Structural characterization of two prototypical repressors of SorC family reveals tetrameric assemblies on DNA and mechanism of function.

The SorC family of transcriptional regulators plays a crucial role in controlling the carbohydrate metabolism and quorum sensing. We employed an integrative approach combining X-ray crystallography and cryo-electron microscopy to investigate architecture and functional mechanism of two prototypical representatives of two sub-classes of the SorC family: DeoR and CggR from Bacillus subtilis. Despite possessing distinct DNA-binding domains, both proteins form similar tetrameric assemblies when bound to their respective DNA operators. Structural analysis elucidates the process by which the CggR-regulated gapA operon is derepressed through the action of two effectors: fructose-1,6-bisphosphate and newly confirmed dihydroxyacetone phosphate. Our findings provide the first comprehensive understanding of the DNA binding mechanism of the SorC-family proteins, shedding new light on their functional characteristics.

An evolutionary molecular adaptation of an unusual stefin from the liver fluke Fasciola hepatica redefines the cystatin superfamily.

Fasciolosis is a worldwide parasitic disease of ruminants and an emerging human disease caused by the liver fluke Fasciola hepatica. The cystatin superfamily of cysteine protease inhibitors is composed of distinct families of intracellular stefins and secreted true cystatins. FhCyLS-2 from F. hepatica is an unusual member of the superfamily, where our sequence and 3D structure analyses in this study revealed that it combines characteristics of both families. The protein architecture demonstrates its relationship to stefins, but FhCyLS-2 also contains the secretion signal peptide and disulfide bridges typical of true cystatins. The secretion status was confirmed by detecting the presence of FhCyLS-2 in excretory/secretory products, supported by immunolocalization. Our high-resolution crystal structure of FhCyLS-2 showed a distinct disulfide bridging pattern and functional reactive center. We determined that FhCyLS-2 is a broad specificity inhibitor of cysteine cathepsins from both the host and F. hepatica, suggesting a dual role in the regulation of exogenous and endogenous proteolysis. Based on phylogenetic analysis that identified several FhCyLS-2 homologues in liver/intestinal foodborne flukes, we propose a new group within the cystatin superfamily called cystatin-like stefins.

ABNOH-Linked Nucleotides and DNA for Bioconjugation and Cross-linking with Tryptophan-Containing Peptides and Proteins.

Reactive N-hydroxy-9-azabicyclo[3.3.1]nonane (ABNOH) linked 2'-deoxyuridine 5'-O-mono- and triphosphates were synthesized through a CuAAC click reaction of ABNO-H-PEG4-N3 with 5-ethynyl-dUMP or -dUTP. The modified triphosphate was used as substrate for enzymatic synthesis of modified DNA probes with KOD XL DNA polymerase. The keto-ABNO radical reacted with tryptophan (Trp) and Trp-containing peptides to form a stable 3-fused hexahydropyrrolo-indole conjugates. Similarly modified ABNO-H-linked nucleotides reacted with Trp-containing peptides to form a stable conjugate in the presence but surprisingly even in the absence of NaNO2 (presumably through activation by O2). The reactive ABNO-H-modified DNA probe was used for bioconjugations and crosslinking with Trp-containing peptides or proteins.

B-H⋯π and C-H⋯π interactions in protein-ligand complexes: carbonic anhydrase II inhibition by carborane sulfonamides.

Among non-covalent interactions, B-H⋯π and C-H⋯π hydrogen bonding is rather weak and less studied. Nevertheless, since both can affect the energetics of protein-ligand binding, their understanding is an important prerequisite for reliable predictions of affinities. Through a combination of high-resolution X-ray crystallography and quantum-chemical calculations on carbonic anhydrase II/carborane-based inhibitor systems, this paper provides the first example of B-H⋯π hydrogen bonding in a protein-ligand complex. It shows that the B-H⋯π interaction is stabilized by dispersion, followed by electrostatics. Furthermore, it demonstrates that the similar C-H⋯π interaction is twice as strong, with a slightly smaller contribution of dispersion and a slightly higher contribution of electrostatics. Such a detailed insight will facilitate the rational design of future protein ligands, controlling these types of non-covalent interactions.

Inhibitors of CA IX Enzyme Based on Polyhedral Boron Compounds.

This review describes recent progress in the design and development of inhibitors of human carbonic anhydrase IX (CA IX) based on space-filling carborane and cobalt bis(dicarbollide) clusters. CA IX enzyme is known to play a crucial role in cancer cell proliferation and metastases. The new class of potent and selective CA IX inhibitors combines the structural motif of a bulky inorganic cluster with an alkylsulfamido or alkylsulfonamido anchor group for Zn2+ ion in the enzyme active site. Detailed structure-activity relationship (SAR) studies of a large series containing 50 compounds uncovered structural features of the cluster-containing inhibitors that are important for efficient and selective inhibition of CA IX activity. Preclinical evaluation of selected compounds revealed low toxicity, favorable pharmacokinetics and ability to reduce tumor growth. Cluster-containing inhibitors of CA IX can thus be considered as promising candidates for drug development and/or for combination therapy in boron neutron capture therapy (BNCT).

Affinity switching of the LEDGF/p75 IBD interactome is governed by kinase-dependent phosphorylation.

Lens epithelium-derived growth factor/p75 (LEDGF/p75, or PSIP1) is a transcriptional coactivator that tethers other proteins to gene bodies. The chromatin tethering function of LEDGF/p75 is hijacked by HIV integrase to ensure viral integration at sites of active transcription. LEDGF/p75 is also important for the development of mixed-lineage leukemia (MLL), where it tethers the MLL1 fusion complex at aberrant MLL targets, inducing malignant transformation. However, little is known about how the LEDGF/p75 protein interaction network is regulated. Here, we obtained solution structures of the complete interfaces between the LEDGF/p75 integrase binding domain (IBD) and its cellular binding partners and validated another binding partner, Mediator subunit 1 (MED1). We reveal that structurally conserved IBD-binding motifs (IBMs) on known LEDGF/p75 binding partners can be regulated by phosphorylation, permitting switching between low- and high-affinity states. Finally, we show that elimination of IBM phosphorylation sites on MLL1 disrupts the oncogenic potential of primary MLL1-rearranged leukemic cells. Our results demonstrate that kinase-dependent phosphorylation of MLL1 represents a previously unknown oncogenic dependency that may be harnessed in the treatment of MLL-rearranged leukemia.

Mialostatin, a Novel Midgut Cystatin from Ixodes ricinus Ticks: Crystal Structure and Regulation of Host Blood Digestion.

The hard tick Ixodes ricinus is a vector of Lyme disease and tick-borne encephalitis. Host blood protein digestion, essential for tick development and reproduction, occurs in tick midgut digestive cells driven by cathepsin proteases. Little is known about the regulation of the digestive proteolytic machinery of I. ricinus. Here we characterize a novel cystatin-type protease inhibitor, mialostatin, from the I. ricinus midgut. Blood feeding rapidly induced mialostatin expression in the gut, which continued after tick detachment. Recombinant mialostatin inhibited a number of I. ricinus digestive cysteine cathepsins, with the greatest potency observed against cathepsin L isoforms, with which it co-localized in midgut digestive cells. The crystal structure of mialostatin was determined at 1.55 Å to explain its unique inhibitory specificity. Finally, mialostatin effectively blocked in vitro proteolysis of blood proteins by midgut cysteine cathepsins. Mialostatin is likely to be involved in the regulation of gut-associated proteolytic pathways, making midgut cystatins promising targets for tick control strategies.

Direct Introduction of an Alkylsulfonamido Group on C-sites of Isomeric Dicarba-closo-dodecaboranes: The Influence of Stereochemistry on Inhibitory Activity against the Cancer-Associated Carbonic Anhydrase IX Isoenzyme.

Carbonic anhydrase IX (CA IX), a tumor-associated metalloenzyme, represents a validated target for cancer therapy and diagnostics. Herein, we report the inhibition properties of isomeric families of sulfonamidopropyl-dicarba-closo-dodecaboranes group(s) prepared using a new direct five-step synthesis from the corresponding parent cages. The protocol offers a reliable solution for synthesis of singly and doubly substituted dicarba-closo-dodecaboranes with a different geometric position of carbon atoms. The closo-compounds from the ortho- and meta-series were then degraded to corresponding 11-vertex dicarba-nido-undecaborate(1-) anions. All compounds show in vitro enzymatic activity against CA IX in the low nanomolar or subnanomolar range. This is accompanied by clear isomer dependence of the inhibition constant (Ki ) and selectivity towards CA IX. Decreasing trends in Ki and selectivity index (SI ) values are observed with increasing separation of the cage carbon atoms. Interactions of compounds with the active sites of CA IX were explored with X-ray crystallography, and eight high-resolution crystal structures uncovered the structural basis of inhibition potency and selectivity.

The structure and function of Iristatin, a novel immunosuppressive tick salivary cystatin.

To successfully feed, ticks inject pharmacoactive molecules into the vertebrate host including cystatin cysteine protease inhibitors. However, the molecular and cellular events modulated by tick saliva remain largely unknown. Here, we describe and characterize a novel immunomodulatory cystatin, Iristatin, which is upregulated in the salivary glands of feeding Ixodes ricinus ticks. We present the crystal structure of Iristatin at 1.76 Å resolution. Purified recombinant Iristatin inhibited the proteolytic activity of cathepsins L and C and diminished IL-2, IL-4, IL-9, and IFN-γ production by different T-cell populations, IL-6 and IL-9 production by mast cells, and nitric oxide production by macrophages. Furthermore, Iristatin inhibited OVA antigen-induced CD4+ T-cell proliferation and leukocyte recruitment in vivo and in vitro. Our results indicate that Iristatin affects wide range of anti-tick immune responses in the vertebrate host and may be exploitable as an immunotherapeutic.

The structural basis for the selectivity of sulfonamido dicarbaboranes toward cancer-associated carbonic anhydrase IX.

Human carbonic anhydrase IX (CA IX), a protein specifically expressed on the surface of solid tumour cells, represents a validated target both for anticancer therapy and diagnostics. We recently identified sulfonamide dicarbaboranes as promising inhibitors of CA IX with favourable activities both in vitro and in vivo. To explain their selectivity and potency, we performed detailed X-ray structural analysis of their interactions within the active sites of CA IX and CA II. Series of compounds bearing various aliphatic linkers between the dicarbaborane cluster and sulfonamide group were examined. Preferential binding towards the hydrophobic part of the active site cavity was observed. Selectivity towards CA IX lies in the shape complementarity of the dicarbaborane cluster with a specific CA IX hydrophobic patch containing V131 residue. The bulky side chain of F131 residue in CA II alters the shape of the catalytic cavity, disrupting favourable interactions of the spherical dicarbaborane cluster.

Investigation of flexibility of neuraminidase 150-loop using tamiflu derivatives in influenza A viruses H1N1 and H5N1.

This study focuses on design, synthesis and in vitro evaluation of inhibitory potency of two series of sialylmimetic that target an exosite ("150-cavity") adjacent to the active site of influenza neuraminidases from A/California/07/2009 (H1N1) pandemic strain and A/chicken/Nakorn-Patom/Thailand/CU-K2-2004 (H5N1). The structure-activity analysis as well as 3-D structure of the complex of parental compound with the pandemic neuraminidase p09N1 revealed high flexibility of the 150-cavity towards various modification of the neuraminidase inhibitors. Furthermore, our comparison of two methods for inhibition constant determination performed at slightly different pH values suggest that the experimental conditions of the measurement could dramatically influence the outcome of the analysis in the compound-dependent manner. Therefore, previously reported Ki values determined at non-physiological pH should be carefully scrutinized.

Capturing a dynamically interacting inhibitor by paramagnetic NMR spectroscopy.

Transient and fuzzy intermolecular interactions are fundamental to many biological processes. Despite their importance, they are notoriously challenging to characterize. Effects induced by paramagnetic ligands in the NMR spectra of interacting biomolecules provide an opportunity to amplify subtle manifestations of weak intermolecular interactions observed for diamagnetic ligands. Here, we present an approach to characterizing dynamic interactions between a partially flexible dimeric protein, HIV-1 protease, and a metallacarborane-based ligand, a system for which data obtained by standard NMR approaches do not enable detailed structural interpretation. We show that for the case where the experimental data are significantly averaged to values close to zero the standard fitting of pseudocontact shifts cannot provide reliable structural information. We based our approach on generating a large ensemble of full atomic models, for which the experimental data can be predicted, ensemble averaged and finally compared to the experiment. We demonstrate that a combination of paramagnetic NMR experiments, quantum chemical calculations, and molecular dynamics simulations offers a route towards structural characterization of dynamic protein-ligand complexes.

DNA-linked inhibitor antibody assay (DIANA) as a new method for screening influenza neuraminidase inhibitors.

Influenza neuraminidase is responsible for the escape of new viral particles from the infected cell surface. Several neuraminidase inhibitors are used clinically to treat patients or stockpiled for emergencies. However, the increasing development of viral resistance against approved inhibitors has underscored the need for the development of new antivirals effective against resistant influenza strains. A facile, sensitive, and inexpensive screening method would help achieve this goal. Recently, we described a multiwell plate-based DNA-linked inhibitor antibody assay (DIANA). This highly sensitive method can quantify femtomolar concentrations of enzymes. DIANA also has been applied to high-throughput enzyme inhibitor screening, allowing the evaluation of inhibition constants from a single inhibitor concentration. Here, we report the design, synthesis, and structural characterization of a tamiphosphor derivative linked to a reporter DNA oligonucleotide for the development of a DIANA-type assay to screen potential influenza neuraminidase inhibitors. The neuraminidase is first captured by an immobilized antibody, and the test compound competes for binding to the enzyme with the oligo-linked detection probe, which is then quantified by qPCR. We validated this novel assay by comparing it with the standard fluorometric assay and demonstrated its usefulness for sensitive neuraminidase detection as well as high-throughput screening of potential new neuraminidase inhibitors.

Ranking Power of the SQM/COSMO Scoring Function on Carbonic Anhydrase†II-Inhibitor Complexes.

Accurate prediction of protein-ligand binding affinities is essential for hit-to-lead optimization and virtual screening. The reliability of scoring functions can be improved by including quantum effects. Here, we demonstrate the ranking power of the semiempirical quantum mechanics (SQM)/implicit solvent (COSMO) scoring function by using a challenging set of 10 inhibitors binding to carbonic anhydrase II through Zn2+ in the active site. This new dataset consists of the high-resolution (1.1-1.4 Å) crystal structures and experimentally determined inhibitory constant (Ki ) values. It allows for evaluation of the common approximations, such as representing the solvent implicitly or by using a single target conformation combined with a set of ligand docking poses. SQM/COSMO attained a good correlation of R2 of 0.56-0.77 with the experimental inhibitory activities, benefiting from careful handling of both noncovalent interactions (e.g. charge transfer) and solvation. This proof-of-concept study of SQM/COSMO ranking for metalloprotein-ligand systems demonstrates its potential for hit-to-lead applications.

Oligomeric interface modulation causes misregulation of purine 5´-nucleotidase in relapsed leukemia.

BACKGROUND: Relapsed acute lymphoblastic leukemia (ALL) is one of the main causes of mortality in childhood malignancies. Previous genetic studies demonstrated that chemoresistant ALL is driven by activating mutations in NT5C2, the gene encoding cytosolic 5´-nucleotidase (cN-II). However, molecular mechanisms underlying this hyperactivation are still unknown. Here, we present kinetic and structural properties of cN-II variants that represent 75 % of mutated alleles in patients who experience relapsed ALL (R367Q, R238W and L375F). RESULTS: Enzyme kinetics measurements revealed that the mutants are consitutively active without need for allosteric activators. This shows that hyperactivity is not caused by a direct catalytic effect but rather by misregulation of cN-II. X-ray crystallography combined with mass spectrometry-based techniques demonstrated that this misregulation is driven by structural modulation of the oligomeric interface within the cN-II homotetrameric assembly. These specific conformational changes are shared between the studied variants, despite the relatively random spatial distribution of the mutations. CONCLUSIONS: These findings define a common molecular mechanism for cN-II hyperactivity, which provides a solid basis for targeted therapy of leukemia. Our study highlights the cN-II oligomerization interface as an attractive pharmacological target.

Molecular mechanism for the action of the anti-CD44 monoclonal antibody MEM-85.

The hyaluronate receptor CD44 plays role in cell adhesion and migration and is involved in tumor metastasis. The extracellular domain of CD44 comprises the hyaluronate-binding domain (HABD) and the membrane-proximal stem region; the short intracellular portion interacts with adaptor proteins and triggers signaling pathways. Binding of hyaluronate to CD44 HABD induces an allosteric conformational change, which results in CD44 shedding. A poorly characterized epitope in human CD44 HABD is recognized by the murine monoclonal antibody MEM-85, which cross-blocks hyaluronate binding to CD44 and also induces CD44 shedding. MEM-85 is of therapeutic interest, as it inhibits growth of lung cancer cells in murine models. In this work, we employed a combination of biophysical methods to determine the MEM-85 binding epitope in CD44 HABD and to provide detailed insight into the mechanism of MEM-85 action. In particular, we constructed a single-chain variable fragment (scFv) of MEM-85 as a tool for detailed characterization of the CD44 HABD-antibody complex and identified residues within CD44 HABD involved in the interaction with scFv MEM-85 by NMR spectroscopy and mutational analysis. In addition, we built a rigid body model of the CD44 HABD-scFv MEM-85 complex using a low-resolution structure obtained by small-angle X-ray scattering. The MEM-85 epitope is situated in the C-terminal part of CD44 HABD, rather than the hyaluronate-binding groove, and the binding of MEM-85 induces a structural reorganization similar to that induced by hyaluronate. Therefore, the mechanism of MEM-85 cross-blocking of hyaluronate binding is likely of an allosteric, relay-like nature.

Atomic resolution crystal structure of Sapp2p, a secreted aspartic protease from Candida parapsilosis.

The virulence of the Candida pathogens is enhanced by the production of secreted aspartic proteases, which therefore represent possible targets for drug design. Here, the crystal structure of the secreted aspartic protease Sapp2p from Candida parapsilosis was determined. Sapp2p was isolated from its natural source and crystallized in complex with pepstatin A, a classical aspartic protease inhibitor. The atomic resolution of 0.83 Šallowed the protonation states of the active-site residues to be inferred. A detailed comparison of the structure of Sapp2p with the structure of Sapp1p, the most abundant C. parapsilosis secreted aspartic protease, was performed. The analysis, which included advanced quantum-chemical interaction-energy calculations, uncovered molecular details that allowed the experimentally observed equipotent inhibition of both isoenzymes by pepstatin A to be rationalized.

Kinetic and structural characterization of an alternatively spliced variant of human mitochondrial 5'(3')-deoxyribonucleotidase.

Human mitochondrial 5'(3')-deoxyribonucleotidase (mdN) catalyzes dephosphorylation of nucleoside monophosphates, and thus helps maintain homeostasis of deoxynucleosides required for mitochondrial DNA synthesis. Mature mdN is a 23-kDa dimeric protein with highest expression levels in the heart, brain and skeletal muscle. We have identified an alternative splice variant of the mdN gene containing an 18-nucleotide insertion encoding 6 amino acids (GKWPAT) at the 3'-end of the penultimate exon 4. We recombinantly expressed this enzyme variant and characterized its biochemical and kinetic properties as well as its three-dimensional structure. Our high-resolution (1.27 Å) crystal structure revealed that the insertion forms a loop located in the vicinity of the active site pocket and affects enzyme kinetic parameters as well as protein thermal stability.

Structures of human cytosolic and mitochondrial nucleotidases: implications for structure-based design of selective inhibitors.

The human 5'(3')-deoxyribonucleotidases catalyze the dephosphorylation of deoxyribonucleoside monophosphates to the corresponding deoxyribonucleosides and thus help to maintain the balance between pools of nucleosides and nucleotides. Here, the structures of human cytosolic deoxyribonucleotidase (cdN) at atomic resolution (1.08 Å) and mitochondrial deoxyribonucleotidase (mdN) at near-atomic resolution (1.4 Å) are reported. The attainment of an atomic resolution structure allowed interatomic distances to be used to assess the probable protonation state of the phosphate anion and the side chains in the enzyme active site. A detailed comparison of the cdN and mdN active sites allowed the design of a cdN-specific inhibitor.

Structural and functional analysis of a novel haloalkane dehalogenase with two halide-binding sites.

The crystal structure of the novel haloalkane dehalogenase DbeA from Bradyrhizobium elkanii USDA94 revealed the presence of two chloride ions buried in the protein interior. The first halide-binding site is involved in substrate binding and is present in all structurally characterized haloalkane dehalogenases. The second halide-binding site is unique to DbeA. To elucidate the role of the second halide-binding site in enzyme functionality, a two-point mutant lacking this site was constructed and characterized. These substitutions resulted in a shift in the substrate-specificity class and were accompanied by a decrease in enzyme activity, stability and the elimination of substrate inhibition. The changes in enzyme catalytic activity were attributed to deceleration of the rate-limiting hydrolytic step mediated by the lower basicity of the catalytic histidine.