Biology subjects

Huiskonen, J. T.

Publications and source records attributed to Huiskonen, J. T..

18 recordsLinked to original sources

bioRxiv2026

AAV2 Crosslinks Actin Filaments: Implications for AAV Gene Therapy Vector Design

Adeno-associated virus (AAV) capsids are important gene therapy vectors, allowing for the one-time treatment of monogenetic disorders, with durable gene expression lasting for years. However, despite the clinical success of AAV usage, low transduction efficiencies require high dosage to achieve therapeutic efficacy, resulting in prohibitive costs and rare, but life-threatening immune responses. One key knowledge gap is how the capsids traffic to the nucleus following endocytosis. Here, we identify a direct interaction between AAV2 and actin filaments. Our results show that AAV2 bundles multiple actin filaments in a highly periodic hexagonal lattice. Therefore, we propose that AAV2 is the founding member of a new class of actin binding proteins, the actin bundling viruses (ABVs). Using cryogenic electron microscopy, we determined the structure of AAV2-actin filaments, identified the interaction interfaces, and engineered capsid variants that no longer bundle actin. Together, our results suggest that the AAV2-actin interaction may be responsible for trapping capsids and mediating peri-nuclear accumulation. Overall, this interaction expands the current understanding of AAV2 biology and offers new directions for capsid engineering.

biochemistry↗

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↗

bioRxiv2024

Structural characterization of antibody-responses from Zolgensma treatment provides the blueprint for the engineering of an AAV capsid suitable for redosing

Monoclonal antibodies (mAbs) are useful tools to dissect the neutralizing antibody response against the adeno-associated virus (AAV) capsids used as gene therapy delivery vectors. This study structurally characterizes the interactions of 21 human-derived antibodies from patients treated with the AAV9 vector, Zolgensma(R), utilizing high-resolution cryo-electron microscopy. The majority of the bound antibodies do not conform to the icosahedral symmetry of the capsid, thus requiring localized reconstructions. These complex structures provide unprecedented details of the mAbs binding interfaces, with some antibodies inducing structural perturbations of the capsid upon binding. Key surface capsid amino acid residues were identified facilitating the design of capsid variants with an antibody escape phenotype, with the potential to expand the patient cohort treatable with AAV9 vectors to include those that were previously excluded due to their pre-existing neutralizing antibodies, and possibly also to those requiring redosing.

immunology↗

bioRxiv2023

Snapshots from Cryo-ET of active SARS-CoV-2 virions

Understanding the molecular properties of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is crucial for tackling future outbreaks. Current structural knowledge of the trimeric spike protein relies on truncated recombinant proteins and/or inactivated full-length forms, which may suffer from overstabilization. Here, we apply cryo-electron tomography (cryo-ET) at a Biosafety Level 3 facility to study the virus structure in its native, active state. The virus particles exhibit variable shapes and sizes with diffusible spikes, with the majority in typical prefusion conformations. Notably, we identified unprecedented, a transient open-trimer prefusion states, revealing a hidden flexibility with opened S1 conformation. Subtomogram averaging of the prefusion spikes indicates a loosely packed trimeric architecture that may facilitate the formation of open-trimer state. A cryo-EM map of recombinant Omicron BA.2.75 spike protein further confirms the presence of this loosely packed trimer as a minor conformational state. The observed dynamics uncover conserved cryptic regions that can be targeted for broadly effective vaccines. Structural analysis of active viruses profoundly impacts our understanding of the overlooked fusion mechanism and vaccine, antibody/drug design.

microbiology↗

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↗

bioRxiv2023

Global analysis of aging-related protein structural changes uncovers enzyme polymerization-based control of longevity

Aging is associated with progressive phenotypic changes over time. Virtually all cellular phenotypes are produced by proteins and structural alterations in proteins can lead to age-related diseases. Nonetheless, comprehensive knowledge of proteins undergoing structural-functional changes during cellular aging and their contribution to age-related phenotypes is lacking. Here, we conducted proteome-wide analysis of early age-related protein structural changes in budding yeast using limited proteolysis-mass spectrometry. The results, compiled in online ProtAge-catalog, unravelled age-related functional changes in regulators of translation, protein folding and amino acid metabolism. Mechanistically, we found that folded glutamate synthase Glt1 polymerizes into supramolecular self-assemblies during aging causing breakdown of cellular amino acid homeostasis. Inhibiting Glt1 polymerization by mutating the polymerization interface restored amino acid levels in aged cells, attenuated mitochondrial dysfunction and led to life span extension. Altogether, this comprehensive map of protein structural changes enables identifying novel mechanisms of age-related phenotypes and offers opportunities for their reversal.

cell biology↗

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

Molecular view of ER membrane remodeling by the Sec61/TRAP translocon

Protein translocation across the endoplasmic reticulum (ER) membrane is an essential initial step in protein entry into the secretory pathway. The conserved Sec61 protein translocon facilitates polypeptide translocation and coordinates cotranslational polypeptide processing events. In cells, the majority of Sec61 is stably associated with a heterotetrameric membrane protein complex, the translocon associated protein complex (TRAP), yet the mechanism by which TRAP assists in polypeptide translocation or cotranslational modifications such as N-glycosylation remains unknown. Here, we demonstrate the structure of the core Sec61/TRAP complex bound to a mammalian ribosome by Cryo-EM. The interactions with ribosome anchor the Sec61/TRAP complex in a conformation that renders the ER membrane locally thinner by significantly curving its the lumenal leaflet. We propose a model for how TRAP stabilizes the ribosome exit tunnel to assist nascent polypeptide insertion through Sec61 and provides a ratcheting mechanism into the ER lumen by direct polypeptide interactions.

biochemistry↗

bioRxiv2022

Nanobody engineering for SARS-CoV-2 neutralization and detection

In response to the ongoing SARS-CoV-2 pandemic, the quest for coronavirus inhibitors has inspired research on a variety of small proteins beyond conventional antibodies, including robust single-domain antibody fragments, nanobodies. Here, we explore the potential of nanobody engineering in the development of antivirals and diagnostic tools. Through fusion of nanobody domains that target distinct binding sites, we engineered multimodular nanobody constructs that neutralize wild-type SARS-CoV-2 and the Alpha and Delta variants with high potency, with IC50 values up to 50 pM. However, we observed a limitation in the efficacy of multimodular nanobodies against the Beta (B.1.351) and Omicron variants (B.1.1.529), underlining the importance of accounting for viral evolution in the design of biologics. To further explore the applications of nanobody engineering in outbreak management, we present a novel detection assay, based on fusions of nanobodies with fragments of NanoLuc luciferase that can detect sub-nanomolar quantities of the SARS-CoV-2 spike protein in a single step. Our work showcases the potential of nanobody engineering to combat emerging infectious disease.

microbiology↗

bioRxiv2022

Signal peptide mimicry primes Sec61 for client-selective inhibition

Preventing the biogenesis of disease-relevant proteins is an attractive therapeutic strategy, but attempts to target essential protein biogenesis factors have been hampered by excessive toxicity. Here, we describe KZR-8445, a cyclic depsipeptide that targets the Sec61 translocon and selectively disrupts secretory and membrane protein biogenesis in a signal peptide-dependent manner. KZR-8445 potently inhibits the secretion of proinflammatory cytokines in primary immune cells and is highly efficacious in a mouse model of rheumatoid arthritis. A cryo-EM structure reveals that KZR-8445 occupies the fully opened Se61 lateral gate and blocks access to the lumenal plug domain. KZR-8445 binding stabilizes the lateral gate helices in a manner that traps select signal peptides in the Sec61 channel and prevents their movement into the lipid bilayer. Our results establish a framework for the structure-guided discovery of novel therapeutics that selectively modulate Sec61-mediated protein biogenesis.

biochemistry↗

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↗

bioRxiv2022

New Vista into Origins of Viruses from a Prototypic ssDNA Phage

Viruses play a central role in all ecological niches; the origin of viruses, however, remains an open question. While phylogenetic analysis of distantly related viruses is hampered by a lack of detectable sequence similarity, structural biology can reveal conserved capsid protein structures that facilitate the study of distant evolutionary relationships. Here, we characterize the lipid-containing ssDNA temperate bacteriophage {Phi}CjT23, which is infecting Flavobacterium sp. (Bacteroidetes). We further detected {Phi}CjT23-like sequences in the genome of strains belonging to several Flavobacterium species. The virion structure determined by cryogenic electron microscopy revealed similarities to members of the viral kingdom Bamfordvirae that currently consists solely of dsDNA viruses. Common to these viruses, infecting hosts from all domains of life, is a major capsid protein composed of two upright {beta}-sandwiches. The minimalistic structure of {Phi}CjT23 suggests that this phage serves as a model for the last common ancestor between ssDNA and dsDNA viruses in the Bamfordvirae. Both {Phi}CjT23 and the related phage FLiP infect Flavobacterium species found in several environments, suggesting that these types of viruses have a global distribution and shared evolutionary origin. Detailed comparisons to related, more complex viruses not only expand our knowledge about this group of viruses but also provide a rare glimpse into early virus evolution.

microbiology↗

bioRxiv2022

Structural basis underlying specific biochemical activities of non-muscle tropomyosin isoforms

The actin cytoskeleton is critical for cell migration, morphogenesis, endocytosis, organelle dynamics, and cytokinesis. To support diverse cellular processes, actin filaments form a variety of structures with specific architectures and dynamic properties. Key proteins specifying actin filaments are tropomyosins. Non-muscle cells express several functionally non-redundant tropomyosin isoforms, which differentially control the interactions of other proteins, including myosins and ADF/cofilin, with actin filaments. However, the underlying molecular mechanisms have remained elusive. By determining the cryogenic electron microscopy structures of actin filaments decorated by two functionally distinct non-muscle tropomyosin isoforms, Tpm1.6 and Tpm3.2, we reveal that actin filament conformation remains unaffected upon binding. However, Tpm1.6 and Tpm3.2 follow different paths along the major groove of the actin filament, providing an explanation for their incapability to co-polymerize on actin filaments. The structures and biochemical work also elucidate the molecular basis underlying specific roles of Tpm1.6 and Tpm3.2 in myosin II activation and protecting actin filaments from ADF/cofilin-catalysed severing.

biochemistry↗

bioRxiv2021

Intranasal inhibitor blocks Omicron and other variants of SARS-CoV-2

The recent emergence of novel SARS-CoV-2 variants capable of efficiently escaping neutralizing antibodies emphasizes the need for prophylactic strategies to complement vaccination in fighting the COVID-19 pandemic. Nasal epithelium is rich in the ACE2 receptor and important for SARS-CoV-2 transmission by supporting early viral replication before seeding to the lung1. Intranasal administration of SARS-CoV-2 neutralizing antibodies or antibody fragments has shown encouraging potential as a protective measure in animal models2-5. However, there remains a dire need for SARS-CoV-2 blocking agents that are less vulnerable to mutational variation in the neutralization epitopes of the viral spike glycoprotein and more economical to produce in large scale. Here we describe TriSb92, a highly manufacturable and extremely stable trimeric human SH3 domain-derived antibody mimetic targeted against a conserved region in the receptor-binding domain of spike. TriSb92 potently neutralizes SARS-CoV-2 and its variants of concern, including Omicron BA.5 as well as the latest and most immunoevasive variants like BF.7, XBB, and BQ.1.1. Intranasal administration of a modest dose of TriSb92 (5 or 50 micrograms) as early as eight hours before a challenge with SARS-CoV-2 efficiently protected mice from infection, and was still effective even when given 4 h after the viral challenge. The target epitope of TriSb92 was defined by cryo-EM, which revealed triggering of a conformational shift in the spike trimer rather than competition for ACE2 binding as the molecular basis of its strong inhibitory action. The high potency and robust biochemical properties of TriSb92 together with the remarkable resistance of its inhibitory action against viral sequence evolution suggest that TriSb92 could be useful as a nasal spray for protecting susceptible individuals from SARS-CoV-2 infection.

microbiology↗

bioRxiv2020

Molecular rationale for hantavirus neutralization by a reservoir host-derived monoclonal antibody

The intricate lattice of Gn and Gc glycoprotein spike complexes at the surface of hantaviruses facilitates host-cell entry and is the primary target of the neutralizing antibody-mediated immune response. Here, through study of a neutralizing monoclonal antibody (mAb 4G2) generated in a bank vole reservoir host following infection with Puumala virus (PUUV), we provide molecular-level insights into how antibody-mediated targeting of the hantaviral glycoprotein lattice effectively neutralizes the virus. Crystallographic analysis reveals that mAb 4G2 binds to a multi-domain site on Gc in the pre-fusion state, and that Fab binding is incompatible with the conformational changes of the Gc that are required for host cell entry. Cryo-electron microscopy of PUUV-like particles treated with Fab 4G2 demonstrates that the antibody binds to monomeric Gc at breaks in the Gn-Gc lattice, highlighting the immunological accessibility of Gc monomers on the mature hantavirus surface and the plastic nature of the higher-order lattice assembly. This work provides a structure-based blueprint for rationalizing antibody-mediated targeting of hantaviruses.

microbiology↗

bioRxiv2018

The structural basis of lipid scrambling and inactivation in the endoplasmic reticulum scramblase TMEM16K

Membranes in cells have defined distributions of lipids in each leaflet, controlled by lipid scramblases and flip/floppases. However, for some intracellular membranes such as the endoplasmic reticulum the scramblases have not been identified. Members of the TMEM16 family have either lipid scramblase and ion channel activity, or specific chloride channel activity. Although TMEM16K is widely distributed and associated with the neurological disorder autosomal recessive spinocerebellar ataxia type 10 (SCAR10), its location in cells, function and structure are largely uncharacterised. Here we show that TMEM16K is an ER-resident calcium-regulated lipid scramblase. Our crystal structures of TMEM16K show a scramblase fold, with an open lipid transporting groove. Additional structures solved by cryo-EM reveal extensive conformational changes extending from the cytoplasmic to the ER side of the membrane, giving a state with a closed lipid permeation pathway. Molecular dynamics simulations showed that the open-groove conformation is necessary for scramblase activity. Our results suggest mechanisms by which missense variants of TMEM16K could cause SCAR10 ataxia, providing new hypotheses to explore for therapy.

biochemistry↗