Biology subjects

Kumpula, E.-P.

Publications and source records attributed to Kumpula, E.-P..

8 recordsLinked to original sources

bioRxiv2026

Gating Mechanism of the Human Connexin 45 Gap Junction Channel

Gap junction channels formed by the 21-member human connexin family enable direct intercellular exchange of ions and small signaling metabolites, coordinating electrical coupling across cardiac, neural and epithelial tissues. Connexin 45 (Cx45), encoded by GJC1, mediates impulse conduction in the atrioventricular node, His bundle, and Purkinje fibers, where disease-linked mutations cause progressive atrioventricular block and familial atrial fibrillation, yet no experimental structure has been reported, and its regulatory mechanism remains undefined. Here, we determine the structural basis of Cx45 gating and Ca2+ regulation using cryo-electron microscopy, mutational analysis, and molecular dynamics simulations. Cryo-EM structures of the apo (2.76 [A]), Ca2+-bound (2.65 [A]), and E41A mutant (3.55 [A]) channel reveal a neck constriction formed by Y45, establishing a steric gate distinct from other connexins. Ca2+ associates with E41, stabilizing the neck via electrostatic remodeling without global conformational change. Together, these data define a dual steric-electrostatic mechanism for Cx45 regulation and provide a structural framework for isoform-specific connexin gating relevant to cardiac physiology and conduction disease.

biochemistry↗

bioRxiv2025

Capsid Restructuring Activates Semi-Conservative dsRNA Transcription in Cystovirus Φ6

Double-stranded (ds)RNA viruses replicate and transcribe their genome within a proteinaceous viral capsid to evade host cell defenses. While Reovirales members use conservative transcription, most dsRNA viruses, including cystoviruses, utilize semi-conservative transcription, where the positive strand of the genome functions as mRNA. Here, we visualize semi-conservative transcription activation in cystovirus {phi}6 double-layered particles using cryogenic electron microscopy. We observe nucleotide-triggered disassembly of the domain-swapped outer capsid layer, subsequent expansion of the inner capsid layer, and stepwise assembly of transcription complexes at the opposing poles of the spooled dsRNA genome. These complexes consist of the viral polymerases embedded into a triskelion formed by the minor protein P7, which we show as essential for continuous transcription. The packaging hexamers proximal to the transcription sites channel the viral mRNA exit. Our results define the complex molecular pathway from the quiescent state to activated semi-conservative transcription.

molecular biology↗

bioRxiv2025

In situ Structure of the Human Gap Junction

Gap junction plaques (GJPs) enable direct intercellular communication and consist of connexin channels arranged into two-dimensional lattices. While structures of purified connexin channels have informed models of gating, they omit key intracellular regions and lack native context. Here, we use cryogenic electron tomography and focused ion beam milling to determine the in situ structure of human connexin-43 (Cx43) GJPs in HEK293 cells at 14 [A] resolution. We discover a previously unresolved structural role for the large C-terminal domain in mediating lateral channel-channel interactions critical for plaque assembly. Coarse-grained molecular dynamics simulations reveal how lipids and cholesterol occupy the space between adjacent connexins. These findings resolve a decades-old question regarding gap junction organization and highlight a mechanistic function for the C-terminal domain, likely regulated by phosphorylation. Our study provides a structural blueprint for understanding how connexin diversity and regulation shape tissue-level communication in health and disease.

cell biology↗

bioRxiv2023

Structure and Interactions of the Endogenous Human Commander Complex

The Commander complex, a 16-protein subunit assembly, plays multiple roles in various intracellular events, including regulation of cell homeostasis, cell cycle, and immune response. The complex is composed of COMMD1-10, CCDC22, CCDC93, DENND10, VPS26C, VPS29, and VPS35L. These proteins are expressed ubiquitously in the human body and have been linked to diseases including Wilsons disease, atherosclerosis, and several cancers. Despite its importance, the structure and molecular functions of the Commander complex are poorly understood. Here, we report the structure and key interactions of the endogenous human Commander complex by cryogenic electron microscopy (cryo-EM) and mass spectrometry-based proteomics. Our results show that the complex is asymmetric, consisting of a stable core of a pseudo-symmetric ring of COMMD proteins 1-10 and a mobile effector consisting of DENND10 and the Retriever sub-complex, constituted by VPS35L, VPS29 and VPS26C. The two halves are scaffolded together by CCDC22 and CCDC93. This study directly confirms the cellular composition of Commander and identifies major interaction interfaces, defining the structure and interaction landscape of the complex. These findings offer new insights into its known roles in endosomal processes and intracellular transport, and uncovers a strong association with cilium assembly, and centrosome and centriole functions. HighlightsO_LIHigh-resolution structure of the Commander complex determined by cryo-EM shows a rigid COMMD1-10 core decorated by mobile effectors DENND10 and the Retriever sub-complex. C_LIO_LIComprehensive interactome analysis uncovers a plethora of novel interactions, implicating the Commander complex in previously unidentified processes, such as cilium assembly, cell cycle, and organelle biogenesis. C_LIO_LIStructural organization and identified interaction partners of the Commander complex provide a basis for further research into its molecular functions, related diseases, and potential therapeutic targets. C_LI eTOC blurbWe provide a comprehensive structural analysis of the endogenous human Commander complex, revealing its functional organization and novel interactions involved in diverse cellular processes. These findings uncover new interaction surfaces and pave the way for further research into the molecular mechanisms of the Commander complex.

biophysics↗

bioRxiv2022

DNA origami directed virus capsid polymorphism

Most known viruses protect their genome by encapsulating it inside a protein capsid. Viral capsids can adopt various geometries, most iconically characterized by icosahedral or helical symmetries. The assembly process of native capsids is highly cooperative and governed by the protein geometry, protein-protein as well as protein-nucleic acid interactions. Importantly, the absolute control over the size and shape of virus capsids would have imminent advantages in the development of new vaccines and delivery systems. However, tools to direct the assembly process in a programmable manner are exceedingly elusive or strictly limited to specific structures. Here, we introduce a modular approach by demonstrating DNA origami directed polymorphism of single protein subunit capsids. We achieve control over the capsid shape, size, and topology by employing user-defined DNA origami nanostructures as binding and assembly platforms for the capsid proteins. Binding assays and single-particle cryo-electron microscopy reconstruction show that the DNA origami nanoshapes are efficiently encapsulated within the capsid. Further, we observe that helical arrangement of hexameric capsomers is the preferred mode of packing, while a negative curvature of the origami structure is not well tolerated. The capsid proteins assemble on DNA origami in single or double layer configurations depending on the applied stoichiometry. In addition, the obtained viral capsid coatings are able to efficiently shield the encapsulated DNA origami from nuclease degradation and prevent the structures from aggregation. Therefore, these findings may in addition find direct implementations in DNA nanotechnology-based bioengineering by paving the way for the next-generation cargo protection and targeting strategies.

bioengineering↗

bioRxiv2022

Mechanistic insights into the activation of lecithin-cholesterol acyltransferase in therapeutic nanodiscs composed of apolipoprotein A-I mimetic peptides and phospholipids

The mechanistic details behind the activation of lecithin-cholesterol acyltransferase (LCAT) by apolipoprotein A-I (apoA-I) and its mimetic peptides are still enigmatic. Resolving the fundamental principles behind the LCAT activation will facilitate the design of advanced HDL-mimetic therapeutic nanodiscs for LCAT deficiencies and coronary heart disease, and for several targeted drug delivery applications. Here, we have combined coarse-grained molecular dynamics simulations with complementary experiments to gain mechanistic insight into how apoA-I mimetic peptide 22A and its variants attune LCAT activity in peptide-lipid nanodiscs. Results highlight that peptide 22A forms transient antiparallel dimers in the rim of nanodiscs. The dimerization tendency considerably decreases with the removal of C-terminal lysine K22, which has also been shown to reduce the cholesterol esterification activity of LCAT. In addition, our simulations revealed that LCAT prefers to localize to the rim of nanodiscs in a manner that shields the membrane-binding domain (MBD), A-A, and the lid amino acids from the water phase, following the previous experimental evidence. Meanwhile, the location and conformation of LCAT in the rim of nanodisc are spatially more restricted when the active site covering lid of LCAT is in the open form. The average location and spatial dimensions of LCAT in its open form were highly compatible with the electron microscopy images. All peptide 22A variants studied here had a specific interaction site in the open LCAT structure flanked by the lid and MBD domain. The bound peptides showed different tendencies to form antiparallel dimers and, interestingly, the temporal binding site occupancies of the peptide variants affected their in vitro ability to promote LCAT-mediated cholesterol esterification.

biophysics↗

bioRxiv2020

MANF regulates unfolded protein response and neuronal survival through its ER-located receptor IRE1α

Mesencephalic astrocyte-derived neurotrophic factor (MANF) is an endoplasmic reticulum (ER)-located protein with cytoprotective effects in numerous cell types in vitro and in models of neurodegeneration and diabetes in vivo. So far, the exact mode of its action has remained elusive and plasma membrane or ER-located receptors of MANF have not been identified. We have found that MANF can directly interact with transmembrane unfolded protein response (UPR) receptor IRE1 and compete with the major ER chaperone BiP (GRP78) for the interaction with IRE1. With lower affinities MANF can also interact with other UPR receptors, PERK and ATF6. Using molecular modeling and mutagenesis analysis, we have identified the exact structural MANF regions involved in its binding to the luminal domain of IRE1. MANF attenuates UPR signaling by decreasing IRE1 oligomerization and IRE1 phosphorylation. MANF mutant deficient in IRE1 binding cannot regulate IRE1 oligomerization and fails to protect neurons from ER stress induced death. Importantly, we found that MANF-IRE1 interaction is also crucial for the survival promoting action of MANF for dopamine neurons in an animal model of Parkinsons disease. Our data reveal a novel mechanism of IRE1 regulation during ER stress and demonstrate the intracellular mode of action of MANF as a modulator of UPR and neuronal cell survival through the direct interaction with IRE1 and regulation of its activity. Furthermore, our data explain why MANF in contrast to other growth factors has no effects on naive cells and rescues only ER stressed or injured cells.

cell biology↗

bioRxiv2018

Atomic view into Plasmodium actin polymerization, ATP hydrolysis, and phosphate release

Plasmodium actins form very short filaments and have a non-canonical link between ATP hydrolysis and polymerization. Long filaments are detrimental to the parasites, but the structural factors constraining Plasmodium microfilament lengths are currently unknown. Using high-resolution crystallography, we show that magnesium binding activates the Plasmodium actin I monomer before polymerization by a slight flattening, which is reversed upon phosphate release. A coordinated potassium ion resides in the active site during hydrolysis and leaves together with the phosphate, a process governed by the position of the Arg178/Asp180-containing A-loop. Asp180 interacts with either Lys270 or His74, depending on protonation, while Arg178 links the inner and outer domains. Hence, the A-loop is a switch between stable and non-stable filament conformations. Our data provide a comprehensive model for polymerization, phosphate release, and the inherent instability of parasite microfilaments.

biochemistry↗