The reversible inhibitor SR-4835 binds Cdk12/cyclin K in a noncanonical G-loop conformation.

Inhibition of cyclin-dependent kinases (CDKs) has evolved as an emerging anticancer strategy. In addition to the cell cycle-regulating CDKs, the transcriptional kinases Cdk12 and Cdk13 have become the focus of interest as they mediate a variety of functions, including the transition from transcription initiation to elongation and termination, precursor mRNA splicing, and intronic polyadenylation. Here, we determine the crystal structure of the small molecular inhibitor SR-4835 bound to the Cdk12/cyclin K complex at 2.68 Å resolution. The compound's benzimidazole moiety is embedded in a unique hydrogen bond network mediated by the kinase hinge region with flanking hydroxy groups of the Y815 and D819 side chains. Whereas the SR-4835 head group targets the adenine-binding pocket, the kinase's glycine-rich loop is shifted down toward the activation loop. Additionally, the αC-helix adopts an inward conformation, and the phosphorylated T-loop threonine interacts with all three canonical arginines, a hallmark of CDK activation that is altered in Cdk12 and Cdk13. Dose-response inhibition measurements with recombinant CMGC kinases show that SR-4835 is highly specific for Cdk12 and Cdk13 following a 10-fold lower potency for Cdk10. Whereas other CDK-targeting compounds exhibit tighter binding affinities and higher potencies for kinase inhibition, SR-4835 can be considered a selective transcription elongation antagonist. Our results provide the basis for a rational improvement of SR-4835 toward Cdk12 inhibition and a gain in selectivity over other transcription regulating CDKs.

A conserved isoleucine in the binding pocket of RIG-I controls immune tolerance to mitochondrial RNA.

RIG-I is a cytosolic receptor of viral RNA essential for the immune response to numerous RNA viruses. Accordingly, RIG-I must sensitively detect viral RNA yet tolerate abundant self-RNA species. The basic binding cleft and an aromatic amino acid of the RIG-I C-terminal domain(CTD) mediate high-affinity recognition of 5'triphosphorylated and 5'base-paired RNA(dsRNA). Here, we found that, while 5'unmodified hydroxyl(OH)-dsRNA demonstrated residual activation potential, 5'-monophosphate(5'p)-termini, present on most cellular RNAs, prevented RIG-I activation. Determination of CTD/dsRNA co-crystal structures and mutant activation studies revealed that the evolutionarily conserved I875 within the CTD sterically inhibits 5'p-dsRNA binding. RIG-I(I875A) was activated by both synthetic 5'p-dsRNA and endogenous long dsRNA within the polyA-rich fraction of total cellular RNA. RIG-I(I875A) specifically interacted with long, polyA-bearing, mitochondrial(mt) RNA, and depletion of mtRNA from total RNA abolished its activation. Altogether, our study demonstrates that avoidance of 5'p-RNA recognition is crucial to prevent mtRNA-triggered RIG-I-mediated autoinflammation.

Interactome map uncovers phosphatidylserine transport by oxysterol-binding proteins.

The internal organization of eukaryotic cells into functionally specialized, membrane-delimited organelles of unique composition implies a need for active, regulated lipid transport. Phosphatidylserine (PS), for example, is synthesized in the endoplasmic reticulum and then preferentially associates--through mechanisms not fully elucidated--with the inner leaflet of the plasma membrane. Lipids can travel via transport vesicles. Alternatively, several protein families known as lipid-transfer proteins (LTPs) can extract a variety of specific lipids from biological membranes and transport them, within a hydrophobic pocket, through aqueous phases. Here we report the development of an integrated approach that combines protein fractionation and lipidomics to characterize the LTP-lipid complexes formed in vivo. We applied the procedure to 13 LTPs in the yeast Saccharomyces cerevisiae: the six Sec14 homology (Sfh) proteins and the seven oxysterol-binding homology (Osh) proteins. We found that Osh6 and Osh7 have an unexpected specificity for PS. In vivo, they participate in PS homeostasis and the transport of this lipid to the plasma membrane. The structure of Osh6 bound to PS reveals unique features that are conserved among other metazoan oxysterol-binding proteins (OSBPs) and are required for PS recognition. Our findings represent the first direct evidence, to our knowledge, for the non-vesicular transfer of PS from its site of biosynthesis (the endoplasmic reticulum) to its site of biological activity (the plasma membrane). We describe a new subfamily of OSBPs, including human ORP5 and ORP10, that transfer PS and propose new mechanisms of action for a protein family that is involved in several human pathologies such as cancer, dyslipidaemia and metabolic syndrome.

Structural basis of Cdk7 activation by dual T-loop phosphorylation.

Cyclin-dependent kinase 7 (Cdk7) occupies a central position in cell-cycle and transcriptional regulation owing to its function as both a CDK-activating kinase (CAK) and part of the general transcription factor TFIIH. Cdk7 forms an active complex upon association with Cyclin H and Mat1, and its catalytic activity is regulated by two phosphorylations in the activation segment (T loop): the canonical activating modification at T170 and another at S164. Here we report the crystal structure of the fully activated human Cdk7/Cyclin H/Mat1 complex containing both T-loop phosphorylations. Whereas pT170 coordinates a set of basic residues conserved in other CDKs, pS164 nucleates an arginine network involving all three subunits that is unique to the ternary Cdk7 complex. We identify differential dependencies of kinase activity and substrate recognition on individual phosphorylations within the Cdk7 T loop. The CAK function of Cdk7 is not affected by T-loop phosphorylation, whereas activity towards non-CDK substrates is increased several-fold by phosphorylation at T170. Moreover, dual T-loop phosphorylation at both T170 and S164 stimulates multi-site phosphorylation of transcriptional substrates-the RNA polymerase II (RNAPII) carboxy-terminal domain (CTD) and the SPT5 carboxy-terminal repeat (CTR) region. In human cells, Cdk7-regulatory phosphorylation is a two-step process in which phosphorylation of S164 precedes, and may prime, T170 phosphorylation. Thus, dual T-loop phosphorylation can regulate Cdk7 through multiple mechanisms, with pS164 supporting tripartite complex formation and possibly influencing Cdk7 processivity, while the canonical pT170 enhances kinase activity towards critical substrates involved in transcription.

Formation of Tat-TAR containing ribonucleoprotein complexes for biochemical and structural analyses.

Viruses manipulate multiple processes of the host cell machinery in order to replicate successfully in the infected cell. Among these, stimulation of transcription of the viral genes is crucial for lentiviruses such as HIV for increased protein expression levels and generation of escape mutants. The transactivation response (TAR) element at the 5'-end of HIV, SIV, BIV, EIAV or JDV retroviruses forms a unique RNA based promoter element that together with the transcription activator protein Tat stimulates viral gene expression at the level of transcription elongation. TAR is a double stranded non-coding RNA of typically 24-40 nucleotides length. Together with Tat it interacts with the Cyclin T subunit of the positive transcription elongation factor P-TEFb to recruit Cyclin T and its corresponding Cyclin-dependent kinase Cdk9 to the RNA polymerase II. In vitro formations of these Tat-TAR containing ribonucleoprotein complexes are a key requisite for biochemical characterizations and interaction studies that eventually will allow structural analyses. Here, we describe purification methods of the different factors employed and chromatography techniques that yield highly specific complex assemblies suitable for crystallization.

Abemaciclib is a potent inhibitor of DYRK1A and HIP kinases involved in transcriptional regulation.

Homeodomain-interacting protein kinases (HIPKs) belong to the CMGC kinase family and are closely related to dual-specificity tyrosine phosphorylation-regulated kinases (DYRKs). HIPKs are regulators of various signaling pathways and involved in the pathology of cancer, chronic fibrosis, diabetes, and multiple neurodegenerative diseases. Here, we report the crystal structure of HIPK3 in its apo form at 2.5 Å resolution. Recombinant HIPKs and DYRK1A are auto-activated and phosphorylate the negative elongation factor SPT5, the transcription factor c-Myc, and the C-terminal domain of RNA polymerase II, suggesting a direct function in transcriptional regulation. Based on a database search, we identified abemaciclib, an FDA-approved Cdk4/Cdk6 inhibitor used for the treatment of metastatic breast cancer, as potent inhibitor of HIPK2, HIPK3, and DYRK1A. We determined the crystal structures of HIPK3 and DYRK1A bound to abemaciclib, showing a similar binding mode to the hinge region of the kinase as observed for Cdk6. Remarkably, DYRK1A is inhibited by abemaciclib to the same extent as Cdk4/Cdk6 in vitro, raising the question of whether targeting of DYRK1A contributes to the transcriptional inhibition and therapeutic activity of abemaciclib.

Structure-activity relationship study of THZ531 derivatives enables the discovery of BSJ-01-175 as a dual CDK12/13 covalent inhibitor with efficacy in Ewing sarcoma.

Development of inhibitors targeting CDK12/13 is of increasing interest as a potential therapy for cancers as these compounds inhibit transcription of DNA damage response (DDR) genes. We previously described THZ531, a covalent inhibitor with selectivity for CDK12/13. In order to elucidate structure-activity relationship (SAR), we have undertaken a medicinal chemistry campaign and established a focused library of THZ531 analogs. Among these analogs, BSJ-01-175 demonstrates exquisite selectivity, potent inhibition of RNA polymerase II phosphorylation, and downregulation of CDK12-targeted genes in cancer cells. A 3.0 Å co-crystal structure with CDK12/CycK provides a structural rational for selective targeting of Cys1039 located in a C-terminal extension from the kinase domain. With moderate pharmacokinetic properties, BSJ-01-175 exhibits efficacy against an Ewing sarcoma tumor growth in a patient-derived xenograft (PDX) mouse model following 10 mg/kg once a day, intraperitoneal administration. Taken together, BSJ-01-175 represents the first selective CDK12/13 covalent inhibitor with in vivo efficacy reported to date.

Structural studies of phosphoglucose isomerase from Mycobacterium tuberculosis H37Rv.

Phosphoglucose isomerase (PGI) plays a key role in both glycolysis and gluconeogenesis inside the cell, whereas outside the cell it exhibits cytokine properties. PGI is also known to act as an autocrine motility factor, a neuroleukin agent and a differentiation and maturation mediator. Here, the first crystal structure of PGI from Mycobacterium tuberculosis H37Rv (Mtb) is reported. The structure was refined at 2.25 A resolution and revealed the presence of one molecule in the asymmetric unit with two globular domains. As known previously, the active site of Mtb PGI contains conserved residues including Glu356, Glu216 and His387 (where His387 is from the neighbouring molecule). The crystal structure of Mtb PGI was observed to be rather more similar to human PGI than other nonbacterial PGIs, with only a few differences being detected in the loops, arm and hook regions of the human and Mtb PGIs, suggesting that the M. tuberculosis enzyme uses the same enzyme mechanism.

Crystal structure of the human NLRP9 pyrin domain suggests a distinct mode of inflammasome assembly.

Inflammasomes are cytosolic multimeric signaling complexes of the innate immune system that induce activation of caspases. The NOD-like receptor NLRP9 recruits the adaptor protein ASC to form an ASC-dependent inflammasome to limit rotaviral replication in intestinal epithelial cells, but only little is known about the molecular mechanisms regulating and driving its assembly. Here, we present the crystal structure of the human NLRP9 pyrin domain (PYD). We show that NLRP9PYD is not able to self-polymerize nor to nucleate ASC specks in HEK293T cells. A comparison with filament-forming PYDs revealed that NLRP9PYD adopts a conformation compatible with filament formation, but several charge inversions of interfacing residues might cause repulsive effects that prohibit self-oligomerization. These results propose that inflammasome assembly of NLRP9 might differ largely from what we know of other inflammasomes.

Cyclin box structure of the P-TEFb subunit cyclin T1 derived from a fusion complex with EIAV tat.

The positive transcription elongation factor b (P-TEFb) is an essential regulator of viral gene expression during the life cycle of human immunodeficiency virus type 1 (HIV-1). Its cyclin T1 subunit forms a ternary complex with the viral transcriptional transactivator (Tat) protein and the transactivation response (TAR) RNA element thereby activating cyclin dependent kinase 9 (Cdk9), which stimulates transcription at the level of chain elongation. We report the structure of the cyclin box domain of human cyclin T1 at a resolution of 2.67 A. The structure was obtained by crystallographic analysis of a fusion protein composed of cyclin T1 linked to the transactivator protein Tat from equine infectious anemia virus (EIAV), which is functionally and structurally related to HIV-1 Tat. The conserved cyclin box domain of cyclin T1 exhibits structural features for interaction with physiological binding partners such as Cdk9. A recognition site for Cdk/Cyclin substrates is partly covered by a cyclin T-specific insert, suggesting specific interactions with regulatory factors. The previously identified Tat/TAR recognition motif (TRM) forms a C-terminal helix that is partly occluded in the cyclin box repeat interface, while cysteine 261 is accessible to form an intermolecular zinc finger with Tat. Residues of the TRM contribute to a positively charged groove that may directly attract RNA molecules. The EIAV Tat protein instead appeared undefined from the electron density map suggesting that it is highly disordered. Functional experiments confirmed the TAR binding properties of the fusion protein and suggested residues on the second cyclin box repeat to contribute to Tat stimulated transcription.

Crystallization and preliminary X-ray characterization of phosphoglucose isomerase from Mycobacterium tuberculosis H37Rv.

Phosphoglucose isomerase is a ubiquitous enzyme that catalyzes the isomerization of D-glucopyranose-6-phosphate to D-fructofuranose-6-phosphate. The present investigation reports the expression, purification, crystallization and preliminary crystallographic studies of the phosphoglucose isomerase from Mycobacterium tuberculosis H37Rv, which shares 46% sequence identity with that of its human host. The recombinant protein, which was prepared using an Escherichia coli expression system, was crystallized by the hanging-drop vapour-diffusion method. The crystals diffracted to a resolution of 2.8 A and belonged to the orthorhombic space group I2(1)2(1)2(1), with unit-cell parameters a = 109.0, b = 119.8, c = 138.9 A.

Endocytic sorting motif interactions involved in Nef-mediated downmodulation of CD4 and CD3.

Lentiviral Nefs recruit assembly polypeptide complexes and target sorting motifs in cellular receptors to induce their internalization. While Nef-mediated CD4 downmodulation is conserved, the ability to internalize CD3 was lost in HIV-1 and its precursors. Although both functions play key roles in lentiviral replication and pathogenicity, the underlying structural requirements are poorly defined. Here, we determine the structure of SIVmac239 Nef bound to the ExxxLM motif of another Nef molecule at 2.5 Å resolution. This provides a basis for a structural model, where a hydrophobic crevice in simian immunodeficiency virus (SIV) Nef targets a dileucine motif in CD4 and a tyrosine-based motif in CD3. Introducing key residues into this crevice of HIV-1 Nef enables CD3 binding but an additional N-terminal tyrosine motif is required for internalization. Our resolution of the CD4/Nef/AP2 complex and generation of HIV-1 Nefs capable of CD3 downregulation provide insights into sorting motif interactions and target discrimination of Nef.HIV and simian immunodeficiency virus (SIV) Nef proteins both stimulate the clathrin-mediated endocytosis of CD4 but differ in downmodulation of the immune receptor CD3. Here, the authors present the structure of SIV Nef bound to the ExxxLM motif of another Nef molecule, which allows them to propose a model how Nef recognizes these motifs in CD3 and CD4.

pH-dependent conformational flexibility of the SARS-CoV main proteinase (M(pro)) dimer: molecular dynamics simulations and multiple X-ray structure analyses.

The SARS coronavirus main proteinase (M(pro)) is a key enzyme in the processing of the viral polyproteins and thus an attractive target for the discovery of drugs directed against SARS. The enzyme has been shown by X-ray crystallography to undergo significant pH-dependent conformational changes. Here, we assess the conformational flexibility of the M(pro) by analysis of multiple crystal structures (including two new crystal forms) and by molecular dynamics (MD) calculations. The MD simulations take into account the different protonation states of two histidine residues in the substrate-binding site and explain the pH-activity profile of the enzyme. The low enzymatic activity of the M(pro) monomer and the need for dimerization are also discussed.

Structural and Functional Analysis of the Cdk13/Cyclin K Complex.

Cyclin-dependent kinases regulate the cell cycle and transcription in higher eukaryotes. We have determined the crystal structure of the transcription kinase Cdk13 and its Cyclin K subunit at 2.0 Å resolution. Cdk13 contains a C-terminal extension helix composed of a polybasic cluster and a DCHEL motif that interacts with the bound ATP. Cdk13/CycK phosphorylates both Ser5 and Ser2 of the RNA polymerase II C-terminal domain (CTD) with a preference for Ser7 pre-phosphorylations at a C-terminal position. The peptidyl-prolyl isomerase Pin1 does not change the phosphorylation specificities of Cdk9, Cdk12, and Cdk13 but interacts with the phosphorylated CTD through its WW domain. Using recombinant proteins, we find that flavopiridol inhibits Cdk7 more potently than it does Cdk13. Gene expression changes after knockdown of Cdk13 or Cdk12 are markedly different, with enrichment of growth signaling pathways for Cdk13-dependent genes. Together, our results provide insights into the structure, function, and activity of human Cdk13/CycK.

Lipid Cooperativity as a General Membrane-Recruitment Principle for PH Domains.

Many cellular processes involve the recruitment of proteins to specific membranes, which are decorated with distinctive lipids that act as docking sites. The phosphoinositides form signaling hubs, and we examine mechanisms underlying recruitment. We applied a physiological, quantitative, liposome microarray-based assay to measure the membrane-binding properties of 91 pleckstrin homology (PH) domains, the most common phosphoinositide-binding target. 10,514 experiments quantified the role of phosphoinositides in membrane recruitment. For most domains examined, the observed binding specificity implied cooperativity with additional signaling lipids. Analyses of PH domains with similar lipid-binding profiles identified a conserved motif, mutations in which-including some found in human cancers-induced discrete changes in binding affinities in vitro and protein mislocalization in vivo. The data set reveals cooperativity as a key mechanism for membrane recruitment and, by enabling the interpretation of disease-associated mutations, suggests avenues for the design of small molecules targeting PH domains.

The structure and substrate specificity of human Cdk12/Cyclin K.

Phosphorylation of the RNA polymerase II C-terminal domain (CTD) by cyclin-dependent kinases is important for productive transcription. Here we determine the crystal structure of Cdk12/CycK and analyse its requirements for substrate recognition. Active Cdk12/CycK is arranged in an open conformation similar to that of Cdk9/CycT but different from those of cell cycle kinases. Cdk12 contains a C-terminal extension that folds onto the N- and C-terminal lobes thereby contacting the ATP ribose. The interaction is mediated by an HE motif followed by a polybasic cluster that is conserved in transcriptional CDKs. Cdk12/CycK showed the highest activity on a CTD substrate prephosphorylated at position Ser7, whereas the common Lys7 substitution was not recognized. Flavopiridol is most potent towards Cdk12 but was still 10-fold more potent towards Cdk9. T-loop phosphorylation of Cdk12 required coexpression with a Cdk-activating kinase. These results suggest the regulation of Pol II elongation by a relay of transcriptionally active CTD kinases.

Structural analyses of the Slm1-PH domain demonstrate ligand binding in the non-canonical site.

BACKGROUND: Pleckstrin homology (PH) domains are common membrane-targeting modules and their best characterized ligands are a set of important signaling lipids that include phosphatidylinositol phosphates (PtdInsPs). PH domains recognize PtdInsPs through two distinct mechanisms that use different binding pockets on opposite sides of the ß-strands 1 and 2: i) a canonical binding site delimited by the ß1-ß2 and ß3-ß4loops and ii) a non-canonical binding site bordered by the ß1-ß2 and ß5-ß6loops. The PH domain-containing protein Slm1 from budding yeast Saccharomyces cerevisiae is required for actin cytoskeleton polarization and cell growth. We recently reported that this PH domain binds PtdInsPs and phosphorylated sphingolipids in a cooperative manner. PRINCIPAL FINDINGS: To study the structural basis for the Slm1-PH domain (Slm1-PH) specificity, we co-crystallized this domain with different soluble compounds that have structures analogous to anionic lipid head groups of reported Slm1 ligands: inositol 4-phosphate, which mimics phosphatidylinositol-4-phosphate (PtdIns(4)P), and phosphoserine as a surrogate for dihydrosphingosine 1-phosphate (DHS1-P). We found electron densities for the ligands within the so-called non-canonical binding site. An additional positively charged surface that contacts a phosphate group was identified next to the canonical binding site. CONCLUSIONS: Our results suggest that Slm1-PH utilizes a non-canonical binding site to bind PtdInsPs, similar to that described for the PH domains of ß-spectrin, Tiam1 and ArhGAP9. Additionally, Slm1-PH may have retained an active canonical site. We propose that the presence of both a canonical and a non-canonical binding pocket in Slm1-PH may account for the cooperative binding to PtdInsPs and DHS-1P.

Structural insights into the cyclin T1-Tat-TAR RNA transcription activation complex from EIAV.

The replication of many retroviruses is mediated by a transcriptional activator protein, Tat, which activates RNA polymerase II at the level of transcription elongation. Tat interacts with Cyclin T1 of the positive transcription-elongation factor P-TEFb to recruit the transactivation-response TAR RNA, which acts as a promoter element in the transcribed 5' end of the viral long terminal repeat. Here we present the structure of the cyclin box domain of Cyclin T1 in complex with the Tat protein from the equine infectious anemia virus and its corresponding TAR RNA. The basic RNA-recognition motif of Tat adopts a helical structure whose flanking regions interact with a cyclin T-specific loop in the first cyclin box repeat. Together, both proteins coordinate the stem-loop structure of TAR. Our findings show that Tat binds to a surface on Cyclin T1 similar to where recognition motifs from substrate and inhibitor peptides were previously found to interact within Cdk-cyclin pairs.

The crystal structure of thrombin-activable fibrinolysis inhibitor (TAFI) provides the structural basis for its intrinsic activity and the short half-life of TAFIa.

Mature thrombin-activable fibrinolysis inhibitor (TAFIa) is a highly unstable metallocarboxypeptidase that stabilizes blood clots by clipping C-terminal lysine residues from partially degraded fibrin. In accordance with its in vitro antifibrinolytic activity, animal studies have reported that inhibition of mature TAFI aids in the prevention of thrombosis. The level of TAFI activity is stringently regulated through (i) controlled proteolytic truncation of the zymogen (TAFI), generating the mature enzyme, TAFIa, and (ii) the short half-life of TAFIa. TAFI itself exhibits an intrinsic enzymatic activity, which is likely required to provide a baseline level of antifibrinolytic activity. The novel crystal structure presented here reveals that the active site of TAFI is accessible, providing the structural explanation for the its intrinsic activity. It also supports the notion that an "instability region" exists, in agreement with site-directed mutagenesis studies. Sulfate ions, bound to this region, point toward a potential heparin-binding site and could explain how heparin stabilizes TAFIa.