High-speed AFM imaging reveals DNA capture and loop extrusion dynamics by cohesin-NIPBL.

3D chromatin organization plays a critical role in regulating gene expression, DNA replication, recombination, and repair. While initially discovered for its role in sister chromatid cohesion, emerging evidence suggests that the cohesin complex (SMC1, SMC3, RAD21, and SA1/SA2), facilitated by NIPBL, mediates topologically associating domains and chromatin loops through DNA loop extrusion. However, information on how conformational changes of cohesin-NIPBL drive its loading onto DNA, initiation, and growth of DNA loops is still lacking. In this study, high-speed atomic force microscopy imaging reveals that cohesin-NIPBL captures DNA through arm extension, assisted by feet (shorter protrusions), and followed by transfer of DNA to its lower compartment (SMC heads, RAD21, SA1, and NIPBL). While binding at the lower compartment, arm extension leads to the capture of a second DNA segment and the initiation of a DNA loop that is independent of ATP hydrolysis. The feet are likely contributed by the C-terminal domains of SA1 and NIPBL and can transiently bind to DNA to facilitate the loading of the cohesin complex onto DNA. Furthermore, high-speed atomic force microscopy imaging reveals distinct forward and reverse DNA loop extrusion steps by cohesin-NIPBL. These results advance our understanding of cohesin by establishing direct experimental evidence for a multistep DNA-binding mechanism mediated by dynamic protein conformational changes.

CTCF and R-loops are boundaries of cohesin-mediated DNA looping.

Cohesin and CCCTC-binding factor (CTCF) are key regulatory proteins of three-dimensional (3D) genome organization. Cohesin extrudes DNA loops that are anchored by CTCF in a polar orientation. Here, we present direct evidence that CTCF binding polarity controls cohesin-mediated DNA looping. Using single-molecule imaging, we demonstrate that a critical N-terminal motif of CTCF blocks cohesin translocation and DNA looping. The cryo-EM structure of the cohesin-CTCF complex reveals that this CTCF motif ahead of zinc fingers can only reach its binding site on the STAG1 cohesin subunit when the N terminus of CTCF faces cohesin. Remarkably, a C-terminally oriented CTCF accelerates DNA compaction by cohesin. DNA-bound Cas9 and Cas12a ribonucleoproteins are also polar cohesin barriers, indicating that stalling may be intrinsic to cohesin itself. Finally, we show that RNA-DNA hybrids (R-loops) block cohesin-mediated DNA compaction in vitro and are enriched with cohesin subunits in vivo, likely forming TAD boundaries.

Cryo-EM structures of human p97 double hexamer capture potentiated ATPase-competent state.

The conserved ATPase p97 (Cdc48 in yeast) and adaptors mediate diverse cellular processes through unfolding polyubiquitinated proteins and extracting them from macromolecular assemblies and membranes for disaggregation and degradation. The tandem ATPase domains (D1 and D2) of the p97/Cdc48 hexamer form stacked rings. p97/Cdc48 can unfold substrates by threading them through the central pore. The pore loops critical for substrate unfolding are, however, not well-ordered in substrate-free p97/Cdc48 conformations. How p97/Cdc48 organizes its pore loops for substrate engagement is unclear. Here we show that p97/Cdc48 can form double hexamers (DH) connected through the D2 ring. Cryo-EM structures of p97 DH reveal an ATPase-competent conformation with ordered pore loops. The C-terminal extension (CTE) links neighboring D2s in each hexamer and expands the central pore of the D2 ring. Mutations of Cdc48 CTE abolish substrate unfolding. We propose that the p97/Cdc48 DH captures a potentiated state poised for substrate engagement.

Cryo-EM structure of VASH1-SVBP bound to microtubules.

The dynamic tyrosination-detyrosination cycle of α-tubulin regulates microtubule functions. Perturbation of this cycle impairs mitosis, neural physiology, and cardiomyocyte contraction. The carboxypeptidases vasohibins 1 and 2 (VASH1 and VASH2), in complex with the small vasohibin-binding protein (SVBP), mediate α-tubulin detyrosination. These enzymes detyrosinate microtubules more efficiently than soluble αß-tubulin heterodimers. The structural basis for this substrate preference is not understood. Using cryo-electron microscopy (cryo-EM), we have determined the structure of human VASH1-SVBP bound to microtubules. The acidic C-terminal tail of α-tubulin binds to a positively charged groove near the active site of VASH1. VASH1 forms multiple additional contacts with the globular domain of α-tubulin, including contacts with a second α-tubulin in an adjacent protofilament. Simultaneous engagement of two protofilaments by VASH1 can only occur within the microtubule lattice, but not with free αß heterodimers. These lattice-specific interactions enable preferential detyrosination of microtubules by VASH1.

Cryo-EM structure of the human cohesin-NIPBL-DNA complex.

As a ring-shaped adenosine triphosphatase (ATPase) machine, cohesin organizes the eukaryotic genome by extruding DNA loops and mediates sister chromatid cohesion by topologically entrapping DNA. How cohesin executes these fundamental DNA transactions is not understood. Using cryo-electron microscopy (cryo-EM), we determined the structure of human cohesin bound to its loader NIPBL and DNA at medium resolution. Cohesin and NIPBL interact extensively and together form a central tunnel to entrap a 72-base pair DNA. NIPBL and DNA promote the engagement of cohesin's ATPase head domains and ATP binding. The hinge domains of cohesin adopt an "open washer" conformation and dock onto the STAG1 subunit. Our structure explains the synergistic activation of cohesin by NIPBL and DNA and provides insight into DNA entrapment by cohesin.

Human cohesin compacts DNA by loop extrusion.

Cohesin is a chromosome-bound, multisubunit adenosine triphosphatase complex. After loading onto chromosomes, it generates loops to regulate chromosome functions. It has been suggested that cohesin organizes the genome through loop extrusion, but direct evidence is lacking. Here, we used single-molecule imaging to show that the recombinant human cohesin-NIPBL complex compacts both naked and nucleosome-bound DNA by extruding DNA loops. DNA compaction by cohesin requires adenosine triphosphate (ATP) hydrolysis and is force sensitive. This compaction is processive over tens of kilobases at an average rate of 0.5 kilobases per second. Compaction of double-tethered DNA suggests that a cohesin dimer extrudes DNA loops bidirectionally. Our results establish cohesin-NIPBL as an ATP-driven molecular machine capable of loop extrusion.

Architecture, substructures, and dynamic assembly of STRIPAK complexes in Hippo signaling.

Striatin-interacting phosphatases and kinases (STRIPAKs) are evolutionarily conserved supramolecular complexes, which have been implicated in the Hippo signaling pathway. Yet the topological structure and dynamic assembly of STRIPAK complexes remain elusive. Here, we report the overall architecture and substructures of a Hippo kinase-containing STRIPAK complex. PP2Aa/c-bound STRN3 directly contacts the Hippo kinase MST2 and also controls the loading of MST2 via two "arms" in a phosphorylation-dependent manner, one arm being STRIP1 and the other SIKE1-SLMAP. A decreased cell density triggered the dissociation of the STRIP1 arm from STRIPAK, reflecting the dynamic assembly of the complex upon sensing upstream signals. Crystallographic studies defined at atomic resolution the interface between STRN3 and SIKE1, and that between SIKE1 and SLMAP. Disrupting the complex assembly abrogated the regulatory effect of STRIPAK towards Hippo signaling. Collectively, our study revealed a "two-arm" assembly of STRIPAK with context-dependent dynamics, offering a framework for further studies on Hippo signaling and biological processes involving MST kinases.

The MST4-MOB4 complex disrupts the MST1-MOB1 complex in the Hippo-YAP pathway and plays a pro-oncogenic role in pancreatic cancer.

The mammalian STE20-like protein kinase 1 (MST1)-MOB kinase activator 1 (MOB1) complex has been shown to suppress the oncogenic activity of Yes-associated protein (YAP) in the mammalian Hippo pathway, which is involved in the development of multiple tumors, including pancreatic cancer (PC). However, it remains unclear whether other MST-MOB complexes are also involved in regulating Hippo-YAP signaling and have potential roles in PC. Here, we report that mammalian STE20-like kinase 4 (MST4), a distantly related ortholog of the MST1 kinase, forms a complex with MOB4 in a phosphorylation-dependent manner. We found that the overall structure of the MST4-MOB4 complex resembles that of the MST1-MOB1 complex, even though the two complexes exhibited opposite biological functions in PC. In contrast to the tumor-suppressor effect of the MST1-MOB1 complex, the MST4-MOB4 complex promoted growth and migration of PANC-1 cells. Moreover, expression levels of MST4 and MOB4 were elevated in PC and were positively correlated with each other, whereas MST1 expression was down-regulated. Because of divergent evolution of key interface residues, MST4 and MOB4 could disrupt assembly of the MST1-MOB1 complex through alternative pairing and thereby increased YAP activity. Collectively, these findings identify the MST4-MOB4 complex as a noncanonical regulator of the Hippo-YAP pathway with an oncogenic role in PC. Our findings highlight that although MST-MOB complexes display some structural conservation, they functionally diverged during their evolution.

Exosome cofactor hMTR4 competes with export adaptor ALYREF to ensure balanced nuclear RNA pools for degradation and export.

The exosome is a key RNA machine that functions in the degradation of unwanted RNAs. Here, we found that significant fractions of precursors and mature forms of mRNAs and long noncoding RNAs are degraded by the nuclear exosome in normal human cells. Exosome-mediated degradation of these RNAs requires its cofactor hMTR4. Significantly, hMTR4 plays a key role in specifically recruiting the exosome to its targets. Furthermore, we provide several lines of evidence indicating that hMTR4 executes this role by directly competing with the mRNA export adaptor ALYREF for associating with ARS2, a component of the cap-binding complex (CBC), and this competition is critical for determining whether an RNA is degraded or exported to the cytoplasm. Together, our results indicate that the competition between hMTR4 and ALYREF determines exosome recruitment and functions in creating balanced nuclear RNA pools for degradation and export.

MST kinases in innate immune signaling.

The mammalian STE20-like (MST) protein kinases are composed of MST1, MST2, MST3, MST4 and YSK1. They play crucial roles in cell growth, migration, polarity and apoptosis. Dysfunction of these kinases often leads to diseases. MST kinases are extensively involved in development and function of immune system. Here, we review recent progresses on the regulatory function of MST kinases in innate immune signaling.

Disruption of the RAG2 zinc finger motif impairs protein stability and causes immunodeficiency.

Although the RAG2 core domain is the minimal region required for V(D)J recombination, the noncore region also plays important roles in the regulation of recombination, and mutations in this region are often related to severe combined immunodeficiency. A complete understanding of the functions of the RAG2 noncore region and the potential contributions of its individual residues has not yet been achieved. Here, we show that the zinc finger motif within the noncore region of RAG2 is indispensable for maintaining the stability of the RAG2 protein. The zinc finger motif in the noncore region of RAG2 is highly conserved from zebrafish to humans. Knock-in mice carrying a zinc finger mutation (C478Y) exhibit decreased V(D)J recombination efficiency and serious impairment in T/B-cell development due to RAG2 instability. Further studies also reveal the importance of the zinc finger motif for RAG2 stability. Moreover, mice harboring a RAG2 noncore region mutation (N474S), which is located near C478 but is not zinc-binding, exhibit no impairment in either RAG2 stability or T/B-cell development. Taken together, our findings contribute to defining critical functions of the RAG2 zinc finger motif and provide insights into the relationships between the mutations within this motif and immunodeficiency diseases.

A non-canonical role of the p97 complex in RIG-I antiviral signaling.

RIG-I is a well-studied sensor of viral RNA that plays a key role in innate immunity. p97 regulates a variety of cellular events such as protein quality control, membrane reassembly, DNA repair, and the cell cycle. Here, we report a new role for p97 with Npl4-Ufd1 as its cofactor in reducing antiviral innate immune responses by facilitating proteasomal degradation of RIG-I. The p97 complex is able to directly bind both non-ubiquitinated RIG-I and the E3 ligase RNF125, promoting K48-linked ubiquitination of RIG-I at residue K181. Viral infection significantly strengthens the interaction between RIG-I and the p97 complex by a conformational change of RIG-I that exposes the CARDs and through K63-linked ubiquitination of these CARDs. Disruption of the p97 complex enhances RIG-I antiviral signaling. Consistently, administration of compounds targeting p97 ATPase activity was shown to inhibit viral replication and protect mice from vesicular stomatitis virus (VSV) infection. Overall, our study uncovered a previously unrecognized role for the p97 complex in protein ubiquitination and revealed the p97 complex as a potential drug target in antiviral therapy.

Structural Insights into mitochondrial antiviral signaling protein (MAVS)-tumor necrosis factor receptor-associated factor 6 (TRAF6) signaling.

In response to viral infection, cytosolic retinoic acid-inducible gene I-like receptors sense viral RNA and promote oligomerization of mitochondrial antiviral signaling protein (MAVS), which then recruits tumor necrosis factor receptor-associated factor (TRAF) family proteins, including TRAF6, to activate an antiviral response. Currently, the interaction between MAVS and TRAF6 is only partially understood, and atomic details are lacking. Here, we demonstrated that MAVS directly interacts with TRAF6 through its potential TRAF6-binding motif 2 (T6BM2; amino acids 455-460). Further, we solved the crystal structure of MAVS T6BM2 in complex with the TRAF6 TRAF_C domain at 2.95 Å resolution. T6BM2 of MAVS binds to the canonical adaptor-binding groove of the TRAF_C domain. Structure-directed mutational analyses in vitro and in cells revealed that MAVS binding to TRAF6 via T6BM2 instead of T6BM1 is essential but not sufficient for an optimal antiviral response. Particularly, a MAVS mutant Y460E retained its TRAF6-binding ability as predicted but showed significantly impaired signaling activity, highlighting the functional importance of this tyrosine. Moreover, these observations were further confirmed in MAVS(-/-) mouse embryonic fibroblast cells. Collectively, our work provides a structural basis for understanding the MAVS-TRAF6 antiviral response.

The Transitional Endoplasmic Reticulum ATPase p97 Regulates the Alternative Nuclear Factor NF-κB Signaling via Partial Degradation of the NF-κB Subunit p100.

Partial degradation of the p100 subunit to generate p52 subunit is a hallmark of the alternative NF-κB pathway, which has been implicated in cancer. Here, we uncovered a role of the p97-Npl4-Ufd1 complex in mediating p100-to-p52 processing and therefore positively regulating the alternative NF-κB pathway. We observed an elevation of p97 mRNA levels in lymphoma patients, which positively correlates with NFKB2 expression, a downstream target gene of the alternative NF-κB pathway. Moreover, NFKB2 mRNA levels were aberrantly down-regulated in patients with inclusion body myopathy associated with Paget's disease of the bone and frontotemporal dementia (IBMPFD), a disease caused by mutation of p97. Inactivation of p97 or depletion of the p97-Npl4-Ufd1 complex inhibits the processing of p100 into p52, decreasing transcription of the downstream target genes. Further analyses reveal that the p97-Npl4-Ufd1 complex interacts with F-box and WD repeats protein SCF(ßTrCP) complex to regulate the partial degradation of p100, a process involving K48- and K11-linked ubiquitination. In line with this, in LPS-induced lung damage mice model, generation of p52 is significantly decreased in p97-KD mice compared with mock mice. Finally, abrogation of p97 ATPase activity by its specific inhibitor DBeQ, efficiently decreased proliferation of lymphoma cells. Collectively, our study revealed a regulatory role of the p97-Npl4-Ufd1 complex in regulating p100 partial degradation, highlighting the potential of p97 as a drug target for cancers with aberrant activation of the alternative NF-κB pathway.

The kinase MST4 limits inflammatory responses through direct phosphorylation of the adaptor TRAF6.

Immune responses need to be tightly controlled to avoid excessive inflammation and prevent unwanted host damage. Here we report that germinal center kinase MST4 responded dynamically to bacterial infection and acted as a negative regulator of inflammation. We found that MST4 directly interacted with and phosphorylated the adaptor TRAF6 to prevent its oligomerization and autoubiquitination. Accordingly, MST4 did not inhibit lipopolysaccharide-induced cytokine production in Traf6(-/-) embryonic fibroblasts transfected to express a mutant form of TRAF6 that cannot be phosphorylated at positions 463 and 486 (with substitution of alanine for threonine at those positions). Upon developing septic shock, mice in which MST4 was knocked down showed exacerbated inflammation and reduced survival, whereas heterozygous deletion of Traf6 (Traf6(+/-)) alleviated such deleterious effects. Our findings reveal a mechanism by which TRAF6 is regulated and highlight a role for MST4 in limiting inflammatory responses.

Structural dissection of Hippo signaling.

The Hippo pathway controls cell number and organ size by restricting cell proliferation and promoting apoptosis, and thus is a key regulator in development and homeostasis. Dysfunction of the Hippo pathway correlates with many pathological conditions, especially cancer. Hippo signaling also plays important roles in tissue regeneration and stem cell biology. Therefore, the Hippo pathway is recognized as a crucial target for cancer therapy and regeneration medicine. To date, structures of several key components in Hippo signaling have been determined. In this review, we summarize current available structural studies of the Hippo pathway, which may help to improve our understanding of its regulatory mechanisms, as well as to facilitate further functional studies and potential therapeutic interventions.

Structural and biochemical insights into the activation mechanisms of germinal center kinase OSR1.

The oxidative stress-responsive 1 (OSR1) kinase belongs to the mammalian STE20-like kinase family. OSR1 is activated by with no lysine [K] (WNKs) kinases, and then it phosphorylates cation-coupled Cl-cotransporters, regulating ion homeostasis and cell volume in mammalian cells. However, the specific mechanisms of OSR1 activation remains poorly defined, largely due to its extremely low basal activity. Here, we dissect in detail the regulatory mechanisms of OSR1 activation from the aspects of autoinhibition, upstream kinase WNK, and the newly identified master regulator mouse protein-25 (MO25). Based on our structural and biochemical studies, we propose a "double lock" model, accounting for the tight autoinhibition of OSR1, an effect that has to be removed by WNK before MO25 further activates OSR1. Particularly, the conserved C-terminal (CCT) domain and αAL helix act together to strongly suppress OSR1 basal activity. WNKs bind to the CCT and trigger its conformational rearrangement to release the kinase domain of OSR1, allowing for MO25 binding and full activation. Finally, the regulatory mechanisms of OSR1 activation were further corroborated by cellular studies of OSR1-regulated cell volume control through WNK-OSR1 signaling pathway. Collectively, these results provide insights into the OSR1 kinase activation to facilitate further functional study.

Structural insights into the C1q domain of Caprin-2 in canonical Wnt signaling.

Previously, we have identified Caprin-2 as a new regulator in canonical Wnt signaling through a mechanism of facilitating LRP5/6 phosphorylation; moreover, we found that its C-terminal C1q-related domain (Cap2_CRD) is required for this process. Here, we determined the crystal structures of Cap2_CRD from human and zebrafish, which both associate as a homotrimer with calcium located at the symmetric center. Surprisingly, the calcium binding-deficient mutant exists as a more stable trimer than its wild-type counterpart. Further studies showed that this Caprin-2 mutant disabled in binding calcium maintains the activity of promoting LRP5/6 phosphorylation, whereas the mutations disrupting Cap2_CRD homotrimer did impair such activity. Together, our findings suggested that the C-terminal CRD domain of Caprin-2 forms a flexible homotrimer mediated by calcium and that such trimeric assembly is required for Caprin-2 to regulate canonical Wnt signaling.

Structure of the kinase domain of Gilgamesh from Drosophila melanogaster.

The CK1 family kinases regulate multiple cellular aspects and play important roles in Wnt/Wingless and Hedgehog signalling. The kinase domain of Drosophila Gilgamesh isoform I (Gilgamesh-I), a homologue of human CK1-γ, was purified and crystallized. Crystals of methylated Gilgamesh-I kinase domain with a D210A mutation diffracted to 2.85 Šresolution and belonged to space group P43212, with unit-cell parameters a = b = 52.025, c = 291.727 Å. The structure of Gilgamesh-I kinase domain, which was determined by molecular replacement, has conserved catalytic elements and an active conformation. Structural comparison indicates that an extended loop between the α1 helix and the ß4 strand exists in the Gilgamesh-I kinase domain. This extended loop may regulate the activity and function of Gilgamesh-I.