Structure of full-length ERGIC-53 in complex with MCFD2 for cargo transport.

ERGIC-53 transports certain subsets of newly synthesized secretory proteins and membrane proteins from the endoplasmic reticulum to the Golgi apparatus. Despite numerous structural and functional studies since its identification, the overall architecture and mechanism of action of ERGIC-53 remain unclear. Here we present cryo-EM structures of full-length ERGIC-53 in complex with its functional partner MCFD2. These structures reveal that ERGIC-53 exists as a homotetramer, not a homohexamer as previously suggested, and comprises a four-leaf clover-like head and a long stalk composed of three sets of four-helix coiled-coil followed by a transmembrane domain. 3D variability analysis visualizes the flexible motion of the long stalk and local plasticity of the head region. Notably, MCFD2 is shown to possess a Zn2+-binding site in its N-terminal lid, which appears to modulate cargo binding. Altogether, distinct mechanisms of cargo capture and release by ERGIC- 53 via the stalk bending and metal binding are proposed.

Structural basis for lysophosphatidylserine recognition by GPR34.

GPR34 is a recently identified G-protein coupled receptor, which has an immunomodulatory role and recognizes lysophosphatidylserine (LysoPS) as a putative ligand. Here, we report cryo-electron microscopy structures of human GPR34-Gi complex bound with one of two ligands bound: either the LysoPS analogue S3E-LysoPS, or M1, a derivative of S3E-LysoPS in which oleic acid is substituted with a metabolically stable aromatic fatty acid surrogate. The ligand-binding pocket is laterally open toward the membrane, allowing lateral entry of lipidic agonists into the cavity. The amine and carboxylate groups of the serine moiety are recognized by the charged residue cluster. The acyl chain of S3E-LysoPS is bent and fits into the L-shaped hydrophobic pocket in TM4-5 gap, and the aromatic fatty acid surrogate of M1 fits more appropriately. Molecular dynamics simulations further account for the LysoPS-regioselectivity of GPR34. Thus, using a series of structural and physiological experiments, we provide evidence that chemically unstable 2-acyl LysoPS is the physiological ligand for GPR34. Overall, we anticipate the present structures will pave the way for development of novel anticancer drugs that specifically target GPR34.

Dual allosteric modulation of voltage and calcium sensitivities of the Slo1-LRRC channel complex.

Modulation of large conductance intracellular ligand-activated potassium (BK) channel family (Slo1-3) by auxiliary subunits allows diverse physiological functions in excitable and non-excitable cells. Cryoelectron microscopy (cryo-EM) structures of voltage-gated potassium (Kv) channel complexes have provided insights into how voltage sensitivity is modulated by auxiliary subunits. However, the modulation mechanisms of BK channels, particularly as ligand-activated ion channels, remain unknown. Slo1 is a Ca2+-activated and voltage-gated BK channel and is expressed in neurons, muscle cells, and epithelial cells. Using cryo-EM and electrophysiology, we show that the LRRC26-γ1 subunit modulates not only voltage but also Ca2+ sensitivity of Homo sapiens Slo1. LRRC26 stabilizes the active conformation of voltage-senor domains of Slo1 by an extracellularly S4-locking mechanism. Furthermore, it also stabilizes the active conformation of Ca2+-sensor domains of Slo1 intracellularly, which is functionally equivalent to intracellular Ca2+ in the activation of Slo1. Such a dual allosteric modulatory mechanism may be general in regulating the intracellular ligand-activated BK channel complexes.

An AsCas12f-based compact genome-editing tool derived by deep mutational scanning and structural analysis.

SpCas9 and AsCas12a are widely utilized as genome-editing tools in human cells. However, their relatively large size poses a limitation for delivery by cargo-size-limited adeno-associated virus (AAV) vectors. The type V-F Cas12f from Acidibacillus sulfuroxidans is exceptionally compact (422 amino acids) and has been harnessed as a compact genome-editing tool. Here, we developed an approach, combining deep mutational scanning and structure-informed design, to successfully generate two AsCas12f activity-enhanced (enAsCas12f) variants. Remarkably, the enAsCas12f variants exhibited genome-editing activities in human cells comparable with those of SpCas9 and AsCas12a. The cryoelectron microscopy (cryo-EM) structures revealed that the mutations stabilize the dimer formation and reinforce interactions with nucleic acids to enhance their DNA cleavage activities. Moreover, enAsCas12f packaged with partner genes in an all-in-one AAV vector exhibited efficient knock-in/knock-out activities and transcriptional activation in mice. Taken together, enAsCas12f variants could offer a minimal genome-editing platform for in vivo gene therapy.

Mechanistic and evolutionary insights into a type V-M CRISPR-Cas effector enzyme.

RNA-guided type V CRISPR-Cas12 effectors provide adaptive immunity against mobile genetic elements (MGEs) in bacteria and archaea. Among diverse Cas12 enzymes, the recently identified Cas12m2 (CRISPR-Cas type V-M) is highly compact and has a unique RuvC active site. Although the non-canonical RuvC triad does not permit dsDNA cleavage, Cas12m2 still protects against invading MGEs through transcriptional silencing by strong DNA binding. However, the molecular mechanism of RNA-guided genome inactivation by Cas12m2 remains unknown. Here we report cryo-electron microscopy structures of two states of Cas12m2-CRISPR RNA (crRNA)-target DNA ternary complexes and the Cas12m2-crRNA binary complex, revealing structural dynamics during crRNA-target DNA heteroduplex formation. The structures indicate that the non-target DNA strand is tightly bound to a unique arginine-rich cluster in the recognition (REC) domains and the non-canonical active site in the RuvC domain, ensuring strong DNA-binding affinity of Cas12m2. Furthermore, a structural comparison of Cas12m2 with TnpB, a putative ancestor of Cas12 enzymes, suggests that the interaction of the characteristic coiled-coil REC2 insertion with the protospacer-adjacent motif-distal region of the heteroduplex is crucial for Cas12m2 to engage in adaptive immunity. Collectively, our findings improve mechanistic understanding of diverse type V CRISPR-Cas effectors and provide insights into the evolution of TnpB to Cas12 enzymes.

Cryo-EM structure of the transposon-associated TnpB enzyme.

The class 2 type V CRISPR effector Cas12 is thought to have evolved from the IS200/IS605 superfamily of transposon-associated TnpB proteins1. Recent studies have identified TnpB proteins as miniature RNA-guided DNA endonucleases2,3. TnpB associates with a single, long RNA (ωRNA) and cleaves double-stranded DNA targets complementary to the ωRNA guide. However, the RNA-guided DNA cleavage mechanism of TnpB and its evolutionary relationship with Cas12 enzymes remain unknown. Here we report the cryo-electron microscopy (cryo-EM) structure of Deinococcus radiodurans ISDra2 TnpB in complex with its cognate ωRNA and target DNA. In the structure, the ωRNA adopts an unexpected architecture and forms a pseudoknot, which is conserved among all guide RNAs of Cas12 enzymes. Furthermore, the structure, along with our functional analysis, reveals how the compact TnpB recognizes the ωRNA and cleaves target DNA complementary to the guide. A structural comparison of TnpB with Cas12 enzymes suggests that CRISPR-Cas12 effectors acquired an ability to recognize the protospacer-adjacent motif-distal end of the guide RNA-target DNA heteroduplex, by either asymmetric dimer formation or diverse REC2 insertions, enabling engagement in CRISPR-Cas adaptive immunity. Collectively, our findings provide mechanistic insights into TnpB function and advance our understanding of the evolution from transposon-encoded TnpB proteins to CRISPR-Cas12 effectors.

Structural basis of gating modulation of Kv4 channel complexes.

Modulation of voltage-gated potassium (Kv) channels by auxiliary subunits is central to the physiological function of channels in the brain and heart1,2. Native Kv4 tetrameric channels form macromolecular ternary complexes with two auxiliary ß-subunits-intracellular Kv channel-interacting proteins (KChIPs) and transmembrane dipeptidyl peptidase-related proteins (DPPs)-to evoke rapidly activating and inactivating A-type currents, which prevent the backpropagation of action potentials1-5. However, the modulatory mechanisms of Kv4 channel complexes remain largely unknown. Here we report cryo-electron microscopy structures of the Kv4.2-DPP6S-KChIP1 dodecamer complex, the Kv4.2-KChIP1 and Kv4.2-DPP6S octamer complexes, and Kv4.2 alone. The structure of the Kv4.2-KChIP1 complex reveals that the intracellular N terminus of Kv4.2 interacts with its C terminus that extends from the S6 gating helix of the neighbouring Kv4.2 subunit. KChIP1 captures both the N and the C terminus of Kv4.2. In consequence, KChIP1 would prevent N-type inactivation and stabilize the S6 conformation to modulate gating of the S6 helices within the tetramer. By contrast, unlike the reported auxiliary subunits of voltage-gated channel complexes, DPP6S interacts with the S1 and S2 helices of the Kv4.2 voltage-sensing domain, which suggests that DPP6S stabilizes the conformation of the S1-S2 helices. DPP6S may therefore accelerate the voltage-dependent movement of the S4 helices. KChIP1 and DPP6S do not directly interact with each other in the Kv4.2-KChIP1-DPP6S ternary complex. Thus, our data suggest that two distinct modes of modulation contribute in an additive manner to evoke A-type currents from the native Kv4 macromolecular complex.

Axon morphogenesis and maintenance require an evolutionary conserved safeguard function of Wnk kinases antagonizing Sarm and Axed.

The molecular and cellular mechanisms underlying complex axon morphogenesis are still poorly understood. We report a novel, evolutionary conserved function for the Drosophila Wnk kinase (dWnk) and its mammalian orthologs, WNK1 and 2, in axon branching. We uncover that dWnk, together with the neuroprotective factor Nmnat, antagonizes the axon-destabilizing factors D-Sarm and Axundead (Axed) during axon branch growth, revealing a developmental function for these proteins. Overexpression of D-Sarm or Axed results in axon branching defects, which can be blocked by overexpression of dWnk or Nmnat. Surprisingly, Wnk kinases are also required for axon maintenance of adult Drosophila and mouse cortical pyramidal neurons. Requirement of Wnk for axon maintenance is independent of its developmental function. Inactivation of dWnk or mouse Wnk1/2 in mature neurons leads to axon degeneration in the adult brain. Therefore, Wnk kinases are novel signaling components that provide a safeguard function in both developing and adult axons.

Nuclear import of the DSCAM-cytoplasmic domain drives signaling capable of inhibiting synapse formation.

DSCAM and DSCAML1 are immunoglobulin and cell adhesion-type receptors serving important neurodevelopmental functions including control of axon growth, branching, neurite self-avoidance, and neuronal cell death. The signal transduction mechanisms or effectors of DSCAM receptors, however, remain poorly characterized. We used a human ORFeome library to perform a high-throughput screen in mammalian cells and identified novel cytoplasmic signaling effector candidates including the Down syndrome kinase Dyrk1a, STAT3, USP21, and SH2D2A. Unexpectedly, we also found that the intracellular domains (ICDs) of DSCAM and DSCAML1 specifically and directly interact with IPO5, a nuclear import protein of the importin beta family, via a conserved nuclear localization signal. The DSCAM ICD is released by γ-secretase-dependent cleavage, and both the DSCAM and DSCAML1 ICDs efficiently translocate to the nucleus. Furthermore, RNA sequencing confirms that expression of the DSCAM as well as the DSCAML1 ICDs alone can profoundly alter the expression of genes associated with neuronal differentiation and apoptosis, as well as synapse formation and function. Gain-of-function experiments using primary cortical neurons show that increasing the levels of either the DSCAM or the DSCAML1 ICD leads to an impairment of neurite growth. Strikingly, increased expression of either full-length DSCAM or the DSCAM ICD, but not the DSCAML1 ICD, significantly decreases synapse numbers in primary hippocampal neurons. Taken together, we identified a novel membrane-to-nucleus signaling mechanism by which DSCAM receptors can alter the expression of regulators of neuronal differentiation and synapse formation and function. Considering that chromosomal duplications lead to increased DSCAM expression in trisomy 21, our findings may help uncover novel mechanisms contributing to intellectual disability in Down syndrome.

Slit and Receptor Tyrosine Phosphatase 69D Confer Spatial Specificity to Axon Branching via Dscam1.

Axonal branching contributes substantially to neuronal circuit complexity. Studies in Drosophila have shown that loss of Dscam1 receptor diversity can fully block axon branching in mechanosensory neurons. Here we report that cell-autonomous loss of the receptor tyrosine phosphatase 69D (RPTP69D) and loss of midline-localized Slit inhibit formation of specific axon collaterals through modulation of Dscam1 activity. Genetic and biochemical data support a model in which direct binding of Slit to Dscam1 enhances the interaction of Dscam1 with RPTP69D, stimulating Dscam1 dephosphorylation. Single-growth-cone imaging reveals that Slit/RPTP69D are not required for general branch initiation but instead promote the extension of specific axon collaterals. Hence, although regulation of intrinsic Dscam1-Dscam1 isoform interactions is essential for formation of all mechanosensory-axon branches, the local ligand-induced alterations of Dscam1 phosphorylation in distinct growth-cone compartments enable the spatial specificity of axon collateral formation.

Cell-intrinsic requirement of Dscam1 isoform diversity for axon collateral formation.

The isoform diversity of the Drosophila Dscam1 receptor is important for neuronal self-recognition and self-avoidance. A canonical model suggests that homophilic binding of identical Dscam1 receptor isoforms on sister dendrites ensures self-avoidance even when only a single isoform is expressed. We detected a cell-intrinsic function of Dscam1 that requires the coexpression of multiple isoforms. Manipulation of the Dscam1 isoform pool in single neurons caused severe disruption of collateral formation of mechanosensory axons. Changes in isoform abundance led to dominant dosage-sensitive inhibition of branching. We propose that the ratio of matching to nonmatching isoforms within a cell influences the Dscam1-mediated signaling strength, which in turn controls axon growth and growth cone sprouting. Cell-intrinsic use of surface receptor diversity may be of general importance in regulating axonal branching during brain wiring.

Ultra-deep profiling of alternatively spliced Drosophila Dscam isoforms by circularization-assisted multi-segment sequencing.

The Drosophila melanogaster gene Dscam (Down syndrome cell adhesion molecule) can generate thousands of different ectodomains via mutual exclusive splicing of three large exon clusters. The isoform diversity plays a profound role in both neuronal wiring and pathogen recognition. However, the isoform expression pattern at the global level remained unexplored. Here, we developed a novel method that allows for direct quantification of the alternatively spliced exon combinations from over hundreds of millions of Dscam transcripts in one sequencing run. With unprecedented sequencing depth, we detected a total of 18,496 isoforms, out of 19,008 theoretically possible combinations. Importantly, we demonstrated that alternative splicing between different clusters is independent. Moreover, the isoforms were expressed across a broad dynamic range, with significant bias in cell/tissue and developmental stage-specific patterns. Hitherto underappreciated, such bias can dramatically reduce the ability of neurons to display unique surface receptor codes. Therefore, the seemingly excessive diversity encoded in the Dscam locus might nevertheless be essential for a robust self and non-self discrimination in neurons.

Sufu recruits GSK3beta for efficient processing of Gli3.

Hedgehog (Hh) signaling activates the transcription factor Gli by suppressing the function of the suppressor of fused (Sufu) protein in mammals. Here, a novel role of mammalian Sufu is identified where it mediates the phosphorylation of Gli3 by GSK3beta, essential for Gli3 processing to generate a transcriptional repressor for Hh-target genes. Studies using Sufu(-/-) mouse embryonic fibroblasts and siRNA targeting Sufu demonstrate the requirement of Sufu for Gli3 processing. In addition, Sufu can bind to GSK3beta as well as Gli3, and mediates formation of the trimolecular complex Gli3/Sufu/GSK3beta. Thus, Sufu stimulates Gli3 phosphorylation by GSK3beta and Gli3 processing. Furthermore, Sonic Hh stimulation dissociates the Sufu/GSK3beta complex from Gli3, resulting in the blockade of Gli3 processing. Collectively, Sufu presumably functions as a GSK3beta recruiter for Hh-dependent regulation of Gli3 processing. Such a function is very similar to that of Costal2 in Drosophila, suggesting a functional complementation through evolution.

GSK3beta positively regulates Hedgehog signaling through Sufu in mammalian cells.

Hedgehog signaling plays important roles in embryonic patterning of multicellular organisms. This pathway is ultimately transmitted by the zinc-finger transcriptional factor Gli, of which activity is suppressed by Sufu, a negative regulator of this signaling. To clarify this regulation to more detail, we screened for Sufu-binding proteins. We identified GSK3beta as a specific binding partner of Sufu by mass spectrometric analysis. GSK3beta bound to Sufu both in vitro and in vivo. Down-regulation of GSK3beta expression by RNAi in Hedgehog-responsive cells attenuated Hedgehog signaling, suggesting that GSK3beta functions as a positive regulator of Hedgehog signaling. In addition, an in vitro kinase assay showed that GSK3beta phosphorylates Sufu and phosphorylation-mimicking mutant of Sufu showed significantly decreased ability to bind Gli1 and could not suppress the Gli-mediated expression of a reporter gene efficiently. These results strongly suggest that GSK3beta phosphorylates Sufu to positively regulate Hedgehog signaling in mammalian cells.

Fused kinase is stabilized by Cdc37/Hsp90 and enhances Gli protein levels.

Serine/threonine kinase Fused (Fu) is an essential component of Hedgehog (Hh) signaling in Drosophila, but the biochemical functions of Fu remain unclear. Here, we have investigated proteins co-precipitated with mammalian Fu and identified a kinase-specific chaperone complex, Cdc37/Hsp90, as a novel-binding partner of Fu. Inhibition of Hsp90 function by geldanamycin (GA) induces rapid degradation of Fu through a ubiquitin-proteasome pathway. We next show that co-expression of Fu with transcription factors Gli1 and Gli2 significantly increases their protein levels and luciferase reporter activities, which are blocked by GA. These increases can be ascribed to Fu-mediated stabilization of Gli because co-expression of Fu prolongs half-life of Gli1 and reduces polyubiquitination of Gli1. Finally, we show that GA inhibits proliferation of PC3, a Hh signaling-activated prostate cancer cell line. This growth inhibition is partially rescued by expression of ectopic Gli1, suggesting that Fu may contribute to enhance Hh signaling activity in cancer cells.

A short peptide insertion crucial for angiostatic activity of human tryptophanyl-tRNA synthetase.

Human tryptophanyl-tRNA synthetase (TrpRS) is secreted into the extracellular region of vascular endothelial cells. The splice variant form (mini TrpRS) functions in vascular endothelial cell apoptosis as an angiostatic cytokine. In contrast, the closely related human tyrosyl-tRNA synthetase (TyrRS) functions as an angiogenic cytokine in its truncated form (mini TyrRS). Here, we determined the crystal structure of human mini TrpRS at a resolution of 2.3 A and compared the structure with those of prokaryotic TrpRS and human mini TyrRS. Deletion of the tRNA anticodon-binding (TAB) domain insertion, consisting of eight residues in the human TrpRS, abolished the enzyme's apoptotic activity for endothelial cells, whereas its translational catalysis and cell-binding activities remained unchanged. Thus, we have identified the inserted peptide motif that activates the angiostatic signaling.