Biology subjects

Reichow, S. L.

Publications and source records attributed to Reichow, S. L..

14 recordsLinked to original sources

bioRxiv2026

Distinct modes of N-terminal remodeling through a conserved inhibitor pocket in connexin-46/50

Selective pharmacological control of gap junction communication remains challenging because existing small-molecule connexin inhibitors act across multiple isoforms. The structural basis of this broad activity remains incompletely understood. Here, we used single-particle cryo-electron microscopy to examine native connexin-46/50 gap junction channels treated with two commonly used and chemically distinct inhibitors, 2-aminoethoxydiphenyl borate (2-APB) and carbenoxolone (CBX). Both treatments perturb the hydrophobic N-terminal (NT)-docking groove, but produce distinct conformational ensembles. 2-APB yields ordered destabilized-open and gated NT states resembling conformations observed during physiological pH- and Ca2+-dependent gating, with ligand- and lipid-associated densities accompanying the gated state. In contrast, CBX-compatible density occupies the same groove and is associated with loss of an ordered NT conformation, consistent with steric competition and altered physicochemical complementarity at the NT-docking site. Comparison with inhibitor-treated connexin-32 and connexin-36 structures identifies this region as a structurally conserved, pharmacologically sensitive pocket. These findings reveal how chemically diverse inhibitors perturb a conserved element of the native gating machinery while producing divergent NT-gating ensembles, providing a structural rationale for broad cross-isoform activity and identifying sequence-variable pocket margins that may support more selective modulation.

biophysics↗

bioRxiv2026

Multi-ion permeation and dynamic conductance modulation in connexin gap junction channels

Gap junction channels formed by connexins mediate direct intercellular communication and are essential for electrical signaling and tissue homeostasis. Despite their large, solvent-accessible pores, connexin channels exhibit distinct conductance, selectivity, and rectification properties, but the molecular mechanisms underlying these behaviors remain incompletely understood. Here, we performed [~]67 s of all-atom computational electrophysiology simulations of connexin-46 (Cx46), connexin-50 (Cx50), and heterotypic Cx46/50 gap junction channels based on high-resolution open-state structures, enabling characterization of both ion permeation and long-timescale channel dynamics. Simulations reveal a multi-ion, multi-pathway permeation mechanism governed by isoform-specific energetic barriers and transient ion coordination sites that shape conductance and selectivity. In heterotypic Cx46/50 channels, asymmetric energetic landscapes establish a mechanistic basis for rectification. Unexpectedly, the microsecond-timescale simulations further revealed dynamic interactions between the intracellular loop (ICL) region and N-terminal domain (NT) that transiently constrict the pore and attenuate ionic currents. These findings suggest that the open-state comprises an ensemble of rapidly interconverting conductance microstates rather than a single static conformation, providing structural information of potential mechanistic importance beyond what has been learned from cryo-EM studies. Together, our results provide a mechanistic basis for ion permeation and current modulation in gap junction channels and highlight the importance of long-timescale protein dynamics in shaping intercellular communication.

biophysics↗

bioRxiv2026

CaMKII T286 autophosphorylation is not propagated at basal Ca2+ levels and is required only for the induction phase of LTP

The Ca2+/calmodulin-dependent protein kinase II (CaMKII) and its autophosphorylation at T286 (P-T286) mediates long-term potentiation (LTP) of synaptic strength. CaMKII forms stable 12-meric holoenzymes, but exchange of its subunits is proposed to propagate the P-T286 state beyond dephosphorylation or degradation of individual subunits. However, a recent study questioned subunit exchange and instead proposed P-T286 propagation by trans-holoenzyme phosphorylation. We show here that both subunit exchange and trans-holoenzyme P-T286 can occur (although cis-holoenzyme P-T286 is much preferred), but that neither mechanism propagates P-T286 at basal cellular Ca2+ levels (50-100 nM) in absence of further Ca2+-stimuli. Functionally, we show that after theta-burst stimulation (TBS), P-T286 is required only during the first minutes of LTP induction that prime for subsequent LTP expression, but neither for LTP expression itself nor for LTP maintenance. These results indicate that P-T286 of CaMKII does not self-perpetuate and does not mediate storage but computation of synaptic information.

neuroscience↗

bioRxiv2026

Lipids intercalate and mediate multi-channel assemblies of connexin-46/50 gap junctions

Gap junction channels enable direct electrical and metabolic exchange between adjacent cells and tissues, where they organize into dense plaques containing tens to thousands of channels. Although plaque formation is known to modulate junctional conductance, the structural basis for channel-channel organization within a membrane environment remains poorly defined. Here, we reconstitute native lens connexin-46/50 (Cx46/50) gap junction channels into MSP-based lipid nanodiscs that incorporate multiple channels to create miniature plaque-like complexes suitable for single-particle cryo-electron microscopy (cryo-EM). We determine high-resolution structures of dual-channel assemblies in two distinct configurations and find no ordered protein-protein contacts across the interface. Instead, the inter-channel space is occupied by ordered lipid density, indicating that channel packing in these assemblies is lipid mediated. These channel-channel interfaces stabilize discrete lipid populations, including an interstitial lipid intercalated between subunits and positioned near the N-terminal gating domain, suggesting a route by which channel organization could promote lipid occupancy near the pore. Additionally, we leverage this dataset to refine the Cx46/50 single-channel structure to 1.8 [A] resolution, revealing exceptional chemical detail of the pore-lining landscape in the stabilized open-state. Together, these results define principles of lipid-mediated multi-channel organization and suggest how plaque-like packing may tune gap junction function through specific lipid interactions.

biophysics↗

bioRxiv2025

Reversible lipid mediated pH-gating of connexin-46/50 by cryo-EM

Gap junctions, formed by connexin proteins, establish direct electrical and metabolic coupling between cells, enabling coordinated tissue responses. These channels universally respond to intracellular pH changes, closing under acidic conditions to limit the spread of cytotoxic signals during cellular stress, such as ischemia. Using cryo-electron microscopy (cryo-EM), we uncover insights into the structural mechanism of pH-gating in native lens connexin-46/50 (Cx46/50) gap junctions. Mild acidification drives lipid infiltration into the channel pore, displacing the N-terminal (NT) domain and stabilizing pore closure. Lipid involvement is both essential and fully reversible, with structural transitions involving an ensemble of gated-states formed through non-cooperative NT domain movement as well as minor populations of a distinct destabilized open-state. These findings provide molecular insights into pH-gating dynamics, illustrating how structural changes may regulate gap junction function under cellular stress and linking Cx46/50 dysregulation to age-related cataract formation.

biophysics↗

bioRxiv2025

Calcium induced N-terminal gating and pore collapse in connexin-46/50 gap junctions

Gap junctions facilitate electrical and metabolic coupling essential for tissue function. Under ischemic conditions (e.g., heart attack or stroke), elevated intracellular calcium (Ca2+) levels uncouple these cell-to-cell communication pathways to protect healthy cells from cytotoxic signals. Using single-particle cryo-EM, we elucidate details of the Ca2+-induced gating mechanism of native connexin-46/50 (Cx46/50) gap junctions. The resolved structures reveal Ca2+ binding sites within the channel pore that alter the chemical environment of the permeation pathway and induce diverse occluded and gated states through N-terminal domain remodeling. Moreover, subunit rearrangements lead to pore collapse, enabling steric blockade by the N-terminal domains, reminiscent of the "iris model" of gating proposed over four decades ago. These findings unify and expand key elements of previous gating models, providing mechanistic insights into how Ca2+ signaling regulates gap junction uncoupling and broader implications for understanding cell stress responses and tissue protection.

biophysics↗

bioRxiv2024

Mechanism of small heat shock protein client sequestration and induced polydispersity

Small heat shock proteins (sHSPs) act as first responders during cellular stress by recognizing and sequestering destabilized proteins (clients), preventing their aggregation and facilitating downstream refolding or degradation1-3. This chaperone function is critically important to proteostasis, conserved across all kingdoms of life, and associated with various protein misfolding diseases in humans4,5. Mechanistic insights into how sHSPs sequester destabilized clients have been limited due to the extreme molecular plasticity and client-induced polydispersity of sHSP/client complexes6-8. Here, we present high-resolution cryo-EM structures of the sHSP from Methanocaldococcus jannaschii (mjHSP16.5) in both the apo-state and in an ensemble of client-bound states. The ensemble not only reveals key molecular mechanisms by which sHSPs respond to and sequester client proteins, but also provides insights into the cooperative nature of chaperone-client interactions. Engagement with destabilized client induces a polarization of stability across the mjHSP16.5 scaffold, proposed to facilitate higher-order assembly and enhance client sequestration capacity. Some higher-order sHSP oligomers appear to form through simple insertion of dimeric subunits into new geometrical features, while other higher-order states suggest multiple sHSP/client assembly pathways. Together, these results provide long-sought insights into the chaperone function of sHSPs and highlight the relationship between polydispersity and client sequestration under stress conditions.

biophysics↗

bioRxiv2024

Dynamic fibrillar assembly of αB-crystallin induced by perturbation of the conserved NT-IXI motif resolved by cryo-EM.

B-crystallin is an archetypical member of the small heat-shock proteins (sHSPs) vital for cellular proteostasis and mitigating protein misfolding diseases. Gaining insights into the principles defining their molecular organization and chaperone function have been hindered by intrinsic dynamic properties and limited high-resolution structural analysis. To disentangle the mechanistic underpinnings of these dynamical properties, we mutated a conserved IXI-motif located within the N-terminal (NT) domain of human B-crystallin. This resulted in a profound structural transformation, from highly polydispersed caged-like native assemblies into a comparatively well-ordered helical fibril state amenable to high-resolution cryo-EM analysis. The reversible nature of the induced fibrils facilitated interrogation of functional effects due to perturbation of the NT-IXI motif in both the native-like oligomer and fibril states. Together, our investigations unveiled several features thought to be key mechanistic attributes to sHSPs and point to a critical significance of the NT-IXI motif in B-crystallin assembly, dynamics and chaperone activity.

biophysics↗

bioRxiv2023

The α-crystallin chaperones undergo a quasi-ordered co-aggregation process in response to saturating client interaction

Small heat shock proteins (sHSPs) are ATP-independent chaperones vital to cellular proteostasis, preventing protein aggregation events linked to various human diseases including cataract. The -crystallins, A-crystallin (Ac) and B-crystallin (Bc), represent archetypal sHSPs that exhibit complex polydispersed oligomeric assemblies and rapid subunit exchange dynamics. Yet, our understanding of how this plasticity contributes to chaperone function remains poorly understood. This study investigates structural changes in Ac and Bc during client sequestration under varying degree of chaperone saturation. Using biochemical and biophysical analyses combined with single-particle electron microscopy (EM), we examined Ac and Bc in their apo-states and at various stages of client-induced co-aggregation, using lysozyme as a model client. Quantitative single-particle analysis unveiled a continuous spectrum of oligomeric states formed during the co-aggregation process, marked by significant client-triggered expansion and quasi-ordered elongation of the sHSP scaffold. These structural modifications culminated in an apparent amorphous collapse of chaperone-client complexes, resulting in the creation of co-aggregates capable of scattering visible light. Intriguingly, these co-aggregates maintain internal morphological features of highly elongated sHSP scaffolding with striking resemblance to polymeric -crystallin species isolated from aged lens tissue. This mechanism appears consistent across both Ac and Bc, albeit with varying degrees of susceptibility to client-induced co-aggregation. Importantly, our findings suggest that client-induced co-aggregation follows a distinctive mechanistic and quasi-ordered trajectory, distinct from a purely amorphous process. These insights reshape our understanding of the physiological and pathophysiological co-aggregation processes of sHSPs, carrying potential implications for a pathway toward cataract formation.

biophysics↗

bioRxiv2021

Molecular mechanisms underlying enhanced hemichannel function of a cataract-associated Cx50 mutant

Connexin-50 (Cx50) is among the most frequently mutated genes associated with congenital cataracts. While most of these disease-linked variants cause loss-of-function due to misfolding or aberrant trafficking, others directly alter channel properties. The mechanistic bases for such functional defects are mostly unknown. We investigated the functional and structural properties of a cataract-linked mutant, Cx50T39R (T39R), in the Xenopus oocyte system. T39R exhibited greatly enhanced hemichannel currents with altered voltage-gating properties compared to Cx50 and induced cell death. Co-expression of mutant T39R with wild-type Cx50 (to mimic the heterozygous state) resulted in hemichannel currents whose properties were indistinguishable from those induced by T39R alone, suggesting that the mutant had a dominant effect. Co-expression with Cx46 also produced channels with altered voltage-gating properties, particularly at negative potentials. All-atom molecular dynamics simulations indicate that the R39 substitution can form multiple electrostatic salt-bridge interactions between neighboring subunits that could stabilize the open-state conformation of the N-terminal domain, while also neutralizing the voltage-sensing residue D3 as well as residue E42 which participates in loop-gating. Together, these results suggest T39R acts as a dominant gain-of-function mutation that produces leaky hemichannels that may cause cytotoxicity in the lens and lead to development of cataracts. Statement of significanceWe investigated the functional and structural properties of a cataract-linked mutant, Cx50T39R (T39R), in the Xenopus oocyte system and showed that T39R exhibited greatly enhanced hemichannel currents with altered voltage-gating properties compared to Cx50 and induced cell death. Consistent with our experimental findings, all-atom equilibrium state molecular dynamics (MD) simulations of T39R show that R39 stabilized the open-state configuration of the N-terminal (NT) domain from an adjacent subunit. These results suggest that T39R causes disease by preventing the hemichannels from closing when present in the plasma membrane in the undocked state and provide an atomistic rationalization for the Cx50 disease-linked phenotype. They also expand our understanding of how connexin hemichannel channel gating is controlled.

physiology↗

bioRxiv2021

Conserved and divergent features of neuronal CaMKII holoenzyme structure, function, and high-order assembly

Neuronal CaMKII holoenzymes (- and {beta}-isoforms) enable molecular signal computation underlying learning and memory, but also mediate excitotoxic neuronal death. Here, we provide a comparative analysis of these signaling devices, using single particle EM in combination with biochemical and live-cell imaging studies. In the basal state, both isoforms assembled mainly as 12-mers (but also 14-mers, and even 16-mers for the {beta}-isoform). CaMKII and {beta}-isoforms adopted an ensemble of extended activatable states (with average radius of 12.6 versus 16.8 nm, respectively), characterized by multiple transient intra- and inter-holoenzyme interactions associated with distinct functional properties. The extended state of CaMKII{beta} allowed EM analysis to directly resolve intra-holoenzyme kinase-domain dimers that could enable the cooperative activation mechanism by calmodulin, which was found for both isoforms. Surprisingly, high-order CaMKII clustering mediated by inter-holoenzyme kinase-domain dimerization was reduced for the {beta} isoform for both basal and excitotoxicity-induced clusters, both in vitro and in neurons.

molecular biology↗

bioRxiv2020

Connexin 46 and connexin 50 gap junction channel open stability and unitary conductance are shaped by structural and dynamic features of their N-terminal domains

The connexins form intercellular communication channels, known as gap junctions (GJs), that facilitate diverse physiological roles in vertebrate species, ranging from electrical coupling and long-range chemical signaling, to coordinating development and nutrient exchange. GJs formed by different connexins are expressed throughout the body and harbor unique channel properties that have not been fully defined mechanistically. Recent structural studies have implicated the amino-terminal (NT) domain as contributing to isoform-specific functional differences that exist between the lens connexins, Cx50 and Cx46. To better understand the structural and functional differences in the two closely related, yet functionally distinct GJs, we constructed models corresponding to CryoEM-based structures of the wildtype Cx50 and Cx46 GJs, NT domain swapped chimeras (Cx46-50NT and Cx50-46NT), and point variants at the 9th residue (Cx46-R9N and Cx50-N9R) for comparative MD simulation and electrophysiology studies. All of these constructs formed functional GJ channels, except Cx46-50NT, which correlated with increased dynamical behavior (instability) of the NT domain observed by MD simulation. Single channel conductance (γj) also correlated well with free-energy landscapes predicted by MD, where γj of Cx46-R9N was increased from Cx46 and the γjs of Cx50-46NT and Cx50-N9R was decreased from Cx50, but to a surprisingly greater degree. Additionally, we observed significant effects on transjunctional voltage-dependent gating (Vj-gating) and open-state dwell times induced by the designed NT domain variants. Together, these studies indicate that the NT domains of Cx46 and Cx50 play an important role in defining channel properties related to open-state stability and single channel conductance.Competing Interest StatementThe authors have declared no competing interest.AbbreviationsGJgap junctionGjgap junctional coupling conductanceγjsingle gap junction channel conductanceIjmacroscopic transjunctional currentijsingle gap junction channel currentCx46sheep connexin 46Cx50sheep connexin 50Vjtransjunctional voltageMDmolecular dynamicsr.m.s.d.root mean square deviationr.m.s.f.root mean square fluctuationPMFpotential of mean forceView Full Text

biophysics↗

bioRxiv2020

Automated and statistically corrected identification of flexible multivalent IDP-bound assemblies in electron micrographs

Multivalent intrinsically disordered protein (IDP) complexes are prevalent in biology and control diverse cellular functions, including tuning levels of transcription, coordinating cell-signaling events, and regulating the assembly and disassembly of complex macromolecular architectures. These systems pose a significant challenge to structural investigation, due to the continuum dynamics imparted by the IDP and compositional heterogeneity resulting from characteristic low-affinity interactions. Traditional single-particle electron microscopy (EM) is a powerful tool for visualizing IDP complexes. However, the IDPs themselves are typically "invisible" by EM, undermining methods of image analysis and structural interpretation. To overcome these challenges, we developed a pipeline for automated analysis of common beads-on-a-string type of assemblies, composed of IDPs bound at multivalent sites to the ubiquitous ~20 kDa cross-linking hub protein LC8. This approach quantifies conformational and compositional heterogeneity on a single-particle basis, and statistically corrects spurious observations arising from random proximity of bound and unbound LC8. After careful validation of the methodology, the approach was applied to the nuclear pore IDP Nup159 and the transcription factor ASCIZ. The analysis unveiled significant compositional and conformational diversity in both systems that could not be obtained from traditional single particle EM class-averaging strategies, and shed new light on how these architectural properties contribute to their physiological roles in supramolecular assembly and transcriptional regulation. Ultimately, we expect that this approach may be adopted to many other intrinsically disordered systems that have evaded traditional methods of structural characterization. Significance StatementIntrinsically disordered proteins (IDPs) or protein regions (IDRs) represent >30% of the human proteome, but mechanistically remain some of the most poorly understood classes of proteins in biology. This dearth in understanding stems from these very same intrinsic and dynamic properties, which make them difficult targets for quantitative and structural characterization. Here, we present an automated approach for extracting quantitative descriptions of conformational and compositional heterogeneity present in a common beads-on-a-string type of multivalent IDP system from single-particle images in electron micrographs. This promising approach may be adopted to many other intrinsically disordered systems that have evaded traditional ensemble methods of characterization.

biophysics↗

bioRxiv2020

Connexin-46/50 in a dynamic lipid environment resolved by CryoEM at 1.9 A

Gap junctions establish direct pathways for connected cells and tissues to transfer metabolic and electrical messages1. The local lipid environment is known to affect the structure, stability and intercellular channel activity of gap junctions2-5; however, the molecular basis for these effects remains unknown. To gain insight toward how gap junctions interact with their local membrane environment, we used lipid nanodisc technology to incorporate native connexin-46/50 (Cx46/50) intercellular channels into a dual lipid membrane system, closely mimicking a native cell-to-cell junction. Structural characterization of Cx46/50 lipid-embedded channels by single particle CryoEM revealed a lipid-induced stabilization to the channel, resulting in a 3D reconstruction at 1.9 [A] resolution. Together with all-atom molecular dynamics (MD) simulations and 3D heterogeneity analysis of the ensemble CryoEM data, it is shown that Cx46/50 in turn imparts long-range stabilization to the dynamic local lipid environment that is specific to the extracellular lipid leaflet of the two opposed membranes. In addition, nearly 400 water molecules are resolved in the CryoEM map, localized throughout the intercellular permeation pathway and contributing to the channel architecture. These results illustrate how the aqueous-lipid environment is integrated with the architectural stability, structure and function of gap junction communication channels, and demonstrates the ability of CryoEM to effectively characterize dynamical protein-lipid interactions.

biophysics↗