Heparan sulfate selectively inhibits the collagenase activity of cathepsin K.

Cathepsin K (CtsK) is a cysteine protease with potent collagenase activity. CtsK is highly expressed by bone-resorbing osteoclasts and plays an essential role in resorption of bone matrix. Although CtsK is known to bind heparan sulfate (HS), the structural details of the interaction, and how HS regulates the biological functions of CtsK, remains largely unknown. In this report, we discovered that HS is a multifaceted regulator of the structure and function of CtsK. Structurally, HS forms a highly stable complex with CtsK and induces its dimerization. Co-crystal structures of CtsK with bound HS oligosaccharides reveal the location of the HS binding site and suggest how HS may support dimerization. Functionally, HS plays a dual role in regulating the enzymatic activity of CtsK. While it preserves the peptidase activity of CtsK by stabilizing its active conformation, it inhibits the collagenase activity of CtsK in a sulfation level-dependent manner. These opposing effects can be explained by our finding that the HS binding site is remote from the active site, which allows HS to specifically inhibit the collagenase activity without affecting the peptidase activity. At last, we show that structurally defined HS oligosaccharides effectively block osteoclast resorption of bone in vitro without inhibiting osteoclast differentiation, which suggests that HS-based oligosaccharide might be explored as a new class of selective CtsK inhibitor for many diseases involving exaggerated bone resorption.

Heparan sulfate selectively inhibits the collagenase activity of cathepsin K.

Cathepsin K (CtsK) is a cysteine protease with potent collagenase activity. CtsK is highly expressed by bone-resorbing osteoclasts and plays an essential role in bone remodeling. Although CtsK is known to bind heparan sulfate (HS), the structural details of the interaction, and how HS ultimately regulates the biological functions of CtsK, remains largely unknown. In this report, we determined that CtsK preferably binds to larger HS oligosaccharides, such as dodecasaccharides (12mer), and that the12mer can induce monomeric CtsK to form a stable dimer in solution. Interestingly, while HS has no effect on the peptidase activity of CtsK, it greatly inhibits the collagenase activity of CtsK in a manner dependent on sulfation level. By forming a complex with CtsK, HS was able to preserve the full peptidase activity of CtsK for prolonged periods, likely by stabilizing its active conformation. Crystal structures of Ctsk with a bound 12mer, alone and in the presence of the endogenous inhibitor cystatin-C reveal the location of HS binding is remote from the active site. Mutagenesis based on these complex structures identified 6 basic residues of Ctsk that play essential roles in mediating HS-binding. At last, we show that HS 12mers can effectively block osteoclast resorption of bone in vitro. Combined, we have shown that HS can function as a multifaceted regulator of CtsK and that HS-based oligosaccharide might be explored as a new class of selective CtsK inhibitor in many diseases that involve exaggerated bone resorption.

Targeting the Structural Maturation Pathway of HIV-1 Reverse Transcriptase.

Formation of active HIV-1 reverse transcriptase (RT) proceeds via a structural maturation process that involves subdomain rearrangements and formation of an asymmetric p66/p66' homodimer. These studies were undertaken to evaluate whether the information about this maturation process can be used to identify small molecule ligands that retard or interfere with the steps involved. We utilized the isolated polymerase domain, p51, rather than p66, since the initial subdomain rearrangements are largely limited to this domain. Target sites at subdomain interfaces were identified and computational analysis used to obtain an initial set of ligands for screening. Chromatographic evaluations of the p51 homodimer/monomer ratio support the feasibility of this approach. Ligands that bind near the interfaces and a ligand that binds directly to a region of the fingers subdomain involved in subunit interface formation were identified, and the interactions were further characterized by NMR spectroscopy and X-ray crystallography. Although these ligands were found to reduce dimer formation, further efforts will be required to obtain ligands with higher binding affinity. In contrast with previous ligand identification studies performed on the RT heterodimer, subunit interface surfaces are solvent-accessible in the p51 and p66 monomers, making these constructs preferable for identification of ligands that directly interfere with dimerization.

XDSGUI: a graphical user interface for XDS, SHELX and ARCIMBOLDO.

XDSGUI is a lightweight graphical user interface (GUI) for the XDS, SHELX and ARCIMBOLDO program packages that serves both novice and experienced users in obtaining optimal processing and phasing results for X-ray, neutron and electron diffraction data. The design of the program enables data processing and phasing without command line usage, and supports advanced command flows in a simple user-modifiable and user-extensible way. The GUI supplies graphical information based on the tabular log output of the programs, which is more intuitive, comprehensible and efficient than text output can be.

Sen1 architecture: RNA-DNA hybrid resolution, autoregulation, and insights into SETX inactivation in AOA2.

The senataxin (SETX, Sen1 in yeasts) RNA-DNA hybrid resolving helicase regulates multiple nuclear transactions, including DNA replication, transcription, and DNA repair, but the molecular basis for Sen1 activities is ill defined. Here, Sen1 cryoelectron microscopy (cryo-EM) reconstructions reveal an elongated inchworm-like architecture. Sen1 is composed of an amino terminal helical repeat Sen1 N-terminal (Sen1N) regulatory domain that is flexibly linked to its C-terminal SF1B helicase motor core (Sen1Hel) via an intrinsically disordered tether. In an autoinhibited state, the Sen1Sen1N domain regulates substrate engagement by promoting occlusion of the RNA substrate-binding cleft. The X-ray structure of an activated Sen1Hel engaging single-stranded RNA and ADP-SO4 shows that the enzyme encircles RNA and implicates a single-nucleotide power stroke in the Sen1 RNA translocation mechanism. Together, our data unveil dynamic protein-protein and protein-RNA interfaces underpinning helicase regulation and inactivation of human SETX activity by RNA-binding-deficient mutants in ataxia with oculomotor apraxia 2 neurodegenerative disease.

Structural basis for pre-tRNA recognition and processing by the human tRNA splicing endonuclease complex.

Throughout bacteria, archaea and eukarya, certain tRNA transcripts contain introns. Pre-tRNAs with introns require splicing to form the mature anticodon stem loop. In eukaryotes, tRNA splicing is initiated by the heterotetrameric tRNA splicing endonuclease (TSEN) complex. All TSEN subunits are essential, and mutations within the complex are associated with a family of neurodevelopmental disorders known as pontocerebellar hypoplasia (PCH). Here, we report cryo-electron microscopy structures of the human TSEN-pre-tRNA complex. These structures reveal the overall architecture of the complex and the extensive tRNA binding interfaces. The structures share homology with archaeal TSENs but contain additional features important for pre-tRNA recognition. The TSEN54 subunit functions as a pivotal scaffold for the pre-tRNA and the two endonuclease subunits. Finally, the TSEN structures enable visualization of the molecular environments of PCH-causing missense mutations, providing insight into the mechanism of pre-tRNA splicing and PCH.

Cryo-EM reveals the architecture of the PELP1-WDR18 molecular scaffold.

PELP1 (Proline-, Glutamic acid-, Leucine-rich protein 1) is a large scaffolding protein that functions in many cellular pathways including steroid receptor (SR) coactivation, heterochromatin maintenance, and ribosome biogenesis. PELP1 is a proto-oncogene whose expression is upregulated in many human cancers, but how the PELP1 scaffold coordinates its diverse cellular functions is poorly understood. Here we show that PELP1 serves as the central scaffold for the human Rix1 complex whose members include WDR18, TEX10, and SENP3. We reconstitute the mammalian Rix1 complex and identified a stable sub-complex comprised of the conserved PELP1 Rix1 domain and WDR18. We determine a 2.7 Å cryo-EM structure of the subcomplex revealing an interconnected tetrameric assembly and the architecture of PELP1's signaling motifs, including eleven LxxLL motifs previously implicated in SR signaling and coactivation of Estrogen Receptor alpha (ERα) mediated transcription. However, the structure shows that none of these motifs is in a conformation that would support SR binding. Together this work establishes that PELP1 scaffolds the Rix1 complex, and association with WDR18 may direct PELP1's activity away from SR coactivation.

Mechanism by which T7 bacteriophage protein Gp1.2 inhibits Escherichia coli dGTPase.

Levels of the cellular dNTPs, the direct precursors for DNA synthesis, are important for DNA replication fidelity, cell cycle control, and resistance against viruses. Escherichia coli encodes a dGTPase (2'-deoxyguanosine-5'-triphosphate [dGTP] triphosphohydrolase [dGTPase]; dgt gene, Dgt) that establishes the normal dGTP level required for accurate DNA replication but also plays a role in protecting E. coli against bacteriophage T7 infection by limiting the dGTP required for viral DNA replication. T7 counteracts Dgt using an inhibitor, the gene 1.2 product (Gp1.2). This interaction is a useful model system for studying the ongoing evolutionary virus/host "arms race." We determined the structure of Gp1.2 by NMR spectroscopy and solved high-resolution cryo-electron microscopy structures of the Dgt-Gp1.2 complex also including either dGTP substrate or GTP coinhibitor bound in the active site. These structures reveal the mechanism by which Gp1.2 inhibits Dgt and indicate that Gp1.2 preferentially binds the GTP-bound form of Dgt. Biochemical assays reveal that the two inhibitors use different modes of inhibition and bind to Dgt in combination to yield enhanced inhibition. We thus propose an in vivo inhibition model wherein the Dgt-Gp1.2 complex equilibrates with GTP to fully inactivate Dgt, limiting dGTP hydrolysis and preserving the dGTP pool for viral DNA replication.

Communication network within the essential AAA-ATPase Rix7 drives ribosome assembly.

Rix7 is an essential AAA+ ATPase that functions during the early stages of ribosome biogenesis. Rix7 is composed of three domains including an N-terminal domain (NTD) and two AAA+ domains (D1 and D2) that assemble into an asymmetric stacked hexamer. It was recently established that Rix7 is a presumed protein translocase that removes substrates from preribosomes by translocating them through its central pore. However, how the different domains of Rix7 coordinate their activities within the overall hexameric structure was unknown. We captured cryo-electron microscopy (EM) structures of single and double Walker B variants of full length Rix7. The disordered NTD was not visible in the cryo-EM reconstructions, but cross-linking mass spectrometry revealed that the NTD can associate with the central channel in vitro. Deletion of the disordered NTD enabled us to obtain a structure of the Rix7 hexamer to 2.9 Å resolution, providing high resolution details of critical motifs involved in substrate translocation and interdomain communication. This structure coupled with cell-based assays established that the linker connecting the D1 and D2 domains as well as the pore loops lining the central channel are essential for formation of the large ribosomal subunit. Together, our work shows that Rix7 utilizes a complex communication network to drive ribosome biogenesis.

Structural insight and characterization of human Twinkle helicase in mitochondrial disease.

Twinkle is the mammalian helicase vital for replication and integrity of mitochondrial DNA. Over 90 Twinkle helicase disease variants have been linked to progressive external ophthalmoplegia and ataxia neuropathies among other mitochondrial diseases. Despite the biological and clinical importance, Twinkle represents the only remaining component of the human minimal mitochondrial replisome that has yet to be structurally characterized. Here, we present 3-dimensional structures of human Twinkle W315L. Employing cryo-electron microscopy (cryo-EM), we characterize the oligomeric assemblies of human full-length Twinkle W315L, define its multimeric interface, and map clinical variants associated with Twinkle in inherited mitochondrial disease. Cryo-EM, crosslinking-mass spectrometry, and molecular dynamics simulations provide insight into the dynamic movement and molecular consequences of the W315L clinical variant. Collectively, this ensemble of structures outlines a framework for studying Twinkle function in mitochondrial DNA replication and associated disease states.

Method for the structural analysis of Twinkle mitochondrial DNA helicase by cryo-EM.

The mitochondrial replisome replicates the 16.6 kb mitochondria DNA (mtDNA). The proper functioning of this multicomponent protein complex is vital for the integrity of the mitochondrial genome. One of the critical protein components of the mitochondrial replisome is the Twinkle helicase, a member of the Superfamily 4 (SF4) helicases. Decades of research has uncovered common themes among SF4 helicases including self-assembly, ATP-dependent translocation, and formation of protein-protein complexes. Some of the molecular details of these processes are still unknown for the mitochondria SF4 helicase, Twinkle. Here, we describe a protocol for expression, purification, and single-particle cryo-electron microscopy of the Twinkle helicase clinical variant, W315L, which resulted in the first high-resolution structure of Twinkle helicase. The methods described here serve as an adaptable protocol to support future high-resolution studies of Twinkle helicase or other SF4 helicases.

Flipped over U: structural basis for dsRNA cleavage by the SARS-CoV-2 endoribonuclease.

Coronaviruses generate double-stranded (ds) RNA intermediates during viral replication that can activate host immune sensors. To evade activation of the host pattern recognition receptor MDA5, coronaviruses employ Nsp15, which is a uridine-specific endoribonuclease. Nsp15 is proposed to associate with the coronavirus replication-transcription complex within double-membrane vesicles to cleave these dsRNA intermediates. How Nsp15 recognizes and processes dsRNA is poorly understood because previous structural studies of Nsp15 have been limited to small single-stranded (ss) RNA substrates. Here we present cryo-EM structures of SARS-CoV-2 Nsp15 bound to a 52nt dsRNA. We observed that the Nsp15 hexamer forms a platform for engaging dsRNA across multiple protomers. The structures, along with site-directed mutagenesis and RNA cleavage assays revealed critical insight into dsRNA recognition and processing. To process dsRNA Nsp15 utilizes a base-flipping mechanism to properly orient the uridine within the active site for cleavage. Our findings show that Nsp15 is a distinctive endoribonuclease that can cleave both ss- and dsRNA effectively.

Flipped Over U: Structural Basis for dsRNA Cleavage by the SARS-CoV-2 Endoribonuclease.

Coronaviruses generate double-stranded (ds) RNA intermediates during viral replication that can activate host immune sensors. To evade activation of the host pattern recognition receptor MDA5, coronaviruses employ Nsp15, which is uridine-specific endoribonuclease. Nsp15 is proposed to associate with the coronavirus replication-transcription complex within double-membrane vesicles to cleave these dsRNA intermediates. How Nsp15 recognizes and processes dsRNA is poorly understood because previous structural studies of Nsp15 have been limited to small single-stranded (ss) RNA substrates. Here we present cryo-EM structures of SARS-CoV-2 Nsp15 bound to a 52nt dsRNA. We observed that the Nsp15 hexamer forms a platform for engaging dsRNA across multiple protomers. The structures, along with site-directed mutagenesis and RNA cleavage assays revealed critical insight into dsRNA recognition and processing. To process dsRNA Nsp15 utilizes a base-flipping mechanism to properly orient the uridine within the active site for cleavage. Our findings show that Nsp15 is a distinctive endoribonuclease that can cleave both ss- and dsRNA effectively.

GADGETS: a genetic algorithm for detecting epistasis using nuclear families.

MOTIVATION: Epistasis may play an etiologic role in complex diseases, but research has been hindered because identification of interactions among sets of single nucleotide polymorphisms (SNPs) requires exploration of immense search spaces. Current approaches using nuclear families accommodate at most several hundred candidate SNPs. RESULTS: GADGETS detects epistatic SNP-sets by applying a genetic algorithm to case-parent or case-sibling data. To allow for multiple epistatic sets, island subpopulations of SNP-sets evolve separately under selection for evident joint relevance to disease risk. The software evaluates the identified SNP-sets via permutation testing and provides graphical visualization. GADGETS correctly identified epistatic SNP-sets in realistically simulated case-parent triads with 10 000 candidate SNPs, far more SNPs than competitors can handle, and it outperformed competitors in simulations with many fewer SNPs. Applying GADGETS to family-based oral-clefting data from dbGaP identified SNP-sets with possible epistatic effects on risk. AVAILABILITY AND IMPLEMENTATION: GADGETS is part of the epistasisGA package at https://github.com/mnodzenski/epistasisGA. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Deciphering the substrate recognition mechanisms of the heparan sulfate 3-O-sulfotransferase-3.

The sulfation at the 3-OH position of a glucosamine saccharide is a rare modification, but is critically important for the biological activities of heparan sulfate polysaccharides. Heparan sulfate 3-O-sulfotransferase (3-OST), the enzyme responsible for completing this modification, is present in seven different isoforms in humans. Individual isoforms display substrate selectivity to uniquely sulfated saccharide sequences present in heparan sulfate polysaccharides. Here, we report two ternary crystal structures of heparan sulfate 3-OST isoform 3 (3-OST-3) with PAP (3'-phosphoadenosine 5'-phosphate) and two octasaccharide substrates: non 6-O-sulfated octasaccharide (8-mer 1) and 6-O-sulfated octasaccharide (8-mer 3). The 8-mer 1 is a known favorable substrate for 3-OST-3, whereas the 8-mer 3 is an unfavorable one. Unlike the 8-mer 1, we discovered that the 8-mer 3 displays two binding orientations to the enzyme: productive binding and non-productive binding. Results from the enzyme activity studies demonstrate that 8-mer 3 can contribute to either substrate or product inhibition, possibly attributed to a non-productive binding mode. Our results suggest that heparan sulfate substrates interact with the 3-OST-3 enzyme in more than one orientation, which may regulate the activity of the enzyme. Our findings also suggest that different binding orientations between polysaccharides and their protein binding partners could influence biological outcomes.

Characterization of SARS2 Nsp15 nuclease activity reveals it's mad about U.

Nsp15 is a uridine specific endoribonuclease that coronaviruses employ to cleave viral RNA and evade host immune defense systems. Previous structures of Nsp15 from across Coronaviridae revealed that Nsp15 assembles into a homo-hexamer and has a conserved active site similar to RNase A. Beyond a preference for cleaving RNA 3' of uridines, it is unknown if Nsp15 has any additional substrate preferences. Here, we used cryo-EM to capture structures of Nsp15 bound to RNA in pre- and post-cleavage states. The structures along with molecular dynamics and biochemical assays revealed critical residues involved in substrate specificity, nuclease activity, and oligomerization. Moreover, we determined how the sequence of the RNA substrate dictates cleavage and found that outside of polyU tracts, Nsp15 has a strong preference for purines 3' of the cleaved uridine. This work advances our understanding of how Nsp15 recognizes and processes viral RNA, and will aid in the development of new anti-viral therapeutics.

Predicting tumor response to drugs based on gene-expression biomarkers of sensitivity learned from cancer cell lines.

BACKGROUND: Human cancer cell line profiling and drug sensitivity studies provide valuable information about the therapeutic potential of drugs and their possible mechanisms of action. The goal of those studies is to translate the findings from in vitro studies of cancer cell lines into in vivo therapeutic relevance and, eventually, patients' care. Tremendous progress has been made. RESULTS: In this work, we built predictive models for 453 drugs using data on gene expression and drug sensitivity (IC50) from cancer cell lines. We identified many known drug-gene interactions and uncovered several potentially novel drug-gene associations. Importantly, we further applied these predictive models to ~ 17,000 bulk RNA-seq samples from The Cancer Genome Atlas (TCGA) and the Genotype-Tissue Expression (GTEx) database to predict drug sensitivity for both normal and tumor tissues. We created a web site for users to visualize and download our predicted data ( https://manticore.niehs.nih.gov/cancerRxTissue ). Using trametinib as an example, we showed that our approach can faithfully recapitulate the known tumor specificity of the drug. CONCLUSIONS: We demonstrated that our approach can predict drugs that 1) are tumor-type specific; 2) elicit higher sensitivity from tumor compared to corresponding normal tissue; 3) elicit differential sensitivity across breast cancer subtypes. If validated, our prediction could have relevance for preclinical drug testing and in phase I clinical design.

Cryo-EM structures of the SARS-CoV-2 endoribonuclease Nsp15 reveal insight into nuclease specificity and dynamics.

Nsp15, a uridine specific endoribonuclease conserved across coronaviruses, processes viral RNA to evade detection by host defense systems. Crystal structures of Nsp15 from different coronaviruses have shown a common hexameric assembly, yet how the enzyme recognizes and processes RNA remains poorly understood. Here we report a series of cryo-EM reconstructions of SARS-CoV-2 Nsp15, in both apo and UTP-bound states. The cryo-EM reconstructions, combined with biochemistry, mass spectrometry, and molecular dynamics, expose molecular details of how critical active site residues recognize uridine and facilitate catalysis of the phosphodiester bond. Mass spectrometry revealed the accumulation of cyclic phosphate cleavage products, while analysis of the apo and UTP-bound datasets revealed conformational dynamics not observed by crystal structures that are likely important to facilitate substrate recognition and regulate nuclease activity. Collectively, these findings advance understanding of how Nsp15 processes viral RNA and provide a structural framework for the development of new therapeutics.