3]. In plants, the physical cell wall-plasma membrane-microtubule continuum is proposed to be integral in transducing mechanical signals from the exterior to intracellular components [4-6]. Cortical microtubules (CMTs) rapidly reorient in response to mechanical stress to align with the maximal tensile stress direction [7,8]. Several studies proposed that CMTs themselves may act as stress sensors; the precise mechanisms involved in the regulation of CMTs and the modes of sensing, however, are still not clearly understood. Here, we show that IQD2 and KLCR1 are enriched at CMTs in proximity to the plasma membrane. IQD2, which is a bona fide microtubule-associated protein, promotes microtubule localization of KLCR1. By combining cross-linking mass spectrometry (XL-MS) and computational modeling with structure-function studies, we present first experimental insights into the composition and structure of IQD2-KLCR1 complexes. Further, we demonstrate that the IQD2-KLCR1 module is a positive regulator of microtubule mechano-responses in pavement cells. Collectively, our work identifies the IQD2-KLCR1 module as novel regulator of mechanostress-mediated CMT reorientation and provides a framework for future mechanistic studies aimed at a functional dissection of mechanotransduction at the plasma membrane-CMT interface during growth and plant morphogenesis. HighlightsO_LIIQD2 and KLCR1 localize to the plasma membrane-microtubule nexus C_LIO_LIIQD2 is required for efficient microtubule targeting of KLCR1 in planta C_LIO_LIIQD2 physically interacts with KLCR1 and microtubules C_LIO_LIThe IQD2-KLCR1 module promotes mechano-stress induced microtubule reorganization C_LI

plant biology↗

bioRxiv2022

Structural Assessment of the Full-Length Wild-Type Tumor Suppressor Protein p53 by Mass Spectrometry-Guided Computational Modeling

The tetrameric tumor suppressor p53 represents a great challenge for 3D-structural analysis due to its high degree of intrinsic disorder (ca. 40%). We aim to shed light on the structural and functional roles of p53s C-terminal region in full-length, wild-type human p53 tetramer and their importance for DNA binding. For this, we employed complementary techniques of structural mass spectrometry (MS) in an integrated approach with AI-based computational modeling. Our results show no major conformational differences in p53 between DNA-bound and DNA-free states, but reveal a substantial compaction of p53s C-terminal region. This supports the proposed mechanism of unspecific DNA binding to the C-terminal region of p53 prior to transcription initiation by specific DNA binding to the core domain of p53. The synergies between complementary structural MS techniques and computational modeling as pursued in our integrative approach is envisioned to serve as general strategy for studying intrinsically disordered proteins (IDPs) and intrinsically disordered region (IDRs).

biochemistry↗

bioRxiv2022

AI-guided cryo-EM probes a thermophilic cell-free system with succinyl-coA manufacturing capability

Cell-free systems display tremendous potential for biotechnological applications, complementing in vitro reconstituted enzymatic processes and traditional expression systems. However, they often represent "black boxes" without much insight into their components. Here, we characterize a thermophilic cell-free system that produces succinyl-CoA and discern its intrinsic, non-stochastic organization. By employing biochemical, biophysical, and bioinformatic methods we resolve its molecular composition, 3D architecture and molecular function at atomic resolution. We further report the high-resolution cryo-EM structure of the reactions main component, the oxoglutarate dehydrogenase complex core (E2o), which displays various structural adaptations. These include hydrogen bonding patterns confining interactions of participating enzymes (E1o-E2o-E3), electrostatic tunneling that drives inter-communication between subunits, and the presence of a flexible subunit, the E3BPo connecting E2o and E3. This multi-scale analysis of a cell-free system provides a blueprint for structure-function studies of complex mixtures of biotechnological value.

biochemistry↗

bioRxiv2022

Delineating organizational principles of the endogenous L-A virus by cryo-EM and computational analysis of native cell extracts

The high abundance of most viruses in infected host cells benefits their structural characterization; endogenous viruses are present in low copy numbers, however, and are therefore challenging to investigate. Here, we retrieve cell extracts enriched with an endogenous virus, the yeast L-A virus. The determined cryo-EM structure discloses capsid-stabilizing cation-{pi} stacking and an interplay of non-covalent interactions from ten distinct capsomere interfaces. The capsid-embedded mRNA decapping active site trench is supported by a constricting movement of two opposite-facing loops. tRNA-loaded polysomes and other biomacromolecules, presumably mRNA, are found in virus proximity while stacked dsRNA bundles and the sub-stoichiometric polymerase localize underneath the capsid surface. Mature viruses participate in larger viral communities resembling their rare in-cell equivalents in terms of size, composition, and inter-virus distances. Our results collectively describe a 3D-architecture of a viral milieu, opening the door to cellextract-based high-resolution structural virology.

biochemistry↗

bioRxiv2022

Exotic alleles of EARLY FLOWERING 3 determine plant development and grain yield in barley

EARLY FLOWERING 3 (ELF3) is an important regulator of various physiological and developmental processes and hence may serve to improve plant adaptation which will be substantial for future plant breeding. To expand the limited knowledge on barley ELF3 in determining agronomic traits, we conducted field studies with heterogeneous inbred families (HIFs) derived from selected lines of the wild barley nested association mapping population HEB-25. During two growing seasons, phenotypes of nearly isogenic HIF sister lines, segregating for exotic and cultivated alleles at the ELF3 locus, were compared for ten developmental and yield-related traits. We determine novel exotic ELF3 alleles and show that HIF lines, carrying the exotic ELF3 allele, accelerated plant development compared to the cultivated ELF3 allele, depending on the genetic background. Remarkably, the most extreme phenotypic effects could be attributed to one exotic ELF3 allele, differing in only one SNP from the cultivated Barke ELF3 allele. This SNP causes an amino acid change, which predictively has an impact on the protein structure of ELF3, thereby possibly affecting phase separation behaviour and nano-compartment formation of ELF3 and, potentially, also affecting its local cellular interactions causing significant trait differences between HIF sister lines.

genetics↗

bioRxiv2022

Osmotic stress response in BetP: How lipids and K+ team up to overcome downregulation

The trimeric betaine symporter BetP senses an osmotic upshift via its osmosensory C-terminal domain and responds rapidly with a steep increase in transport rate. Full activation requires both an elevated internal K+ concentration and a yet unknown membrane stimulus. Moreover, the molecular mechanisms of stress sensing and upregulation remain unclear. Here, we show that K+ binding to BetP in vivo is highly cooperative. Using X-ray crystallography, we identify four putative K+ interaction sites at the C-terminal domains of BetP. Single particle CryoEM on BetP reconstituted in amphipols (AMP-BetP), in the absence of K+, revealed that the three C-terminal domains are oriented symmetrically near the membrane surface, representing a new downregulated state. The presence of K+ resulted in asymmetric partial unfolding of the C-terminal domains, which was assigned as an intermediate between the downregulated state and the conformation observed in crystal structures. Attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy in 2D crystals of BetP reveal glutamate/aspartate and tyrosine responses to K+, in agreement with the identified K+ interaction sites, as well as specific unfolding events in the C-terminal domain upon activation. A rearrangement of the relative protomer orientations confers upregulation through key structural elements involved in the alternating access of BetP affecting sodium and betaine binding affinities. Although K+-specific regulation is unique to BetP we discuss unfolding/refolding of sensory domains as a unifying element in hyperosmotic stress response of osmoregulated transporters.

biophysics↗

bioRxiv2022

Cryo-EM structure of the Rhodobaca bogoriensis RC-LH1-PufX dimeric complex at 2.9 angstrom

The reaction centre-light harvesting 1 (RC-LH1) complex is essential for converting light into proton motive force in photosynthetic bacteria. RC-LH1 is a monomer in most purple bacteria, but in Rhodobacter species, it is a dimer. Its assembly depends on an accessory polypeptide (PufX) and, ultimately, on photosynthetic growth. To date, knowledge on the RC-LH1-PufX structure, where the dimer has two incomplete C-shaped antenna rings surrounding an RC, is mainly limited to the model organism Rhodobacter sphaeroides. Here we present a cryo-electron microscopy structure at 2.9 [A] from Rhodobaca bogorensis strain LBB1. RCs are surrounded by 30 antennas and incorporate protein Y and PufX. RCs are stably connected by PufX, which self-interacts, electrostatically attracts cytochrome c2 (cyt c2) and forms extensive networks with co-factors. This structure underlines coordinated energy transfer in a combinatorial manner, providing a basis to describe bacterial photosynthesis within a dimeric photosynthetic apparatus.

biophysics↗

bioRxiv2020

First 3D-Structural Data of Full-length Rod-Outer-Segment Guanylyl Cyclase 1 in Bovine Retina by Cross-linking/Mass Spectrometry

The rod-outer-segment guanylyl cyclase 1 (ROS-GC1) is a key transmembrane protein for retinal phototransduction. Mutations of ROS-GC1 correlate with different retinal diseases that often lead to blindness. No structural data are available for ROS-GC1 so far. We performed a 3D-structural analysis of native ROS-GC1 from bovine retina by cross-linking/mass spectrometry (XL-MS) and computational modeling. Absolute quantification and activity measurements of native ROS-GC1 were performed by MS-based assays directly in bovine retina samples. Our data present the first 3D-structural analysis of active, full-length ROS-GC1 in bovine retina. We propose a novel domain organization for the intracellular domain ROS-GC1. Our XL-MS data reveal that the -helical domain connecting the kinase homology and catalytic domains can acquire different conformations. Also, the XL-MS data of native ROS-GC1 in bovine retina agree with a dimeric architecture. Our integrated approach can serve as a blueprint for conducting 3D-structural studies of membrane proteins in their native environment.

biochemistry↗