Biophysical characterization of the CXC chemokine receptor 2 ligands.

The chemokines of the immune system act as first responders by operating as chemoattractants, directing immune cells to specific locations of inflamed tissues. This promiscuous network is comprised of 50 ligands and 18 receptors where the ligands may interact with the receptors in various oligomeric states i.e., monomers, homodimers, and heterodimers. Chemokine receptors are G-protein coupled receptors (GPCRs) present in the membrane of immune cells. The migration of immune cells occurs in response to a concentration gradient of the ligands. Chemotaxis of neutrophils is directed by CXC-ligand (CXCL) activation of the membrane bound CXC chemokine receptor 2 (CXCR2). CXCR2 plays an important role in human health and is linked to disorders such as autoimmune disorders, inflammation, and cancer. Yet, despite their important role, little is known about the biophysical characteristics controlling ligand:ligand and ligand:receptor interaction essential for biological activity. In this work, we study the homodimers of three of the CXCR2 cognate ligands, CXCL1, CXCL5, and CXCL8. The ligands share high structural integrity but a low sequence identity. We show that the sequence diversity has evolved different binding affinities and stabilities for the CXC-ligands resulting in diverse agonist/antagonist behavior. Furthermore, CXC-ligands fold through a three-state mechanism, populating a folded monomeric state before associating into an active dimer.

Mimicking Protein Kinase C Phosphorylation Inhibits Arc/Arg3.1 Palmitoylation and Its Interaction with Nucleic Acids.

Activity-regulated cytoskeleton-associated protein (Arc) plays essential roles in diverse forms of synaptic plasticity, including long-term potentiation (LTP), long-term depression (LTD), and homeostatic plasticity. In addition, it assembles into virus-like particles that may deliver mRNAs and/or other cargo between neurons and neighboring cells. Considering this broad range of activities, it is not surprising that Arc is subject to regulation by multiple types of post-translational modification, including phosphorylation, palmitoylation, SUMOylation, ubiquitylation, and acetylation. Here we explore the potential regulatory role of Arc phosphorylation by protein kinase C (PKC), which occurs on serines 84 and 90 within an α-helical segment in the N-terminal domain. To mimic the effect of PKC phosphorylation, we mutated the two serines to negatively charged glutamic acid. A consequence of introducing these phosphomimetic mutations is the almost complete inhibition of Arc palmitoylation, which occurs on nearby cysteines and contributes to synaptic weakening. The mutations also inhibit the binding of nucleic acids and destabilize high-order Arc oligomers. Thus, PKC phosphorylation of Arc may limit the full expression of LTD and may suppress the interneuronal transport of mRNAs.

Oligomer-to-monomer transition underlies the chaperone function of AAGAB in AP1/AP2 assembly.

Assembly of protein complexes is facilitated by assembly chaperones. Alpha and gamma adaptin-binding protein (AAGAB) is a chaperone governing the assembly of the heterotetrameric adaptor complexes 1 and 2 (AP1 and AP2) involved in clathrin-mediated membrane trafficking. Here, we found that before AP1/2 binding, AAGAB exists as a homodimer. AAGAB dimerization is mediated by its C-terminal domain (CTD), which is critical for AAGAB stability and is missing in mutant proteins found in patients with the skin disease punctate palmoplantar keratoderma type 1 (PPKP1). We solved the crystal structure of the dimerization-mediating CTD, revealing an antiparallel dimer of bent helices. Interestingly, AAGAB uses the same CTD to recognize and stabilize the γ subunit in the AP1 complex and the α subunit in the AP2 complex, forming binary complexes containing only one copy of AAGAB. These findings demonstrate a dual role of CTD in stabilizing resting AAGAB and binding to substrates, providing a molecular explanation for disease-causing AAGAB mutations. The oligomerization state transition mechanism may also underlie the functions of other assembly chaperones.

Self-assembling Peptides with Internal Ionizable Unnatural Amino Acids: A General Approach to pH-responsive Peptide Materials.

Self-assembled peptides are an emerging family of biomaterials that show great promise for a range of biomedical and biotechnological applications. Introducing and tuning the pH-responsiveness of the assembly is highly desirable for improving their biological activities. Inspired by proteins with internal ionizable residues, we report a simple but effective approach to constructing pH-responsive peptide assembly containing unnatural ionic amino acids with an aliphatic tertiary amine side chain. Through a combined experimental and computational investigation, we demonstrate that these residues can be accommodated and stabilized within the internal hydrophobic compartment of the peptide assembly. The hydrophobic microenvironment shifts their pKa significantly from a basic pH typically found for free amines to a more biologically relevant pH in the weakly acidic range. The pH-induced ionization and ionization-dependent self-assembly and disassembly are thoroughly investigated and correlated with the biological activity of the assembly. This new approach has unique advantages in tuning the pH-responsiveness of self-assembled peptides across a large pH range in a complex biological environment. We anticipate the ionizable amino acids developed here can be widely applicable to the synthesis and self-assembly of many amphiphilic peptides with endowed pH-responsive properties to enhance their biological activities toward applications ranging from targeted therapeutic delivery to proton transport.

A Protein Semisynthesis-Based Strategy to Investigate the Functional Impact of Linker Histone Serine ADP-Ribosylation.

Recently developed chemical and enzyme-based technologies to install serine ADP-ribosylation onto synthetic peptides have enabled new approaches to study poly(ADP-ribose) polymerase (PARP) biology. Here, we establish a generalizable strategy to prepare ADP-ribosylated peptides that are compatible with N-terminal, C-terminal, and sequential protein ligation reactions. Two unique protein-assembly routes are employed to generate full-length linker histone constructs that are homogeneously ADP-ribosylated at known DNA damage-dependent modification sites. We found that serine mono-ADP-ribosylation is sufficient to alleviate linker histone-dependent chromatin compaction and that this effect is amplified by ADP-ribose chain elongation. Our work will greatly expand the scope of ADP-ribose-modified proteins that can be constructed via semisynthesis, which is rapidly emerging as a robust approach to elucidate the direct effects that site-specific serine mono- and poly-ADP-ribosylation have on protein function.

Mechanism of karyopherin-ß2 binding and nuclear import of ALS variants FUS(P525L) and FUS(R495X).

Mutations in the RNA-binding protein FUS cause familial amyotropic lateral sclerosis (ALS). Several mutations that affect the proline-tyrosine nuclear localization signal (PY-NLS) of FUS cause severe juvenile ALS. FUS also undergoes liquid-liquid phase separation (LLPS) to accumulate in stress granules when cells are stressed. In unstressed cells, wild type FUS resides predominantly in the nucleus as it is imported by the importin Karyopherin-ß2 (Kapß2), which binds with high affinity to the C-terminal PY-NLS of FUS. Here, we analyze the interactions between two ALS-related variants FUS(P525L) and FUS(R495X) with importins, especially Kapß2, since they are still partially localized to the nucleus despite their defective/missing PY-NLSs. The crystal structure of the Kapß2·FUS(P525L)PY-NLS complex shows the mutant peptide making fewer contacts at the mutation site, explaining decreased affinity for Kapß2. Biochemical analysis revealed that the truncated FUS(R495X) protein, although missing the PY-NLS, can still bind Kapß2 and suppresses LLPS. FUS(R495X) uses its C-terminal tandem arginine-glycine-glycine regions, RGG2 and RGG3, to bind the PY-NLS binding site of Kapß2 for nuclear localization in cells when arginine methylation is inhibited. These findings suggest the importance of the C-terminal RGG regions in nuclear import and LLPS regulation of ALS variants of FUS that carry defective PY-NLSs.

Biophysical and Biochemical Characterization of TP0037, a d-Lactate Dehydrogenase, Supports an Acetogenic Energy Conservation Pathway in Treponema pallidum.

A longstanding conundrum in Treponema pallidum biology concerns how the spirochete generates sufficient energy to fulfill its complex pathogenesis processes during human syphilitic infection. For decades, it has been assumed that the bacterium relies solely on glucose catabolism (via glycolysis) for generation of its ATP. However, the organism's robust motility, believed to be essential for human tissue invasion and dissemination, would require abundant ATP likely not provided by the parsimony of glycolysis. As such, additional ATP generation, either via a chemiosmotic gradient, substrate-level phosphorylation, or both, likely exists in T. pallidum Along these lines, we have hypothesized that T. pallidum exploits an acetogenic energy conservation pathway that relies on the redox chemistry of flavins. Central to this hypothesis is the apparent existence in T. pallidum of an acetogenic pathway for the conversion of d-lactate to acetate. Herein we have characterized the structural, biophysical, and biochemical properties of the first enzyme (d-lactate dehydrogenase [d-LDH]; TP0037) predicted in this pathway. Binding and enzymatic studies showed that recombinant TP0037 consumed d-lactate and NAD+ to produce pyruvate and NADH. The crystal structure of TP0037 revealed a fold similar to that of other d-acid dehydrogenases; residues in the cofactor-binding and active sites were homologous to those of other known d-LDHs. The crystal structure and solution biophysical experiments revealed the protein's propensity to dimerize, akin to other d-LDHs. This study is the first to elucidate the enzymatic properties of T. pallidum's d-LDH, thereby providing new compelling evidence for a flavin-dependent acetogenic energy conservation (ATP-generating) pathway in T. pallidumIMPORTANCE Because T. pallidum lacks a Krebs cycle and the capability for oxidative phosphorylation, historically it has been difficult to reconcile how the syphilis spirochete generates sufficient ATP to fulfill its energy needs, particularly for its robust motility, solely from glycolysis. We have postulated the existence in T. pallidum of a flavin-dependent acetogenic energy conservation pathway that would generate additional ATP for T. pallidum bioenergetics. In the proposed acetogenic pathway, first d-lactate would be converted to pyruvate. Pyruvate would then be metabolized to acetate in three additional steps, with ATP being generated via substrate-level phosphorylation. This study provides structural, biochemical, and biophysical evidence for the first T. pallidum enzyme in the pathway (TP0037; d-lactate dehydrogenase) requisite for the conversion of d-lactate to pyruvate. The findings represent the first experimental evidence to support a role for an acetogenic energy conservation pathway that would contribute to nonglycolytic ATP production in T. pallidum.

Tissue-Specific Regulation of the Wnt/ß-Catenin Pathway by PAGE4 Inhibition of Tankyrase.

Spatiotemporal control of Wnt/ß-catenin signaling is critical for organism development and homeostasis. The poly-(ADP)-ribose polymerase Tankyrase (TNKS1) promotes Wnt/ß-catenin signaling through PARylation-mediated degradation of AXIN1, a component of the ß-catenin destruction complex. Although Wnt/ß-catenin is a niche-restricted signaling program, tissue-specific factors that regulate TNKS1 are not known. Here, we report prostate-associated gene 4 (PAGE4) as a tissue-specific TNKS1 inhibitor that robustly represses canonical Wnt/ß-catenin signaling in human cells, zebrafish, and mice. Structural and biochemical studies reveal that PAGE4 acts as an optimal substrate decoy that potently hijacks substrate binding sites on TNKS1 to prevent AXIN1 PARylation and degradation. Consistently, transgenic expression of PAGE4 in mice phenocopies TNKS1 knockout. Physiologically, PAGE4 is selectively expressed in stromal prostate fibroblasts and functions to establish a proper Wnt/ß-catenin signaling niche through suppression of autocrine signaling. Our findings reveal a non-canonical mechanism for TNKS1 inhibition that functions to establish tissue-specific control of the Wnt/ß-catenin pathway.

Recognition of nuclear export signals by CRM1 carrying the oncogenic E571K mutation.

The E571K mutation of CRM1 is highly prevalent in some cancers, but its mechanism of tumorigenesis is unclear. Glu571 of CRM1 is located in its nuclear export signal (NES)-binding groove, suggesting that binding of select NESs may be altered. We generated HEK 293 cells with either monoallelic CRM1WT/E571K or biallelic CRM1E571K/E571K using CRISPR/Cas9. We also combined analysis of binding affinities and structures of 27 diverse NESs for wild-type and E571K CRM1 with structure-based bioinformatics. While most NESs bind the two CRM1 similarly, NESs from Mek1, eIF4E-transporter, and RPS2 showed >10-fold affinity differences. These NESs have multiple charged side chains binding close to CRM1 position 571, but this feature alone was not sufficient to predict different binding to CRM1(E571K). Consistent with eIF4E-transporter NES binding weaker to CRM1(E571K), eIF4E-transporter was mislocalized in tumor cells carrying CRM1(E571K). This serves as proof of concept that understanding how CRM1(E571K) affects NES binding provides a platform for identifying cargoes that are mislocalized in cancer upon CRM1 mutation. Finally, we showed that large affinity changes seen with some NES peptides (of Mek1 and RPS2) do not always translate to the full-length cargoes, suggesting limitations with current NES prediction methods. Therefore, comprehensive studies like ours are imperative to identify CRM1 cargoes with real pathogenic potential.

Genetic and structural studies of RABL3 reveal an essential role in lymphoid development and function.

The small GTPase RABL3 is an oncogene of unknown physiological function. Homozygous knockout alleles of mouse Rabl3 were embryonic lethal, but a viable hypomorphic allele (xiamen [xm]) causing in-frame deletion of four amino acids from the interswitch region resulted in profound defects in lymphopoiesis. Impaired lymphoid progenitor development led to deficiencies of B cells, T cells, and natural killer (NK) cells in Rabl3xm/xm mice. T cells and NK cells exhibited impaired cytolytic activity, and mice infected with mouse cytomegalovirus (MCMV) displayed elevated titers in the spleen. Myeloid cells were normal in number and function. Biophysical and crystallographic studies demonstrated that RABL3 formed a homodimer in solution via interactions between the effector binding surfaces on each subunit; monomers adopted a typical small G protein fold. RABL3xm displayed a large compensatory alteration in switch I, which adopted a ß-strand configuration normally provided by the deleted interswitch residues, thereby permitting homodimer formation. Dysregulated effector binding due to conformational changes in the switch I-interswitch-switch II module likely underlies the xm phenotype. One such effector may be GPR89, putatively an ion channel or G protein-coupled receptor (GPCR). RABL3, but not RABL3xm, strongly associated with and stabilized GPR89, and an N-ethyl-N-nitrosourea (ENU)-induced mutation (explorer) in Gpr89 phenocopied Rabl3xm.

Cryo-EM analyses reveal the common mechanism and diversification in the activation of RET by different ligands.

RET is a receptor tyrosine kinase (RTK) that plays essential roles in development and has been implicated in several human diseases. Different from most of RTKs, RET requires not only its cognate ligands but also co-receptors for activation, the mechanisms of which remain unclear due to lack of high-resolution structures of the ligand/co-receptor/receptor complexes. Here, we report cryo-EM structures of the extracellular region ternary complexes of GDF15/GFRAL/RET, GDNF/GFRα1/RET, NRTN/GFRα2/RET and ARTN/GFRα3/RET. These structures reveal that all the four ligand/co-receptor pairs, while using different atomic interactions, induce a specific dimerization mode of RET that is poised to bring the two kinase domains into close proximity for cross-phosphorylation. The NRTN/GFRα2/RET dimeric complex further pack into a tetrameric assembly, which is shown by our cell-based assays to regulate the endocytosis of RET. Our analyses therefore reveal both the common mechanism and diversification in the activation of RET by different ligands.

Fucosylated Molecules Competitively Interfere with Cholera Toxin Binding to Host Cells.

Cholera toxin (CT) enters host intestinal epithelia cells, and its retrograde transport to the cytosol results in the massive loss of fluids and electrolytes associated with severe dehydration. To initiate this intoxication process, the B subunit of CT (CTB) first binds to a cell surface receptor displayed on the apical surface of the intestinal epithelia. While the monosialoganglioside GM1 is widely accepted to be the sole receptor for CT, intestinal epithelial cell lines also utilize fucosylated glycan epitopes on glycoproteins to facilitate cell surface binding and endocytic uptake of the toxin. Further, l-fucose can competively inhibit CTB binding to intestinal epithelia cells. Here, we use competition binding assays with l-fucose analogs to decipher the molecular determinants for l-fucose inhibition of cholera toxin subunit B (CTB) binding. Additionally, we find that mono- and difucosylated oligosaccharides are more potent inhibitors than l-fucose alone, with the LeY tetrasaccharide emerging as the most potent inhibitor of CTB binding to two colonic epithelial cell lines (T84 and Colo205). Finally, a non-natural fucose-containing polymer inhibits CTB binding two orders of magnitude more potently than the LeY glycan when tested against Colo205 cells. This same polymer also inhibits CTB binding to T84 cells and primary human jejunal epithelial cells in a dose-dependent manner. These findings suggest the possibility that polymeric display of fucose might be exploited as a prophylactic or therapeutic approach to block the action of CT toward the human intestinal epithelium.

Using two-site binding models to analyze microscale thermophoresis data.

The emergence of microscale thermophoresis (MST) as a technique for determining the dissociation constants for bimolecular interactions has enabled these quantities to be measured in systems that were previously difficult or impracticable. However, most models for analyses of these data featured the assumption of a simple 1:1 binding interaction. The only model widely used for multiple binding sites was the Hill equation. Here, we describe two new MST analytic models that assume a 1:2 binding scheme: the first features two microscopic binding constants (KD(1) and KD(2)), while the other assumes symmetry in the bivalent molecule, culminating in a model with a single macroscopic dissociation constant (KD,M) and a single factor (α) that accounts for apparent cooperativity in the binding. We also discuss the general applicability of the Hill equation for MST data. The performances of the algorithms on both real and simulated data are assessed, and implementation of the algorithms in the MST analysis program PALMIST is discussed.

Functional clues from the crystal structure of an orphan periplasmic ligand-binding protein from Treponema pallidum.

The spirochete Treponema pallidum is the causative agent of syphilis, a sexually transmitted infection of major global importance. Other closely related subspecies of Treponema also are the etiological agents of the endemic treponematoses, such as yaws, pinta, and bejel. The inability of T. pallidum and its close relatives to be cultured in vitro has prompted efforts to characterize T. pallidum's proteins structurally and biophysically, particularly those potentially relevant to treponemal membrane biology, with the goal of possibly revealing the functions of those proteins. This report describes the structure of the treponemal protein Tp0737; this polypeptide has a fold characteristic of a class of periplasmic ligand-binding proteins associated with ABC-type transporters. Although no ligand for the protein was observed in electron-density maps, and thus the nature of the native ligand remains obscure, the structural data described herein provide a foundation for further efforts to elucidate the ligand and thus the function of this protein in T. pallidum.

Molecular insights into the enzymatic diversity of flavin-trafficking protein (Ftp; formerly ApbE) in flavoprotein biogenesis in the bacterial periplasm.

We recently reported a flavin-trafficking protein (Ftp) in the syphilis spirochete Treponema pallidum (Ftp_Tp) as the first bacterial metal-dependent FAD pyrophosphatase that hydrolyzes FAD into AMP and FMN in the periplasm. Orthologs of Ftp_Tp in other bacteria (formerly ApbE) appear to lack this hydrolytic activity; rather, they flavinylate the redox subunit, NqrC, via their metal-dependent FMN transferase activity. However, nothing has been known about the nature or mechanism of metal-dependent Ftp catalysis in either Nqr- or Rnf-redox-containing bacteria. In the current study, we identified a bimetal center in the crystal structure of Escherichia coli Ftp (Ftp_Ec) and show via mutagenesis that a single amino acid substitution converts it from an FAD-binding protein to a Mg(2+)-dependent FAD pyrophosphatase (Ftp_Tp-like). Furthermore, in the presence of protein substrates, both types of Ftps are capable of flavinylating periplasmic redox-carrying proteins (e.g., RnfG_Ec) via the metal-dependent covalent attachment of FMN. A high-resolution structure of the Ftp-mediated flavinylated protein of Shewanella oneidensis NqrC identified an essential lysine in phosphoester-threonyl-FMN bond formation in the posttranslationally modified flavoproteins. Together, these discoveries broaden our understanding of the physiological capabilities of the bacterial periplasm, and they also clarify a possible mechanism by which flavoproteins are generated.

Secondary PDZ domain-binding site on class B plexins enhances the affinity for PDZ-RhoGEF.

PDZ domains are abundant protein interaction modules and typically recognize a short motif at the C terminus of their ligands, with a few residues in the motif endowing the binding specificity. The sequence-based rules, however, cannot fully account for the specificity between the vast number of PDZ domains and ligands in the cell. Plexins are transmembrane receptors that regulate processes such as axon guidance and angiogenesis. Two related guanine nucleotide exchange factors (GEFs), PDZ-RhoGEF and leukemia-associated RhoGEF (LARG), use their PDZ domains to bind class B plexins and play critical roles in signaling. Here, we present the crystal structure of the full-length cytoplasmic region of PlexinB2 in complex with the PDZ domain of PDZ-RhoGEF. The structure reveals that, in addition to the canonical C-terminal motif/PDZ interaction, the 3D domain of PlexinB2 forms a secondary interface with the PDZ domain. Our biophysical and cell-based assays show that the secondary interface contributes to the specific interaction between plexin and PDZ-RhoGEF and to signaling by plexin in the cell. Formation of secondary interfaces may be a general mechanism for increasing affinity and specificity of modular domain-mediated interactions.

A Moraxella catarrhalis two-component signal transduction system necessary for growth in liquid media affects production of two lysozyme inhibitors.

There are a paucity of data concerning gene products that could contribute to the ability of Moraxella catarrhalis to colonize the human nasopharynx. Inactivation of a gene (mesR) encoding a predicted response regulator of a two-component signal transduction system in M. catarrhalis yielded a mutant unable to grow in liquid media. This mesR mutant also exhibited increased sensitivity to certain stressors, including polymyxin B, SDS, and hydrogen peroxide. Inactivation of the gene (mesS) encoding the predicted cognate sensor (histidine) kinase yielded a mutant with the same inability to grow in liquid media as the mesR mutant. DNA microarray and real-time reverse transcriptase PCR analyses indicated that several genes previously shown to be involved in the ability of M. catarrhalis to persist in the chinchilla nasopharynx were upregulated in the mesR mutant. Two other open reading frames upregulated in the mesR mutant were shown to encode small proteins (LipA and LipB) that had amino acid sequence homology to bacterial adhesins and structural homology to bacterial lysozyme inhibitors. Inactivation of both lipA and lipB did not affect the ability of M. catarrhalis O35E to attach to a human bronchial epithelial cell line in vitro. Purified recombinant LipA and LipB fusion proteins were each shown to inhibit human lysozyme activity in vitro and in saliva. A lipA lipB deletion mutant was more sensitive than the wild-type parent strain to killing by human lysozyme in the presence of human apolactoferrin. This is the first report of the production of lysozyme inhibitors by M. catarrhalis.

Identification of an outer membrane lipoprotein involved in nasopharyngeal colonization by Moraxella catarrhalis in an animal model.

Colonization of the human nasopharynx by Moraxella catarrhalis is presumed to involve attachment of this bacterium to the mucosa. DNA microarray analysis was used to determine whether attachment of M. catarrhalis to human bronchial epithelial (HBE) cells in vitro affected gene expression in this bacterium. Attachment affected expression of at least 454 different genes, with 163 being upregulated and 291 being downregulated. Among the upregulated genes was one (ORF113) previously annotated as encoding a protein with some similarity to outer membrane protein A (OmpA). The protein encoded by ORF113 was predicted to have a signal peptidase II cleavage site, and globomycin inhibition experiments confirmed that this protein was indeed a lipoprotein. The ORF113 protein also contained a predicted peptidoglycan-binding domain in its C-terminal half. The use of mutant and recombinant M. catarrhalis strains confirmed that the ORF113 protein was present in outer membrane preparations, and this protein was also shown to be at least partially exposed on the bacterial cell surface. A mutant unable to produce the ORF113 protein showed little or no change in its growth rate in vitro, in its ability to attach to HBE cells in vitro, or in its autoagglutination characteristics, but it did exhibit a reduced ability to survive in the chinchilla nasopharynx. This is the first report of a lipoprotein essential to the ability of M. catarrhalis to persist in an animal model.

Structural basis for autoactivation of human Mst2 kinase and its regulation by RASSF5.

The tumor-suppressive Hippo pathway controls tissue homeostasis through balancing cell proliferation and apoptosis. Activation of the kinases Mst1 and Mst2 (Mst1/2) is a key upstream event in this pathway and remains poorly understood. Mst1/2 and their critical regulators RASSFs contain Salvador/RASSF1A/Hippo (SARAH) domains that can homo- and heterodimerize. Here, we report the crystal structures of human Mst2 alone and bound to RASSF5. Mst2 undergoes activation through transautophosphorylation at its activation loop, which requires SARAH-mediated homodimerization. RASSF5 disrupts Mst2 homodimer and blocks Mst2 autoactivation. Binding of RASSF5 to already activated Mst2, however, does not inhibit its kinase activity. Thus, RASSF5 can act as an inhibitor or a potential positive regulator of Mst2, depending on whether it binds to Mst2 before or after activation-loop phosphorylation. We propose that these temporally sensitive functions of RASSFs enable the Hippo pathway to respond to and integrate diverse cellular signals.

Structure of the human cohesin inhibitor Wapl.

Cohesin, along with positive regulators, establishes sister-chromatid cohesion by forming a ring to circle chromatin. The wings apart-like protein (Wapl) is a key negative regulator of cohesin and forms a complex with precocious dissociation of sisters protein 5 (Pds5) to promote cohesin release from chromatin. Here we report the crystal structure and functional characterization of human Wapl. Wapl contains a flexible, variable N-terminal region (Wapl-N) and a conserved C-terminal domain (Wapl-C) consisting of eight HEAT (Huntingtin, Elongation factor 3, A subunit, and target of rapamycin) repeats. Wapl-C folds into an elongated structure with two lobes. Structure-based mutagenesis maps the functional surface of Wapl-C to two distinct patches (I and II) on the N lobe and a localized patch (III) on the C lobe. Mutating critical patch I residues weaken Wapl binding to cohesin and diminish sister-chromatid resolution and cohesin release from mitotic chromosomes in human cells and Xenopus egg extracts. Surprisingly, patch III on the C lobe does not contribute to Wapl binding to cohesin or its known regulators. Although patch I mutations reduce Wapl binding to intact cohesin, they do not affect Wapl-Pds5 binding to the cohesin subcomplex of sister chromatid cohesion protein 1 (Scc1) and stromal antigen 2 (SA2) in vitro, which is instead mediated by Wapl-N. Thus, Wapl-N forms extensive interactions with Pds5 and Scc1-SA2. Wapl-C interacts with other cohesin subunits and possibly unknown effectors to trigger cohesin release from chromatin.