ABSTRACT
Among non-covalent interactions, B-Hâ¯π and C-Hâ¯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-Hâ¯π hydrogen bonding in a protein-ligand complex. It shows that the B-Hâ¯π interaction is stabilized by dispersion, followed by electrostatics. Furthermore, it demonstrates that the similar C-Hâ¯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.
Subject(s)
Carbonic Anhydrase II , Sulfonamides , Ligands , Sulfanilamide , Crystallography, X-RayABSTRACT
Fluorescent molecules are like antennas: The rate at which they absorb light depends on their orientation with respect to the incoming light wave, and the apparent intensity of their emission depends on their orientation with respect to the observer. However, the directions along which the most important fluorescent molecules in biology, fluorescent proteins (FPs), absorb and emit light are generally not known. Our optical and X-ray investigations of FP crystals have now allowed us to determine the molecular orientations of the excitation and emission transition dipole moments in the FPs mTurquoise2, eGFP, and mCherry, and the photoconvertible FP mEos4b. Our results will allow using FP directionality in studies of molecular and biological processes, but also in development of novel bioengineering and bioelectronics applications.
Subject(s)
Luminescent Proteins/chemistry , Anisotropy , Crystallography, X-Ray , Green Fluorescent Proteins/chemistry , Green Fluorescent Proteins/genetics , Light , Luminescent Proteins/genetics , Microscopy, Polarization , Red Fluorescent ProteinABSTRACT
Cathepsin K (CatK) is a target for the treatment of osteoporosis, arthritis, and bone metastasis. Peptidomimetics with a cyanohydrazide warhead represent a new class of highly potent CatK inhibitors; however, their binding mechanism is unknown. We investigated two model cyanohydrazide inhibitors with differently positioned warheads: an azadipeptide nitrile Gü1303 and a 3-cyano-3-aza-ß-amino acid Gü2602. Crystal structures of their covalent complexes were determined with mature CatK as well as a zymogen-like activation intermediate of CatK. Binding mode analysis, together with quantum chemical calculations, revealed that the extraordinary picomolar potency of Gü2602 is entropically favoured by its conformational flexibility at the nonprimed-primed subsites boundary. Furthermore, we demonstrated by live cell imaging that cyanohydrazides effectively target mature CatK in osteosarcoma cells. Cyanohydrazides also suppressed the maturation of CatK by inhibiting the autoactivation of the CatK zymogen. Our results provide structural insights for the rational design of cyanohydrazide inhibitors of CatK as potential drugs.
Subject(s)
Cathepsin K/antagonists & inhibitors , Hydrazines/pharmacology , Nitriles/pharmacology , Protease Inhibitors/pharmacology , Cathepsin K/metabolism , Dose-Response Relationship, Drug , Humans , Hydrazines/chemistry , Models, Molecular , Molecular Structure , Nitriles/chemistry , Protease Inhibitors/chemistry , Structure-Activity Relationship , Tumor Cells, CulturedABSTRACT
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).
Subject(s)
Boron Compounds/chemistry , Carbonic Anhydrase IX/antagonists & inhibitors , Carbonic Anhydrase Inhibitors/chemistry , Binding Sites , Boron Compounds/metabolism , Boron Compounds/therapeutic use , Carbonic Anhydrase IX/metabolism , Carbonic Anhydrase Inhibitors/chemical synthesis , Carbonic Anhydrase Inhibitors/metabolism , Carbonic Anhydrase Inhibitors/therapeutic use , Humans , Molecular Dynamics Simulation , Neoplasms/drug therapy , Organometallic Compounds/chemistry , Structure-Activity Relationship , Sulfonamides/chemistryABSTRACT
Capillary electrophoresis (CE) has been applied for determination of the thermodynamic acidity constants (pKa ) of the sulfamidoalkyl and sulfonamidoalkyl groups, the actual and limiting ionic mobilities and hydrodynamic radii of important compounds, eight carborane-based inhibitors of carbonic anhydrases, which are potential new anticancer drugs. Two types of carboranes were investigated, (i) icosahedral cobalt bis(dicarbollide)(1-) ion with sulfamidoalkyl moieties, and (ii) 7,8-nido-dicarbaundecaborate with sulfonamidoalkyl side chains. First, the mixed acidity constants, pKamix , of the sulfamidoalkyl and sulfonamidoalkyl groups of the above carboranes and their actual ionic mobilities were determined by nonlinear regression analysis of the pH dependences of their effective electrophoretic mobility measured by capillary electrophoresis in the pH range 8.00-12.25, at constant ionic strength (25 mM), and constant temperature (25°C). Second, the pKamix were recalculated to the thermodynamic pKa s using the Debye-Hückel theory. The sulfamidoalkyl and sulfonamidoalkyl groups were found to be very weakly acidic with the pKa s in the range 10.78-11.45 depending on the type of carborane cluster and on the position and length of the alkyl chain on the carborane scaffold. These pKa s were in a good agreement with the pKa s (10.67-11.27) obtained by new program AnglerFish (freeware at https://echmet.natur.cuni.cz), which provides thermodynamic pKa s and limiting ionic mobilities directly from the raw CE data. The absolute values of the limiting ionic mobilities of univalent and divalent carborane anions were in the range 18.3-27.8 TU (Tiselius unit, 1 × 10-9 m2 /Vs), and 36.4-45.9 TU, respectively. The Stokes hydrodynamic radii of univalent and divalent carborane anions varied in the range 0.34-0.52 and 0.42-0.52 nm, respectively.
Subject(s)
Carbonic Anhydrases , Hydrodynamics , Acids , Electrophoresis, Capillary , Hydrogen-Ion Concentration , IonsABSTRACT
Pathogenic Candida albicans yeasts frequently cause infections in hospitals. Antifungal drugs lose effectiveness due to other Candida species and resistance. New medications are thus required. Secreted aspartic protease of C. parapsilosis (Sapp1p) is a promising target. We have thus solved the crystal structures of Sapp1p complexed to four peptidomimetic inhibitors. Three potent inhibitors (Ki: 0.1, 0.4, 6.6 nM) resembled pepstatin A (Ki: 0.3 nM), a general aspartic protease inhibitor, in terms of their interactions with Sapp1p. However, the weaker inhibitor (Ki: 14.6 nM) formed fewer nonpolar contacts with Sapp1p, similarly to the smaller HIV protease inhibitor ritonavir (Ki: 1.9 µM), which, moreover, formed fewer H-bonds. The analyses have revealed the structural determinants of the subnanomolar inhibition of C. parapsilosis aspartic protease. Because of the high similarity between Saps from different Candida species, these results can further be used for the design of potent and specific Sap inhibitor-based antimycotic drugs.
Subject(s)
Aspartic Acid Endopeptidases/antagonists & inhibitors , Candida parapsilosis/enzymology , Fungal Proteins/antagonists & inhibitors , Peptidomimetics/pharmacology , Protease Inhibitors/pharmacology , Aspartic Acid Endopeptidases/metabolism , Dose-Response Relationship, Drug , Fungal Proteins/metabolism , Models, Molecular , Molecular Structure , Peptidomimetics/chemical synthesis , Peptidomimetics/chemistry , Protease Inhibitors/chemical synthesis , Protease Inhibitors/chemistry , Structure-Activity RelationshipABSTRACT
The part of the influenza polymerase PA subunit featuring endonuclease activity is a target for anti-influenza therapies, including the FDA-approved drug Xofluza. A general feature of endonuclease inhibitors is their ability to chelate Mg2+ or Mn2+ ions located in the enzyme's catalytic site. Previously, we screened a panel of flavonoids for PA inhibition and found luteolin and its C-glucoside orientin to be potent inhibitors. Through structural analysis, we identified the presence of a 3',4'-dihydroxyphenyl moiety as a crucial feature for sub-micromolar inhibitory activity. Here, we report results from a subsequent investigation exploring structural changes at the C-7 and C-8 positions of luteolin. Experimental IC50 values were determined by AlphaScreen technology. The most potent inhibitors were C-8 derivatives with inhibitory potencies comparable to that of luteolin. Bio-isosteric replacement of the C-7 hydroxyl moiety of luteolin led to a series of compounds with one-order-of-magnitude-lower inhibitory potencies. Using X-ray crystallography, we solved structures of the wild-type PA-N-terminal domain and its I38T mutant in complex with orientin at 1.9 Å and 2.2 Å resolution, respectively.
Subject(s)
Endonucleases/antagonists & inhibitors , Luteolin/chemical synthesis , Luteolin/pharmacology , Orthomyxoviridae/drug effects , Antiviral Agents/chemical synthesis , Antiviral Agents/pharmacology , Catalytic Domain/drug effects , Viral Proteins/antagonists & inhibitorsABSTRACT
Influenza A virus (IAV) encodes a polymerase composed of three subunits: PA, with endonuclease activity, PB1 with polymerase activity and PB2 with host RNA five-prime cap binding site. Their cooperation and stepwise activation include a process called cap-snatching, which is a crucial step in the IAV life cycle. Reproduction of IAV can be blocked by disrupting the interaction between the PB2 domain and the five-prime cap. An inhibitor of this interaction called pimodivir (VX-787) recently entered the third phase of clinical trial; however, several mutations in PB2 that cause resistance to pimodivir were observed. First major mutation, F404Y, causing resistance was identified during preclinical testing, next the mutation M431I was identified in patients during the second phase of clinical trials. The mutation H357N was identified during testing of IAV strains at Centers for Disease Control and Prevention. We set out to provide a structural and thermodynamic analysis of the interactions between cap-binding domain of PB2 wild-type and PB2 variants bearing these mutations and pimodivir. Here we present four crystal structures of PB2-WT, PB2-F404Y, PB2-M431I and PB2-H357N in complex with pimodivir. We have thermodynamically analysed all PB2 variants and proposed the effect of these mutations on thermodynamic parameters of these interactions and pimodivir resistance development. These data will contribute to understanding the effect of these missense mutations to the resistance development and help to design next generation inhibitors.
Subject(s)
Drug Resistance, Viral/drug effects , Influenza A virus/enzymology , Protein Subunits/antagonists & inhibitors , Pyridines/chemistry , Pyridines/pharmacology , Pyrimidines/chemistry , Pyrimidines/pharmacology , Pyrroles/chemistry , Pyrroles/pharmacology , RNA-Dependent RNA Polymerase/metabolism , Viral Proteins/antagonists & inhibitors , Crystallography, X-Ray , Influenza A virus/drug effects , Models, Molecular , Mutant Proteins/metabolism , Mutation/genetics , Protein Domains , Protein Subunits/chemistry , Protein Subunits/metabolism , Quantum Theory , RNA-Dependent RNA Polymerase/antagonists & inhibitors , RNA-Dependent RNA Polymerase/chemistry , Thermodynamics , Viral Proteins/chemistry , Viral Proteins/metabolismABSTRACT
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.
Subject(s)
Antigens, Neoplasm/chemistry , Carbonic Anhydrase IX/chemistry , Carbonic Anhydrase I/chemistry , Carbonic Anhydrase Inhibitors , Neoplasms , Antigens, Neoplasm/metabolism , Carbonic Anhydrase I/metabolism , Carbonic Anhydrase IX/metabolism , Carbonic Anhydrase Inhibitors/pharmacology , Humans , Isoenzymes , Structure-Activity RelationshipABSTRACT
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.
Subject(s)
Antineoplastic Agents/chemistry , Boron Compounds/chemistry , Carbonic Anhydrase IX/antagonists & inhibitors , Carbonic Anhydrase Inhibitors/chemistry , Sulfonamides/chemistry , Amino Acid Sequence , Antigens, Neoplasm/genetics , Antineoplastic Agents/pharmacology , Carbonic Anhydrase IX/genetics , Carbonic Anhydrase Inhibitors/pharmacology , Catalytic Domain , Crystallography, X-Ray , Drug Screening Assays, Antitumor , HEK293 Cells , Humans , Hydrophobic and Hydrophilic Interactions , Protein Binding , Structure-Activity Relationship , Sulfonamides/pharmacologyABSTRACT
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.
Subject(s)
Boranes/chemistry , HIV Protease/chemistry , Metals/chemistry , Molecular Dynamics Simulation , Ligands , Magnetic Resonance Spectroscopy/methods , Protein Binding , Protein Conformation , Quantum TheoryABSTRACT
BACKGROUND: The pathogenic yeast Candida albicans can proliferate in environments with different carbon dioxide concentrations thanks to the carbonic anhydrase CaNce103p, which accelerates spontaneous conversion of carbon dioxide to bicarbonate and vice versa. Without functional CaNce103p, C. albicans cannot survive in atmospheric air. CaNce103p falls into the ß-carbonic anhydrase class, along with its ortholog ScNce103p from Saccharomyces cerevisiae. The crystal structure of CaNce103p is of interest because this enzyme is a potential target for surface disinfectants. RESULTS: Recombinant CaNce103p was prepared in E. coli, and its crystal structure was determined at 2.2 Å resolution. CaNce103p forms a homotetramer organized as a dimer of dimers, in which the dimerization and tetramerization surfaces are perpendicular. Although the physiological role of CaNce103p is similar to that of ScNce103p from baker's yeast, on the structural level it more closely resembles carbonic anhydrase from the saprophytic fungus Sordaria macrospora, which is also tetrameric. Dimerization is mediated by two helices in the N-terminal domain of the subunits. The N-terminus of CaNce103p is flexible, and crystals were obtained only upon truncation of the first 29 amino acids. Analysis of CaNce103p variants truncated by 29, 48 and 61 amino acids showed that residues 30-48 are essential for dimerization. Each subunit contains a zinc atom in the active site and displays features characteristic of type I ß-carbonic anhydrases. Zinc is tetrahedrally coordinated by one histidine residue, two cysteine residues and a molecule of ß-mercaptoethanol originating from the crystallization buffer. The active sites are accessible via substrate tunnels, which are slightly longer and narrower than those observed in other fungal carbonic anhydrases. CONCLUSIONS: CaNce103p is a ß-class homotetrameric metalloenzyme composed of two homodimers. Its structure closely resembles those of other ß-type carbonic anhydrases, in particular CAS1 from Sordaria macrospora. The main differences occur in the N-terminal part and the substrate tunnel. Detailed knowledge of the CaNce103p structure and the properties of the substrate tunnel in particular will facilitate design of selective inhibitors of this enzyme.
Subject(s)
Candida albicans/enzymology , Carbonic Anhydrases/chemistry , Amino Acid Sequence , Catalytic Domain , Crystallography, X-Ray , Models, Molecular , Protein Multimerization , Protein Structure, QuaternaryABSTRACT
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 anhydraseâ II through Zn2+ in the active site. This new dataset consists of the high-resolution (1.1-1.4â 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.
Subject(s)
Carbonic Anhydrase II/metabolism , Carbonic Anhydrase Inhibitors/metabolism , Sulfonamides/metabolism , Carbonic Anhydrase II/chemistry , Carbonic Anhydrase Inhibitors/chemistry , Drug Design , Ligands , Models, Chemical , Molecular Docking Simulation , Protein Binding , Quantum Theory , Sulfonamides/chemistryABSTRACT
Zika virus is considered a major global threat to human kind. Here, we present a crystal structure of one of its essential enzymes, the methyltransferase, with the inhibitor sinefungin. This structure, together with previously solved structures with bound substrates, will provide the information needed for rational inhibitor design. Based on the structural data we suggest the modification of the adenine moiety of sinefungin to increase selectivity and to covalently link it to a GTP analogue, to increase the affinity of the synthesized compounds.
Subject(s)
Adenosine/analogs & derivatives , Enzyme Inhibitors/chemistry , Methyltransferases/antagonists & inhibitors , Zika Virus/enzymology , Adenosine/chemistry , Binding Sites , Models, Molecular , Protein BindingABSTRACT
BACKGROUND: Myristoylation of the matrix (MA) domain mediates the transport and binding of Gag polyproteins to the plasma membrane (PM) and is required for the assembly of most retroviruses. In betaretroviruses, which assemble immature particles in the cytoplasm, myristoylation is dispensable for assembly but is crucial for particle transport to the PM. Oligomerization of HIV-1 MA stimulates the transition of the myristoyl group from a sequestered to an exposed conformation, which is more accessible for membrane binding. However, for other retroviruses, the effect of MA oligomerization on myristoyl group exposure has not been thoroughly investigated. RESULTS: Here, we demonstrate that MA from the betaretrovirus mouse mammary tumor virus (MMTV) forms dimers in solution and that this process is stimulated by its myristoylation. The crystal structure of N-myristoylated MMTV MA, determined at 1.57 Å resolution, revealed that the myristoyl groups are buried in a hydrophobic pocket at the dimer interface and contribute to dimer formation. Interestingly, the myristoyl groups in the dimer are mutually swapped to achieve energetically stable binding, as documented by molecular dynamics modeling. Mutations within the myristoyl binding site resulted in reduced MA dimerization and extracellular particle release. CONCLUSIONS: Based on our experimental, structural, and computational data, we propose a model for dimerization of MMTV MA in which myristoyl groups stimulate the interaction between MA molecules. Moreover, dimer-forming MA molecules adopt a sequestered conformation with their myristoyl groups entirely buried within the interaction interface. Although this differs from the current model proposed for lentiviruses, in which oligomerization of MA triggers exposure of myristoyl group, it appears convenient for intracellular assembly, which involves no apparent membrane interaction and allows the myristoyl group to be sequestered during oligomerization.
Subject(s)
Mammary Tumor Virus, Mouse/chemistry , Mammary Tumor Virus, Mouse/physiology , Protein Multimerization , Protein Processing, Post-Translational , Viral Matrix Proteins/chemistry , Viral Matrix Proteins/metabolism , Animals , Cell Line , Crystallography, X-Ray , Humans , Models, Biological , Models, Molecular , Molecular Dynamics Simulation , RatsABSTRACT
In nonmammalian vertebrates, the functional units of hemostasis are thrombocytes. Thrombocytes are thought to arise from bipotent thrombocytic/erythroid progenitors (TEPs). TEPs have been experimentally demonstrated in avian models of hematopoiesis, and mammals possess functional equivalents known as megakaryocyte/erythroid progenitors (MEPs). However, the presence of TEPs in teleosts has only been speculated. To identify and prospectively isolate TEPs, we identified, cloned, and generated recombinant zebrafish thrombopoietin (Tpo). Tpo mRNA expanded itga2b:GFP(+) (cd41:GFP(+)) thrombocytes as well as hematopoietic stem and progenitor cells (HSPCs) in the zebrafish embryo. Utilizing Tpo in clonal methylcellulose assays, we describe for the first time the prospective isolation and characterization of TEPs from transgenic zebrafish. Combinatorial use of zebrafish Tpo, erythropoietin, and granulocyte colony stimulating factor (Gcsf) allowed the investigation of HSPCs responsible for erythro-, myelo-, and thrombo-poietic differentiation. Utilizing these assays allowed the visualization and differentiation of hematopoietic progenitors ex vivo in real-time with time-lapse and high-throughput microscopy, allowing analyses of their clonogenic and proliferative capacity. These studies indicate that the functional role of Tpo in the differentiation of thrombocytes from HSPCs is well conserved among vertebrate organisms, positing the zebrafish as an excellent model to investigate diseases caused by dysregulated erythro- and thrombo-poietic differentiation.
Subject(s)
Hematopoiesis/genetics , Thrombopoietin/genetics , Zebrafish/physiology , Animals , Animals, Genetically Modified , Blood Platelets/physiology , Cell Differentiation , Cell Proliferation , Cells, Cultured , Embryo, Nonmammalian , Hematopoietic Stem Cells/physiology , Zebrafish/embryologyABSTRACT
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 0.83â Å allowed 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.
Subject(s)
Aspartic Acid Proteases/chemistry , Candida/chemistry , Fungal Proteins/chemistry , Pepstatins/chemistry , Protease Inhibitors/chemistry , Amino Acid Sequence , Aspartic Acid Proteases/genetics , Aspartic Acid Proteases/isolation & purification , Aspartic Acid Proteases/metabolism , Candida/enzymology , Candida/genetics , Catalytic Domain , Crystallography, X-Ray , Fungal Proteins/genetics , Fungal Proteins/isolation & purification , Fungal Proteins/metabolism , Gene Expression , Isoenzymes/chemistry , Isoenzymes/genetics , Isoenzymes/isolation & purification , Isoenzymes/metabolism , Kinetics , Models, Molecular , Molecular Sequence Data , Protein Binding , Protein Structure, Secondary , Quantum Theory , Sequence Alignment , Structural Homology, Protein , Substrate Specificity , ThermodynamicsABSTRACT
Insertions in the protease (PR) region of human immunodeficiency virus (HIV) represent an interesting mechanism of antiviral resistance against HIV PR inhibitors (PIs). Here, we demonstrate the improved ability of a phosphonate-containing experimental HIV PI, GS-8374, relative to that of other PIs, to effectively inhibit patient-derived recombinant HIV strains bearing PR insertions and numerous other mutations. We correlate enzyme inhibition with the catalytic activities of corresponding recombinant PRs in vitro and provide a biochemical and structural analysis of the PR-inhibitor complex.
Subject(s)
HIV Infections/virology , HIV Protease Inhibitors/chemistry , HIV Protease/genetics , HIV-1/drug effects , HIV-1/enzymology , Mutagenesis, Insertional , Amino Acid Sequence , Binding Sites , Crystallography, X-Ray , Drug Resistance, Viral , HIV Infections/drug therapy , HIV Protease/chemistry , HIV Protease/metabolism , HIV Protease Inhibitors/pharmacology , HIV-1/chemistry , HIV-1/genetics , Humans , Models, Molecular , Organophosphonates/analysisABSTRACT
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.
Subject(s)
5'-Nucleotidase/chemistry , Alternative Splicing , Mitochondrial Proteins/chemistry , 5'-Nucleotidase/genetics , Catalytic Domain , Cloning, Molecular , Crystallography, X-Ray , Escherichia coli/genetics , Escherichia coli/metabolism , Exons , Gene Expression , Humans , Isoenzymes/chemistry , Isoenzymes/genetics , Kinetics , Mitochondrial Proteins/genetics , Models, Molecular , Molecular Sequence Data , Mutagenesis, Insertional , Protein Structure, Secondary , Protein Structure, Tertiary , Recombinant Proteins/chemistry , Recombinant Proteins/geneticsABSTRACT
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 (1.08â Å) and mitochondrial deoxyribonucleotidase (mdN) at near-atomic resolution (1.4â 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.