Structure of native lens connexin 46/50 intercellular channels by cryo-EM.

Gap junctions establish direct pathways for cell-to-cell communication through the assembly of twelve connexin subunits that form intercellular channels connecting neighbouring cells. Co-assembly of different connexin isoforms produces channels with unique properties and enables communication across cell types. Here we used single-particle cryo-electron microscopy to investigate the structural basis of connexin co-assembly in native lens gap junction channels composed of connexin 46 and connexin 50 (Cx46/50). We provide the first comparative analysis to connexin 26 (Cx26), which-together with computational studies-elucidates key energetic features governing gap junction permselectivity. Cx46/50 adopts an open-state conformation that is distinct from the Cx26 crystal structure, yet it appears to be stabilized by a conserved set of hydrophobic anchoring residues. 'Hot spots' of genetic mutations linked to hereditary cataract formation map to the core structural-functional elements identified in Cx46/50, suggesting explanations for many of the disease-causing effects.

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. Although most of these disease-linked variants cause loss of function because of 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. Coexpression 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. Furthermore, when T39R was coexpressed with Cx46, it produced hemichannels with increased activity, particularly at negative potentials, which could potentially contribute to its pathogenicity in the lens. In contrast, coexpression of wild-type Cx50 with Cx46 was associated with a marked reduction in hemichannel activity, indicating that it may have a protective effect. 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 (NT) 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.

Connexin 46 and connexin 50 gap junction channel properties are shaped by structural and dynamic features of their N-terminal domains.

KEY POINTS: Gap junctions formed by different connexins are expressed throughout the body and harbour unique channel properties that have not been fully defined mechanistically. Recent structural studies by cryo-electron microscopy have produced high-resolution models of the related but functionally distinct lens connexins (Cx50 and Cx46) captured in a stable open state, opening the door for structure-function comparison. Here, we conducted comparative molecular dynamics simulation and electrophysiology studies to dissect the isoform-specific differences in Cx46 and Cx50 intercellular channel function. We show that key determinants Cx46 and Cx50 gap junction channel open stability and unitary conductance are shaped by structural and dynamic features of their N-terminal domains, in particular the residue at the 9th position and differences in hydrophobic anchoring sites. The results of this study establish the open state Cx46/50 structural models as archetypes for structure-function studies targeted at elucidating the mechanism of gap junction channels and the molecular basis of disease-causing variants. ABSTRACT: Connexins form intercellular communication channels, known as gap junctions (GJs), that facilitate diverse physiological roles, from long-range electrical and chemical coupling to coordinating development and nutrient exchange. GJs formed by different connexin isoforms harbour unique channel properties that have not been fully defined mechanistically. Recent structural studies on Cx46 and Cx50 defined a novel and stable open state and implicated the amino-terminal (NT) domain as a major contributor for isoform-specific functional differences between these closely related lens connexins. To better understand these differences, we constructed models corresponding to wildtype Cx50 and Cx46 GJs, NT domain swapped chimeras, and point variants at the 9th residue for comparative molecular dynamics (MD) simulation and electrophysiology studies. All constructs formed functional GJ channels, except the chimeric Cx46-50NT variant, which correlated with an introduced steric clash and increased dynamical behaviour (instability) of the NT domain observed by MD simulation. Single channel conductance correlated well with free-energy landscapes predicted by MD, but resulted in a surprisingly greater degree of effect. Additionally, we observed significant effects on transjunctional voltage-dependent gating (Vj gating) and/or open state dwell times induced by the designed NT domain variants. Together, these studies indicate intra- and inter-subunit interactions involving both hydrophobic and charged residues within the NT domains of Cx46 and Cx50 play important roles in defining GJ open state stability and single channel conductance, and establish the open state Cx46/50 structural models as archetypes for structure-function studies targeted at elucidating GJ channel mechanisms and the molecular basis of cataract-linked connexin variants.

Tension directly stabilizes reconstituted kinetochore-microtubule attachments.

Kinetochores are macromolecular machines that couple chromosomes to dynamic microtubule tips during cell division, thereby generating force to segregate the chromosomes. Accurate segregation depends on selective stabilization of correct 'bi-oriented' kinetochore-microtubule attachments, which come under tension as the result of opposing forces exerted by microtubules. Tension is thought to stabilize these bi-oriented attachments indirectly, by suppressing the destabilizing activity of a kinase, Aurora B. However, a complete mechanistic understanding of the role of tension requires reconstitution of kinetochore-microtubule attachments for biochemical and biophysical analyses in vitro. Here we show that native kinetochore particles retaining the majority of kinetochore proteins can be purified from budding yeast and used to reconstitute dynamic microtubule attachments. Individual kinetochore particles maintain load-bearing associations with assembling and disassembling ends of single microtubules for >30 min, providing a close match to the persistent coupling seen in vivo between budding yeast kinetochores and single microtubules. Moreover, tension increases the lifetimes of the reconstituted attachments directly, through a catch bond-like mechanism that does not require Aurora B. On the basis of these findings, we propose that tension selectively stabilizes proper kinetochore-microtubule attachments in vivo through a combination of direct mechanical stabilization and tension-dependent phosphoregulation.

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 α-crystallins, αA-crystallin (αAc) and αB-crystallin (αBc), 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. Using biochemical and biophysical analyses combined with single-particle electron microscopy (EM), we examined structural changes in αAc, αBc and native heteromeric lens α-crystallins (αLc) in their apo-states and at varying degree of chaperone saturation leading to co-aggregation, using lysozyme and insulin as model clients. 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 oligomeric scaffold, whereby the native cage-like sHSP assembly displays a directional growth to accommodate saturating conditions of client sequestration. 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 oligomers with striking resemblance to polymeric α-crystallin species isolated from aged lens tissue. This mechanism appears consistent across αAc, αBc and αLc, 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 α-crystallins, carrying potential implications for a pathway toward cataract formation.

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 α-crystallins, αA-crystallin (αAc) and αB-crystallin (αBc), 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 αAc and αBc during client sequestration under varying degree of chaperone saturation. Using biochemical and biophysical analyses combined with single-particle electron microscopy (EM), we examined αAc and αBc 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 α-crystallin species isolated from aged lens tissue. This mechanism appears consistent across both αAc and αBc, 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.

Continuum dynamics and statistical correction of compositional heterogeneity in multivalent IDP oligomers resolved by single-particle EM.

Multivalent intrinsically disordered protein (IDP) complexes are prevalent in biology and act in regulation of diverse processes, including transcription, signaling events, and the assembly and disassembly of complex macromolecular architectures. These systems pose significant challenges to structural investigation, due to continuum dynamics imparted by the IDP and compositional heterogeneity resulting from characteristic low-affinity interactions. Here, we developed a modular pipeline for automated single-particle electron microscopy (EM) distribution analysis of common but relatively understudied semi-ordered systems: 'beads-on-a-string' assemblies, composed of IDPs bound at multivalent sites to the ubiquitous ∼20 kDa cross-linking hub protein LC8. This approach quantifies conformational geometries and compositional heterogeneity on a single-particle basis, and statistically corrects spurious observations arising from random proximity of bound and unbound LC8. The statistical correction is generically applicable to oligomer characterization and not specific to our pipeline. Following validation, the approach was applied to the nuclear pore IDP Nup159 and the transcription factor ASCIZ. This analysis unveiled significant compositional and conformational diversity in both systems that could not be obtained from ensemble single particle EM class-averaging strategies, and new insights for exploring how these architectural properties might contribute to their physiological roles in supramolecular assembly and transcriptional regulation. We expect that this approach may be adopted to many other intrinsically disordered systems that have evaded traditional methods of structural characterization.

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

Neuronal CaMKII holoenzymes (α and ß 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 electron microscopy (EM) in combination with biochemical and live-cell imaging studies. In the basal state, both isoforms assemble mainly as 12-mers (but also 14-mers and even 16-mers for the ß isoform). CaMKIIα and ß isoforms adopt 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 CaMKIIß allows direct resolution of intra-holoenzyme kinase domain dimers. These dimers could enable cooperative activation by calmodulin, which is observed for both isoforms. High-order CaMKII clustering mediated by inter-holoenzyme kinase domain dimerization is reduced for the ß isoform for both basal and excitotoxicity-induced clusters, both in vitro and in neurons.

The Cbf5-Nop10 complex is a molecular bracket that organizes box H/ACA RNPs.

Box H/ACA ribonucleoprotein particles (RNPs) catalyze RNA pseudouridylation and direct processing of ribosomal RNA, and are essential architectural components of vertebrate telomerases. H/ACA RNPs comprise four proteins and a multihelical RNA. Two proteins, Cbf5 and Nop10, suffice for basal enzymatic activity in an archaeal in vitro system. We now report their cocrystal structure at 1.95-A resolution. We find that archaeal Cbf5 can assemble with yeast Nop10 and with human telomerase RNA, consistent with the high sequence identity of the RNP components between archaea and eukarya. Thus, the Cbf5-Nop10 architecture is phylogenetically conserved. The structure shows how Nop10 buttresses the active site of Cbf5, and it reveals two basic troughs that bidirectionally extend the active site cleft. Mutagenesis results implicate an adjacent basic patch in RNA binding. This tripartite RNA-binding surface may function as a molecular bracket that organizes the multihelical H/ACA and telomerase RNAs.

Noncanonical binding of calmodulin to aquaporin-0: implications for channel regulation.

Aquaporins (AQPs) are a family of ubiquitous membrane channels that conduct water across cell membranes. AQPs form homotetramers containing four functional and independent water pores. Aquaporin-0 (AQP0) is expressed in the eye lens, where its water permeability is regulated by calmodulin (CaM). Here we use a combination of biochemical methods and NMR spectroscopy to probe the interaction between AQP0 and CaM. We show that CaM binds the AQP0 C-terminal domain in a calcium-dependent manner. We demonstrate that only two CaM molecules bind a single AQP0 tetramer in a noncanonical fashion, suggesting a form of cooperativity between AQP0 monomers. Based on these results, we derive a structural model of the AQP0/CaM complex, which suggests CaM may be inhibitory to channel permeability by capping the vestibules of two monomers within the AQP0 tetramer. Finally, phosphorylation within AQP0's CaM binding domain inhibits the AQP0/CaM interaction, suggesting a temporal regulatory mechanism for complex formation.

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

Gap junctions establish direct pathways for cells to transfer metabolic and electrical messages. The local lipid environment is known to affect the structure, stability and intercellular channel activity of gap junctions; however, the molecular basis for these effects remains unknown. Here, we incorporate native connexin-46/50 (Cx46/50) intercellular channels into a dual lipid nanodisc system, mimicking a native cell-to-cell junction. Structural characterization by CryoEM reveals a lipid-induced stabilization to the channel, resulting in a 3D reconstruction at 1.9 Å resolution. Together with all-atom molecular dynamics simulations, 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. In addition, ~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.

The structure and function of small nucleolar ribonucleoproteins.

Eukaryotes and archaea use two sets of specialized ribonucleoproteins (RNPs) to carry out sequence-specific methylation and pseudouridylation of RNA, the two most abundant types of modifications of cellular RNAs. In eukaryotes, these protein-RNA complexes localize to the nucleolus and are called small nucleolar RNPs (snoRNPs), while in archaea they are known as small RNPs (sRNP). The C/D class of sno(s)RNPs carries out ribose-2'-O-methylation, while the H/ACA class is responsible for pseudouridylation of their RNA targets. Here, we review the recent advances in the structure, assembly and function of the conserved C/D and H/ACA sno(s)RNPs. Structures of each of the core archaeal sRNP proteins have been determined and their assembly pathways delineated. Furthermore, the recent structure of an H/ACA complex has revealed the organization of a complete sRNP. Combined with current biochemical data, these structures offer insight into the highly homologous eukaryotic snoRNPs.

Nop10 is a conserved H/ACA snoRNP molecular adaptor.

The H/ACA class of small nucleolar ribonucleoproteins (snoRNPs) is primarily responsible for catalyzing the isomerization of uridine to pseudouridine (Psi) in ribosomal and other cellular RNAs. Each H/ACA snoRNP consist of four conserved proteins, Cbf5 (the Psi-synthase), Gar1, Nhp2 (L7Ae in archaea) and Nop10, that assemble onto a unique RNA component (the snoRNA). The smallest of these proteins, Nop10 ( approximately 7 kDa), has an essential role in the assembly and activity of these particles and binds directly to the Psi-synthase to form the minimal active enzyme in archaea. To better understand the conserved function of this protein, we characterized the NMR structure and dynamics of Nop10 proteins from both archaea and yeast. We show that archaeal Nop10 contains a highly stable Zn2+ binding motif that is replaced in eukaryotes by a smaller meta-stable beta-hairpin, while a highly conserved and conformationally dynamic linker connects these motifs to a nascent alpha-helical structure. Our structural analysis and NMR relaxation data show that these motifs do not interact with each other and tumble independently in solution. Several residues within the archaeal Nop10 Zn2+ binding motif have clear structural and functional roles and are conserved in eukaryotes, yet remain disordered in the free yeast Nop10. We propose that the dynamic structure of Nop10 facilitates an induced-fit recognition with the H/ACA Psi-synthase and allows it to act as a molecular adaptor for guiding snoRNP assembly in similar fashion in all archaea and eukaryotic organisms.

High-resolution structural and thermodynamic analysis of extreme stabilization of human procarboxypeptidase by computational protein design.

Recent efforts to design de novo or redesign the sequence and structure of proteins using computational techniques have met with significant success. Most, if not all, of these computational methodologies attempt to model atomic-level interactions, and hence high-resolution structural characterization of the designed proteins is critical for evaluating the atomic-level accuracy of the underlying design force-fields. We previously used our computational protein design protocol RosettaDesign to completely redesign the sequence of the activation domain of human procarboxypeptidase A2. With 68% of the wild-type sequence changed, the designed protein, AYEdesign, is over 10 kcal/mol more stable than the wild-type protein. Here, we describe the high-resolution crystal structure and solution NMR structure of AYEdesign, which show that the experimentally determined backbone and side-chains conformations are effectively superimposable with the computational model at atomic resolution. To isolate the origins of the remarkable stabilization, we have designed and characterized a new series of procarboxypeptidase mutants that gain significant thermodynamic stability with a minimal number of mutations; one mutant gains more than 5 kcal/mol of stability over the wild-type protein with only four amino acid changes. We explore the relationship between force-field smoothing and conformational sampling by comparing the experimentally determined free energies of the overall design and these focused subsets of mutations to those predicted using modified force-fields, and both fixed and flexible backbone sampling protocols.

Electron crystallography of aquaporins.

Aquaporins are a family of ubiquitous membrane proteins that form a pore for the permeation of water. Both electron and X-ray crystallography played major roles in determining the atomic structures of a number of aquaporins. This review focuses on electron crystallography, and its contribution to the field of aquaporin biology. We briefly discuss electron crystallography and the two-dimensional crystallization process. We describe features of aquaporins common to both electron and X-ray crystallographic structures; as well as some structural insights unique to electron crystallography, including aquaporin junction formation and lipid-protein interactions.

Publisher Correction: The CaMKII holoenzyme structure in activation-competent conformations.

This corrects the article DOI: 10.1038/ncomms15742.

Multivalency regulates activity in an intrinsically disordered transcription factor.

The transcription factor ASCIZ (ATMIN, ZNF822) has an unusually high number of recognition motifs for the product of its main target gene, the hub protein LC8 (DYNLL1). Using a combination of biophysical methods, structural analysis by NMR and electron microscopy, and cellular transcription assays, we developed a model that proposes a concerted role of intrinsic disorder and multiple LC8 binding events in regulating LC8 transcription. We demonstrate that the long intrinsically disordered C-terminal domain of ASCIZ binds LC8 to form a dynamic ensemble of complexes with a gradient of transcriptional activity that is inversely proportional to LC8 occupancy. The preference for low occupancy complexes at saturating LC8 concentrations with both human and Drosophila ASCIZ indicates that negative cooperativity is an important feature of ASCIZ-LC8 interactions. The prevalence of intrinsic disorder and multivalency among transcription factors suggests that formation of heterogeneous, dynamic complexes is a widespread mechanism for tuning transcriptional regulation.

The CaMKII holoenzyme structure in activation-competent conformations.

The Ca2+/calmodulin-dependent protein kinase II (CaMKII) assembles into large 12-meric holoenzymes, which is thought to enable regulatory processes required for synaptic plasticity underlying learning, memory and cognition. Here we used single particle electron microscopy (EM) to determine a pseudoatomic model of the CaMKIIα holoenzyme in an extended and activation-competent conformation. The holoenzyme is organized by a rigid central hub complex, while positioning of the kinase domains is highly flexible, revealing dynamic holoenzymes ranging from 15-35 nm in diameter. While most kinase domains are ordered independently, ∼20% appear to form dimers and <3% are consistent with a compact conformation. An additional level of plasticity is revealed by a small fraction of bona-fide 14-mers (<4%) that may enable subunit exchange. Biochemical and cellular FRET studies confirm that the extended state of CaMKIIα resolved by EM is the predominant form of the holoenzyme, even under molecular crowding conditions.

Allosteric mechanism of water-channel gating by Ca2+-calmodulin.

Calmodulin (CaM) is a universal regulatory protein that communicates the presence of calcium to its molecular targets and correspondingly modulates their function. This key signaling protein is important for controlling the activity of hundreds of membrane channels and transporters. However, understanding of the structural mechanisms driving CaM regulation of full-length membrane proteins has remained elusive. In this study, we determined the pseudoatomic structure of full-length mammalian aquaporin-0 (AQP0, Bos taurus) in complex with CaM, using EM to elucidate how this signaling protein modulates water-channel function. Molecular dynamics and functional mutation studies reveal how CaM binding inhibits AQP0 water permeability by allosterically closing the cytoplasmic gate of AQP0. Our mechanistic model provides new insight, only possible in the context of the fully assembled channel, into how CaM regulates multimeric channels by facilitating cooperativity between adjacent subunits.