Juraj Sedláček | Institute of Molecular Genetics (original) (raw)

Papers by Juraj Sedláček

Research paper thumbnail of A dominant epitope of HIV-1 protease recognized by hamster monoclonal antibodies

Journal of acquired immune deficiency syndromes

Monoclonal antibodies (mAbs) against HIV-1 protease (HIV PR), the essential protease of human imm... more Monoclonal antibodies (mAbs) against HIV-1 protease (HIV PR), the essential protease of human immunodeficiency virus type 1 (HIV-1), were produced in Armenian hamsters. Studies of direct binding to synthetic peptides and inhibition of binding to intact protease by peptide competition showed that five mAbs recognized an epitope that includes the sequence LPGRWKPK (residues 38-45), which lies near the region of the protease called the flap. All of the mAbs react specifically with HIV PR in Western blots. Because of structural conservation of the epitope in the proteases of many HIV-1 isolates, mAbs to this epitope are likely to be useful for detection of HIV PR in field isolates of HIV-1. Also, mAbs specific for this epitope, which lies close to the flap of HIV PR, may be useful for functional studies of HIV PR and possibly for the design of inhibitors of protease activity that bind outside the enzyme's catalytic site.

Research paper thumbnail of Processing, purification, and kinetic characterization of the Gag-Pol encoded retroviral proteinase of myeloblastosis associated virus expressed in E. coli

Advances in Experimental Medicine and Biology

Research paper thumbnail of Inhibition of the EF-Tu factor by L-1-tosylamido-2-phenylethyl chloromethyl ketone

Acta biologica et medica Germanica

Research paper thumbnail of The elongation factor Tu.kirromycin complex has two binding sites for tRNA molecules

The EMBO Journal

The interaction of the polypeptide chain elongation factor Tu (EF-Tu) with the antibiotic kirromy... more The interaction of the polypeptide chain elongation factor Tu (EF-Tu) with the antibiotic kirromycin and tRNA has been studied by measuring the extent of protein modification with N-tosyl-L-phenylalanine chloromethylketone (TPCK) and N-ethylmaleimide (NEM). Kirromycin protects both EF-Tu.GDP and EF-Tu.GTP against modification with TPCK. Binding of aminoacyl-tRNA added at increasing concentrations to a solution of 40 microM EF-Tu.GDP.kirromycin complex re-exposes the TPCK target site on the protein. However, when the aminoacyl-tRNA concentration is raised beyond 20 microM, TPCK labeling drops again and is blocked completely at approximately 300 microM aminoacyl-tRNA. By contrast, addition of uncharged tRNA or N- acetylaminoacyl -tRNA enhances TPCK labeling of the protein over the entire tRNA concentration range studied. These data strongly suggest that kirromycin induces in EF-Tu.GDP an additional tRNA binding site that can bind uncharged tRNA, aminoacyl-tRNA, and N- acetylaminoacyl ...

Research paper thumbnail of Structural basis of HIV-1 protease inhibition by a monoclonal antibody

Acta Crystallographica Section A Foundations of Crystallography, 1996

Research paper thumbnail of Peptidomimetic inhibitors complexed with HIV-1 protease: crystallisation for X-ray diffraction studies

General physiology and biophysics, 1998

HIV protease has become one of possible targets of anti AIDS treatment The complexmg abihtv of su... more HIV protease has become one of possible targets of anti AIDS treatment The complexmg abihtv of subnanomolar K, tetrapeptide inhibitors Boc-Phe-<r[(S/R)-CH(OH)CH2NH]-Phe-Glu/Gln-Phe-NH 2 (slashes denote alternatives, the four inhibitors are coded as SE, SQ, RE, RQ) (Konvalinka et al 1997) is a subject of in vestigation by X-ray structure analysis of inhibitor-protease complexes to elucidate high affinity of the inhibitors to the protease dimer, the change of affinity as a result of Glu/Gln alteration at the P2' position and of chirahtv of the tetrahedrally co-ordinated transitionstate-analogue carbon Details of the binding mode of these inhibitors are expected to explain the differences in affinities measured by K, , K^E = 0 15 nM, Kf Q = 33 0 nM, K, RE = 0 12 nM, K RQ = 14 0 nM

Research paper thumbnail of Inactivation of protein-synthesizing T-factor by N-tosyl-L-phenylalanyl chloromethane

Biochimica et biophysica acta, Jan 30, 1971

N-Tosyl-L-phenylalanyl chloromethane causes the irreversible inhibition of protein synthesis in p... more N-Tosyl-L-phenylalanyl chloromethane causes the irreversible inhibition of protein synthesis in poly(U)-and poly(A)-directed cell-free systems from Escherichia coll. The elongation factor T is the component affected by the inhibitor. The T-factor inactivated by the chloromethylketone loses its ability to stimulate the binding of aminoacyl-tRNA to ribosomes. Activities of aminoacyl-tRNA ligases, ribosomes and the elongation factor G are not altered.

Research paper thumbnail of Configurations of Diastereomeric Hydroxyethylene Isosteres Strongly Affect Biological Activities of a Series of Specific Inhibitors of Human-Immunodeficiency-Virus Proteinase

European Journal of Biochemistry, 1997

Human immunodeficiency virus (HIV) proteinase (PR) represents an important target for antiviral c... more Human immunodeficiency virus (HIV) proteinase (PR) represents an important target for antiviral chemotherapy. We present an analysis of inhibitory activities of a series of pseudopeptide inhibitors of HIV-1 PR. All inhibitors were N-protected tetrapeptides with the scissile bond replaced by a nonhydrolysable hydroxyethylene or hydroxyethylamine isostere. To elucidate subtle structural requirements of the PR binding cleft, we synthesised inhibitors with four combinations of configurations at the asymmetric carbons of the isostere. Compounds were tested in vitro using purified recombinant enzyme and a chromogenic peptide substrate. The differences in inhibition constants between individual diastereoisomers reached three orders of magnitude. The most active hydroxyethylene-containing inhibitor possessed the 2R,4S,5S configuration at the isostere. Inhibitor activity was also tested in mammalian cell culture by analysing reduction of viral polyprotein processing and virus infectivity. The results obtained in tissue culture were generally in agreement with the in vitro data, giving a similar order of potency for the individual diastereoisomers. The most active compounds completely blocked production of infectious virus. A simulation method for interaction was employed to build a model of the inhibitors in the PR active site, to identify the interactions responsible for the differences in activities of individual stereoisomers, and to estimate the relative contribution of individual structural features to the overall inhibitory activity.

Research paper thumbnail of Optimization of the crystallizability of a single-chain antibody fragment

Acta Crystallographica Section F Structural Biology Communications, 2014

Single-chain variable antibody fragments (scFvs) are molecules with immense therapeutic and diagn... more Single-chain variable antibody fragments (scFvs) are molecules with immense therapeutic and diagnostic potential. Knowledge of their three-dimensional structure is important for understanding their antigen-binding mode as well as for protein-engineering approaches such as antibody humanization. A major obstacle to the crystallization of single-chain variable antibody fragments is their relatively poor homogeneity caused by spontaneous oligomerization. A new approach to optimization of the crystallizability of single-chain variable antibody fragments is demonstrated using a representative single-chain variable fragment derived from the anti-CD3 antibody MEM-57. A Thermofluor-based assay was utilized to screen for optimal conditions for antibody-fragment stability and homogeneity. Such an optimization of the protein storage buffer led to a significantly improved ability of the scFv MEM-57 to yield crystals.

Research paper thumbnail of Processing, Purification, and Kinetic Characterization of the Gag-Pol Encoded Retroviral Proteinase of Myeloblastosis Associated Virus Expressed in E. Coli

Advances in Experimental Medicine and Biology, 1995

Research paper thumbnail of Role of hydroxyl group and R/S configuration of isostere in binding properties of HIV-1 protease inhibitors

European Journal of Biochemistry, 2004

The crystal structure of the complex between human immunodeficiency virus type 1 (HIV-1) protease... more The crystal structure of the complex between human immunodeficiency virus type 1 (HIV-1) protease and a peptidomimetic inhibitor of ethyleneamine type has been refined to R factor of 0.178 with diffraction limit 2.5 Å . The peptidomimetic inhibitor Boc-Phe-Y[CH 2 CH 2 NH]-Phe-Glu-Phe-NH 2 (denoted here as OE) contains the ethyleneamine replacement of the scissile peptide bond. The inhibitor lacks the hydroxyl group which is believed to mimic tetrahedral transition state of proteolytic reaction and thus is suspected to be necessary for good properties of peptidomimetic HIV-1 protease inhibitors. Despite the missing hydroxyl group the inhibition constant of OE is 1.53 nM and it remains in the nanomolar range also towards several available mutants of HIV-1 protease. The inhibitor was found in the active site of protease in an extended

Research paper thumbnail of The role of aminoacyl-tRNA binding site on the factor EF-T in uncoupled GTPase reaction

Biochimica et Biophysica Acta (BBA) - Nucleic Acids and Protein Synthesis, 1974

The properties of the N-tosyl-L-phenylalanyl chloromethane treated EF-T factor were studied in a ... more The properties of the N-tosyl-L-phenylalanyl chloromethane treated EF-T factor were studied in a ribosomal system in which splitting of GTP occurs. The action of N-tosyl-L-phenylalanyl chloromethane inhibits the binding of aminoacyl-tRNA to the EF-T factor. The binding and exchange of guanosine phosphates continued to be preserved. The inhibited factor is inactive in the GTPase reaction which depends on the participation of ribosomes and aminoacyl-tRNA. The uncoupled GTPase reaction (which is not dependent on the presence of aminoacyl-tRNA) is also sensitive to the effect of the inhibitor. The inhibition of the uncoupled GTPase is incomplete. These findings are attributed to the involvement of the aminoacyl-tRNA binding site of the EF-T factor in the uncoupled GTPase reaction.

Research paper thumbnail of Myeloblastosis Associated Virus (MAV) Proteinase Site-Mutated to Be HIV-like Has a Higher Activity and Allows Production of Infectious but Morphologically Altered Virus

Virology, 1993

We have characterized the structure and infectivity of an avian retrovirus, myeloblastosis associ... more We have characterized the structure and infectivity of an avian retrovirus, myeloblastosis associated virus (MAV), containing a genetically altered proteinase (PR). A site-directed mutant of MAV-PR that shows an increased proteolytic activity in vitro (about 20 times higher kcat/Km) as a consequence of substituting five amino acids from the substrate-binding pocket with those corresponding to the HIV-1 PR was cloned into a full-sized MAV plasmid. In particular, the wild-type MAV-PR gene was replaced with the mutant one. Despite encoding for an enzyme with increased PR activity, mutant plasmid-transfected turkey fibroblasts displayed an unimpaired virus production in cell cultures. Further, the mutant progeny virus was infectious and its pattern of gag processing products appeared identical to that of wild-type virus. However, by electron microscopy we found that the predominant morphology of mutant viral particles was altered. Instead of a centrally collapsed avian retroviral core, a more diffuse core was visualized for wild-type mutant virions, similar to that observed in mammalian C-type retroviruses.

Research paper thumbnail of A Phenylnorstatine Inhibitor Binding to HIV1 Protease:  Geometry, Protonation, and Subsite−Pocket Interactions Analyzed at Atomic Resolution

Journal of Medicinal Chemistry, 2004

The X-ray structure of a complex of HIV-1 protease (PR) with a phenylnorstatine inhibitor Z-Pns-P... more The X-ray structure of a complex of HIV-1 protease (PR) with a phenylnorstatine inhibitor Z-Pns-Phe-Glu-Glu-NH 2 has been determined at 1.03 Å, the highest resolution so far reported for any HIV PR complex. The inhibitor shows subnanomolar K i values for both the wild-type PR and the variant representing one of the most common mutations linked to resistance development. The structure comprising the phenylnorstatine moiety of (2R,3S)-chirality displays a unique pattern of hydrogen bonding to the two catalytic aspartate residues. This high resolution makes it possible to assess the donor and acceptor relations of this hydrogen bonding and to indicate a proton shared by the two catalytic residues. A structural mechanism for the unimpaired inhibition of the protease Val82Ala mutant is also suggested, based on energy calculations and analyses.

Research paper thumbnail of The Gag-Pol Encoded Proteinase of an Avian Retrovirus Expressed in E. coli Can Produce a Novel Proteinase (PR+IIeGly) That Is Two Amino Acids Larger at Its Carboxy-Terminal Region Than the Major Gag Proteinase (PR)

Research paper thumbnail of Structure of HOE/BAY 793 Complexed to Human Immunodeficiency Virus (HIV-1) Protease in Two Different Crystal Forms Structure/Function Relationship and Influence of Crystal Packing

European Journal of Biochemistry, 1997

Human immunodeficiency virus 1 (HIV-1) protease is a prime target in the search for drugs to comb... more Human immunodeficiency virus 1 (HIV-1) protease is a prime target in the search for drugs to combat the AIDS virus. The enzyme functions as a C,-symmetric dimer, cleaving the gag and gag-pol viral polyproteins at distinct sites. The possession of a twofold axis passing through the active site, has led to the design of C,-symmetrical inhibitors in the form of substrate-based transition-state analogs. One of the most active compounds of this class of inhibitors is HOE/BAY 793, which contains a vicinal diol central The structure of this inhibitor bound to HIV-1 protease, in two different crystal forms, has been solved at 0.24-nm resolution using X-ray crystallography. In both forms, the details of the inhibitor-protease interactions revealed an overall asymmetric binding mode, especially between the central diol unit and the active-site aspartates. The main binding interactions comprise several specific H-bonds and hydrophobic contacts, which rationalize many of the characteristics of the structure/ activity relationship in the class of vicinal diol inhibitors. In a general analysis of the mobility of the flap regions, which cover the active site and participate directly in binding, using our structures and the HTV protease models present in the Brookhaven databank, we found that in most structures the flexibility of the flaps is limited by local crystal contacts. However, in one of the structures presented here, no significant crystal contacts to the flap regions were present, and as a result the flexibility of the inhibitor bound flaps increased significantly. This suggests that the mobility and conformational flexibility of the flap residues are important in the functioning of HIV-1 protease, and must be considered in the future design of drugs against HIV protease and in structure-based drug design in general.

Research paper thumbnail of Proteolytic Processing of Particle-Associated Retroviral Polyproteins by Homologous and Heterologous Viral Proteinases

European Journal of Biochemistry, 1995

Research paper thumbnail of Structural studies of the retroviral proteinase from avian myeloblastosis associated virus

Proteins: Structure, Function, and Genetics, 1992

The structure of the retroviral proteinase from avian myeloblastosis associated virus (MAV) has b... more The structure of the retroviral proteinase from avian myeloblastosis associated virus (MAV) has been determined and refined at 2.2 A resolution. This structure is compared with those of homologous proteinases from Rous sarcoma virus (RSV) and hum a n immunodeficiency type 1 virus (HIV). Through comparison with the structure of a proteinase-inhibitor complex from HIV, a model of a complex between MAV proteinase and a peptide substrate has been generated. Examination of this model suggests structural basis for the diverse specifications of viral PrOteinaSeS. 0 1992 Wiley-Liss, Inc.

Research paper thumbnail of Stabilization of antibody structure upon association to a human carbonic anhydrase IX epitope studied by X-ray crystallography, microcalorimetry, and molecular dynamics simulations

Proteins: Structure, Function, and Bioinformatics, 2007

Specific antibodies interfere with the function of human tumor-associated carbonic anhydrase IX (... more Specific antibodies interfere with the function of human tumor-associated carbonic anhydrase IX (CA IX), and show potential as tools for anticancer interventions. In this work, a correlation between structural elements and thermodynamic parameters of the association of antibody fragment Fab M75 to a peptide corresponding to its epitope in the proteoglycan-like domain of CA IX, is presented. Comparisons of the crystal structures of free Fab M75 and its complex with the epitope peptide reveal major readjustments of CDR-H1 and CDR-H3. In contrast, the overall conformations and positions of CDR-H2 and CDR-L2 remain unaltered, and their positively charged residues may thus present a fixed frame for epitope recognition. Adoption of the altered CDR-H3 conformation in the structure of the complex is accompanied by an apparent local stabilization. Analysis of domain mobility with translation-libration-screw (TLS) method shows that librations of the entire heavy chain variable domain (V(H)) decrease and reorient in the complex, which correlates well with participation of the heavy chain in ligand binding. Isothermal titration microcalorimetry (ITC) experiments revealed a highly unfavorable entropy term, which can be attributed mainly to the decrease in the degrees of freedom of the system, the loss of conformational freedom of peptide and partially to a local stabilization of CDR-H3. Moreover, it was observed that one proton is transferred from the environment to the protein-ligand complex upon binding. Molecular dynamics simulations followed by molecular mechanics/generalized Born surface area (MM-GBSA) calculations of the ligand (epitope peptide) binding energy yielded energy values that were in agreement with the ITC measurements and indicated that the charged residues play crucial role in the epitope binding. Theoretical arguments presented in this work indicate that two adjacent arginine residues (ArgH50 and ArgH52) are responsible for the observed proton transfer.

Research paper thumbnail of From nonpeptide toward noncarbon protease inhibitors: Metallacarboranes as specific and potent inhibitors of HIV protease

Proceedings of the National Academy of Sciences, 2005

HIV protease (PR) represents a prime target for rational drug design, and protease inhibitors (PI... more HIV protease (PR) represents a prime target for rational drug design, and protease inhibitors (PI) are powerful antiviral drugs. Most of the current PIs are pseudopeptide compounds with limited bioavailability and stability, and their use is compromised by high costs, side effects, and development of resistant strains. In our search for novel PI structures, we have identified a group of inorganic compounds, icosahedral metallacarboranes, as candidates for a novel class of nonpeptidic PIs. Here, we report the potent, specific, and selective competitive inhibition of HIV PR by substituted metallacarboranes. The most active compound, sodium hydrogen butylimino bis-8,8-[5-(3-oxa-pentoxy)-3-cobalt bis(1,2dicarbollide)]di-ate, exhibited a Ki value of 2.2 nM and a submicromolar EC50 in antiviral tests, showed no toxicity in tissue culture, weakly inhibited human cathepsin D and pepsin, and was inactive against trypsin, papain, and amylase. The structure of the parent cobalt bis(1,2-dicarbollide) in complex with HIV PR was determined at 2.15 Å resolution by protein crystallography and represents the first carborane-protein complex structure determined. It shows the following mode of PR inhibition: two molecules of the parent compound bind to the hydrophobic pockets in the flap-proximal region of the S3 and S3 subsites of PR. We suggest, therefore, that these compounds block flap closure in addition to filling the corresponding binding pockets as conventional PIs. This type of binding and inhibition, chemical and biological stability, low toxicity, and the possibility to introduce various modifications make boron clusters attractive pharmacophores for potent and specific enzyme inhibition. rational drug design ͉ aspartic proteases ͉ carboranes ͉ x-ray structure analysis ͉ virostatics Freely available online through the PNAS open access option.

Research paper thumbnail of A dominant epitope of HIV-1 protease recognized by hamster monoclonal antibodies

Journal of acquired immune deficiency syndromes

Monoclonal antibodies (mAbs) against HIV-1 protease (HIV PR), the essential protease of human imm... more Monoclonal antibodies (mAbs) against HIV-1 protease (HIV PR), the essential protease of human immunodeficiency virus type 1 (HIV-1), were produced in Armenian hamsters. Studies of direct binding to synthetic peptides and inhibition of binding to intact protease by peptide competition showed that five mAbs recognized an epitope that includes the sequence LPGRWKPK (residues 38-45), which lies near the region of the protease called the flap. All of the mAbs react specifically with HIV PR in Western blots. Because of structural conservation of the epitope in the proteases of many HIV-1 isolates, mAbs to this epitope are likely to be useful for detection of HIV PR in field isolates of HIV-1. Also, mAbs specific for this epitope, which lies close to the flap of HIV PR, may be useful for functional studies of HIV PR and possibly for the design of inhibitors of protease activity that bind outside the enzyme's catalytic site.

Research paper thumbnail of Processing, purification, and kinetic characterization of the Gag-Pol encoded retroviral proteinase of myeloblastosis associated virus expressed in E. coli

Advances in Experimental Medicine and Biology

Research paper thumbnail of Inhibition of the EF-Tu factor by L-1-tosylamido-2-phenylethyl chloromethyl ketone

Acta biologica et medica Germanica

Research paper thumbnail of The elongation factor Tu.kirromycin complex has two binding sites for tRNA molecules

The EMBO Journal

The interaction of the polypeptide chain elongation factor Tu (EF-Tu) with the antibiotic kirromy... more The interaction of the polypeptide chain elongation factor Tu (EF-Tu) with the antibiotic kirromycin and tRNA has been studied by measuring the extent of protein modification with N-tosyl-L-phenylalanine chloromethylketone (TPCK) and N-ethylmaleimide (NEM). Kirromycin protects both EF-Tu.GDP and EF-Tu.GTP against modification with TPCK. Binding of aminoacyl-tRNA added at increasing concentrations to a solution of 40 microM EF-Tu.GDP.kirromycin complex re-exposes the TPCK target site on the protein. However, when the aminoacyl-tRNA concentration is raised beyond 20 microM, TPCK labeling drops again and is blocked completely at approximately 300 microM aminoacyl-tRNA. By contrast, addition of uncharged tRNA or N- acetylaminoacyl -tRNA enhances TPCK labeling of the protein over the entire tRNA concentration range studied. These data strongly suggest that kirromycin induces in EF-Tu.GDP an additional tRNA binding site that can bind uncharged tRNA, aminoacyl-tRNA, and N- acetylaminoacyl ...

Research paper thumbnail of Structural basis of HIV-1 protease inhibition by a monoclonal antibody

Acta Crystallographica Section A Foundations of Crystallography, 1996

Research paper thumbnail of Peptidomimetic inhibitors complexed with HIV-1 protease: crystallisation for X-ray diffraction studies

General physiology and biophysics, 1998

HIV protease has become one of possible targets of anti AIDS treatment The complexmg abihtv of su... more HIV protease has become one of possible targets of anti AIDS treatment The complexmg abihtv of subnanomolar K, tetrapeptide inhibitors Boc-Phe-<r[(S/R)-CH(OH)CH2NH]-Phe-Glu/Gln-Phe-NH 2 (slashes denote alternatives, the four inhibitors are coded as SE, SQ, RE, RQ) (Konvalinka et al 1997) is a subject of in vestigation by X-ray structure analysis of inhibitor-protease complexes to elucidate high affinity of the inhibitors to the protease dimer, the change of affinity as a result of Glu/Gln alteration at the P2' position and of chirahtv of the tetrahedrally co-ordinated transitionstate-analogue carbon Details of the binding mode of these inhibitors are expected to explain the differences in affinities measured by K, , K^E = 0 15 nM, Kf Q = 33 0 nM, K, RE = 0 12 nM, K RQ = 14 0 nM

Research paper thumbnail of Inactivation of protein-synthesizing T-factor by N-tosyl-L-phenylalanyl chloromethane

Biochimica et biophysica acta, Jan 30, 1971

N-Tosyl-L-phenylalanyl chloromethane causes the irreversible inhibition of protein synthesis in p... more N-Tosyl-L-phenylalanyl chloromethane causes the irreversible inhibition of protein synthesis in poly(U)-and poly(A)-directed cell-free systems from Escherichia coll. The elongation factor T is the component affected by the inhibitor. The T-factor inactivated by the chloromethylketone loses its ability to stimulate the binding of aminoacyl-tRNA to ribosomes. Activities of aminoacyl-tRNA ligases, ribosomes and the elongation factor G are not altered.

Research paper thumbnail of Configurations of Diastereomeric Hydroxyethylene Isosteres Strongly Affect Biological Activities of a Series of Specific Inhibitors of Human-Immunodeficiency-Virus Proteinase

European Journal of Biochemistry, 1997

Human immunodeficiency virus (HIV) proteinase (PR) represents an important target for antiviral c... more Human immunodeficiency virus (HIV) proteinase (PR) represents an important target for antiviral chemotherapy. We present an analysis of inhibitory activities of a series of pseudopeptide inhibitors of HIV-1 PR. All inhibitors were N-protected tetrapeptides with the scissile bond replaced by a nonhydrolysable hydroxyethylene or hydroxyethylamine isostere. To elucidate subtle structural requirements of the PR binding cleft, we synthesised inhibitors with four combinations of configurations at the asymmetric carbons of the isostere. Compounds were tested in vitro using purified recombinant enzyme and a chromogenic peptide substrate. The differences in inhibition constants between individual diastereoisomers reached three orders of magnitude. The most active hydroxyethylene-containing inhibitor possessed the 2R,4S,5S configuration at the isostere. Inhibitor activity was also tested in mammalian cell culture by analysing reduction of viral polyprotein processing and virus infectivity. The results obtained in tissue culture were generally in agreement with the in vitro data, giving a similar order of potency for the individual diastereoisomers. The most active compounds completely blocked production of infectious virus. A simulation method for interaction was employed to build a model of the inhibitors in the PR active site, to identify the interactions responsible for the differences in activities of individual stereoisomers, and to estimate the relative contribution of individual structural features to the overall inhibitory activity.

Research paper thumbnail of Optimization of the crystallizability of a single-chain antibody fragment

Acta Crystallographica Section F Structural Biology Communications, 2014

Single-chain variable antibody fragments (scFvs) are molecules with immense therapeutic and diagn... more Single-chain variable antibody fragments (scFvs) are molecules with immense therapeutic and diagnostic potential. Knowledge of their three-dimensional structure is important for understanding their antigen-binding mode as well as for protein-engineering approaches such as antibody humanization. A major obstacle to the crystallization of single-chain variable antibody fragments is their relatively poor homogeneity caused by spontaneous oligomerization. A new approach to optimization of the crystallizability of single-chain variable antibody fragments is demonstrated using a representative single-chain variable fragment derived from the anti-CD3 antibody MEM-57. A Thermofluor-based assay was utilized to screen for optimal conditions for antibody-fragment stability and homogeneity. Such an optimization of the protein storage buffer led to a significantly improved ability of the scFv MEM-57 to yield crystals.

Research paper thumbnail of Processing, Purification, and Kinetic Characterization of the Gag-Pol Encoded Retroviral Proteinase of Myeloblastosis Associated Virus Expressed in E. Coli

Advances in Experimental Medicine and Biology, 1995

Research paper thumbnail of Role of hydroxyl group and R/S configuration of isostere in binding properties of HIV-1 protease inhibitors

European Journal of Biochemistry, 2004

The crystal structure of the complex between human immunodeficiency virus type 1 (HIV-1) protease... more The crystal structure of the complex between human immunodeficiency virus type 1 (HIV-1) protease and a peptidomimetic inhibitor of ethyleneamine type has been refined to R factor of 0.178 with diffraction limit 2.5 Å . The peptidomimetic inhibitor Boc-Phe-Y[CH 2 CH 2 NH]-Phe-Glu-Phe-NH 2 (denoted here as OE) contains the ethyleneamine replacement of the scissile peptide bond. The inhibitor lacks the hydroxyl group which is believed to mimic tetrahedral transition state of proteolytic reaction and thus is suspected to be necessary for good properties of peptidomimetic HIV-1 protease inhibitors. Despite the missing hydroxyl group the inhibition constant of OE is 1.53 nM and it remains in the nanomolar range also towards several available mutants of HIV-1 protease. The inhibitor was found in the active site of protease in an extended

Research paper thumbnail of The role of aminoacyl-tRNA binding site on the factor EF-T in uncoupled GTPase reaction

Biochimica et Biophysica Acta (BBA) - Nucleic Acids and Protein Synthesis, 1974

The properties of the N-tosyl-L-phenylalanyl chloromethane treated EF-T factor were studied in a ... more The properties of the N-tosyl-L-phenylalanyl chloromethane treated EF-T factor were studied in a ribosomal system in which splitting of GTP occurs. The action of N-tosyl-L-phenylalanyl chloromethane inhibits the binding of aminoacyl-tRNA to the EF-T factor. The binding and exchange of guanosine phosphates continued to be preserved. The inhibited factor is inactive in the GTPase reaction which depends on the participation of ribosomes and aminoacyl-tRNA. The uncoupled GTPase reaction (which is not dependent on the presence of aminoacyl-tRNA) is also sensitive to the effect of the inhibitor. The inhibition of the uncoupled GTPase is incomplete. These findings are attributed to the involvement of the aminoacyl-tRNA binding site of the EF-T factor in the uncoupled GTPase reaction.

Research paper thumbnail of Myeloblastosis Associated Virus (MAV) Proteinase Site-Mutated to Be HIV-like Has a Higher Activity and Allows Production of Infectious but Morphologically Altered Virus

Virology, 1993

We have characterized the structure and infectivity of an avian retrovirus, myeloblastosis associ... more We have characterized the structure and infectivity of an avian retrovirus, myeloblastosis associated virus (MAV), containing a genetically altered proteinase (PR). A site-directed mutant of MAV-PR that shows an increased proteolytic activity in vitro (about 20 times higher kcat/Km) as a consequence of substituting five amino acids from the substrate-binding pocket with those corresponding to the HIV-1 PR was cloned into a full-sized MAV plasmid. In particular, the wild-type MAV-PR gene was replaced with the mutant one. Despite encoding for an enzyme with increased PR activity, mutant plasmid-transfected turkey fibroblasts displayed an unimpaired virus production in cell cultures. Further, the mutant progeny virus was infectious and its pattern of gag processing products appeared identical to that of wild-type virus. However, by electron microscopy we found that the predominant morphology of mutant viral particles was altered. Instead of a centrally collapsed avian retroviral core, a more diffuse core was visualized for wild-type mutant virions, similar to that observed in mammalian C-type retroviruses.

Research paper thumbnail of A Phenylnorstatine Inhibitor Binding to HIV1 Protease:  Geometry, Protonation, and Subsite−Pocket Interactions Analyzed at Atomic Resolution

Journal of Medicinal Chemistry, 2004

The X-ray structure of a complex of HIV-1 protease (PR) with a phenylnorstatine inhibitor Z-Pns-P... more The X-ray structure of a complex of HIV-1 protease (PR) with a phenylnorstatine inhibitor Z-Pns-Phe-Glu-Glu-NH 2 has been determined at 1.03 Å, the highest resolution so far reported for any HIV PR complex. The inhibitor shows subnanomolar K i values for both the wild-type PR and the variant representing one of the most common mutations linked to resistance development. The structure comprising the phenylnorstatine moiety of (2R,3S)-chirality displays a unique pattern of hydrogen bonding to the two catalytic aspartate residues. This high resolution makes it possible to assess the donor and acceptor relations of this hydrogen bonding and to indicate a proton shared by the two catalytic residues. A structural mechanism for the unimpaired inhibition of the protease Val82Ala mutant is also suggested, based on energy calculations and analyses.

Research paper thumbnail of The Gag-Pol Encoded Proteinase of an Avian Retrovirus Expressed in E. coli Can Produce a Novel Proteinase (PR+IIeGly) That Is Two Amino Acids Larger at Its Carboxy-Terminal Region Than the Major Gag Proteinase (PR)

Research paper thumbnail of Structure of HOE/BAY 793 Complexed to Human Immunodeficiency Virus (HIV-1) Protease in Two Different Crystal Forms Structure/Function Relationship and Influence of Crystal Packing

European Journal of Biochemistry, 1997

Human immunodeficiency virus 1 (HIV-1) protease is a prime target in the search for drugs to comb... more Human immunodeficiency virus 1 (HIV-1) protease is a prime target in the search for drugs to combat the AIDS virus. The enzyme functions as a C,-symmetric dimer, cleaving the gag and gag-pol viral polyproteins at distinct sites. The possession of a twofold axis passing through the active site, has led to the design of C,-symmetrical inhibitors in the form of substrate-based transition-state analogs. One of the most active compounds of this class of inhibitors is HOE/BAY 793, which contains a vicinal diol central The structure of this inhibitor bound to HIV-1 protease, in two different crystal forms, has been solved at 0.24-nm resolution using X-ray crystallography. In both forms, the details of the inhibitor-protease interactions revealed an overall asymmetric binding mode, especially between the central diol unit and the active-site aspartates. The main binding interactions comprise several specific H-bonds and hydrophobic contacts, which rationalize many of the characteristics of the structure/ activity relationship in the class of vicinal diol inhibitors. In a general analysis of the mobility of the flap regions, which cover the active site and participate directly in binding, using our structures and the HTV protease models present in the Brookhaven databank, we found that in most structures the flexibility of the flaps is limited by local crystal contacts. However, in one of the structures presented here, no significant crystal contacts to the flap regions were present, and as a result the flexibility of the inhibitor bound flaps increased significantly. This suggests that the mobility and conformational flexibility of the flap residues are important in the functioning of HIV-1 protease, and must be considered in the future design of drugs against HIV protease and in structure-based drug design in general.

Research paper thumbnail of Proteolytic Processing of Particle-Associated Retroviral Polyproteins by Homologous and Heterologous Viral Proteinases

European Journal of Biochemistry, 1995

Research paper thumbnail of Structural studies of the retroviral proteinase from avian myeloblastosis associated virus

Proteins: Structure, Function, and Genetics, 1992

The structure of the retroviral proteinase from avian myeloblastosis associated virus (MAV) has b... more The structure of the retroviral proteinase from avian myeloblastosis associated virus (MAV) has been determined and refined at 2.2 A resolution. This structure is compared with those of homologous proteinases from Rous sarcoma virus (RSV) and hum a n immunodeficiency type 1 virus (HIV). Through comparison with the structure of a proteinase-inhibitor complex from HIV, a model of a complex between MAV proteinase and a peptide substrate has been generated. Examination of this model suggests structural basis for the diverse specifications of viral PrOteinaSeS. 0 1992 Wiley-Liss, Inc.

Research paper thumbnail of Stabilization of antibody structure upon association to a human carbonic anhydrase IX epitope studied by X-ray crystallography, microcalorimetry, and molecular dynamics simulations

Proteins: Structure, Function, and Bioinformatics, 2007

Specific antibodies interfere with the function of human tumor-associated carbonic anhydrase IX (... more Specific antibodies interfere with the function of human tumor-associated carbonic anhydrase IX (CA IX), and show potential as tools for anticancer interventions. In this work, a correlation between structural elements and thermodynamic parameters of the association of antibody fragment Fab M75 to a peptide corresponding to its epitope in the proteoglycan-like domain of CA IX, is presented. Comparisons of the crystal structures of free Fab M75 and its complex with the epitope peptide reveal major readjustments of CDR-H1 and CDR-H3. In contrast, the overall conformations and positions of CDR-H2 and CDR-L2 remain unaltered, and their positively charged residues may thus present a fixed frame for epitope recognition. Adoption of the altered CDR-H3 conformation in the structure of the complex is accompanied by an apparent local stabilization. Analysis of domain mobility with translation-libration-screw (TLS) method shows that librations of the entire heavy chain variable domain (V(H)) decrease and reorient in the complex, which correlates well with participation of the heavy chain in ligand binding. Isothermal titration microcalorimetry (ITC) experiments revealed a highly unfavorable entropy term, which can be attributed mainly to the decrease in the degrees of freedom of the system, the loss of conformational freedom of peptide and partially to a local stabilization of CDR-H3. Moreover, it was observed that one proton is transferred from the environment to the protein-ligand complex upon binding. Molecular dynamics simulations followed by molecular mechanics/generalized Born surface area (MM-GBSA) calculations of the ligand (epitope peptide) binding energy yielded energy values that were in agreement with the ITC measurements and indicated that the charged residues play crucial role in the epitope binding. Theoretical arguments presented in this work indicate that two adjacent arginine residues (ArgH50 and ArgH52) are responsible for the observed proton transfer.

Research paper thumbnail of From nonpeptide toward noncarbon protease inhibitors: Metallacarboranes as specific and potent inhibitors of HIV protease

Proceedings of the National Academy of Sciences, 2005

HIV protease (PR) represents a prime target for rational drug design, and protease inhibitors (PI... more HIV protease (PR) represents a prime target for rational drug design, and protease inhibitors (PI) are powerful antiviral drugs. Most of the current PIs are pseudopeptide compounds with limited bioavailability and stability, and their use is compromised by high costs, side effects, and development of resistant strains. In our search for novel PI structures, we have identified a group of inorganic compounds, icosahedral metallacarboranes, as candidates for a novel class of nonpeptidic PIs. Here, we report the potent, specific, and selective competitive inhibition of HIV PR by substituted metallacarboranes. The most active compound, sodium hydrogen butylimino bis-8,8-[5-(3-oxa-pentoxy)-3-cobalt bis(1,2dicarbollide)]di-ate, exhibited a Ki value of 2.2 nM and a submicromolar EC50 in antiviral tests, showed no toxicity in tissue culture, weakly inhibited human cathepsin D and pepsin, and was inactive against trypsin, papain, and amylase. The structure of the parent cobalt bis(1,2-dicarbollide) in complex with HIV PR was determined at 2.15 Å resolution by protein crystallography and represents the first carborane-protein complex structure determined. It shows the following mode of PR inhibition: two molecules of the parent compound bind to the hydrophobic pockets in the flap-proximal region of the S3 and S3 subsites of PR. We suggest, therefore, that these compounds block flap closure in addition to filling the corresponding binding pockets as conventional PIs. This type of binding and inhibition, chemical and biological stability, low toxicity, and the possibility to introduce various modifications make boron clusters attractive pharmacophores for potent and specific enzyme inhibition. rational drug design ͉ aspartic proteases ͉ carboranes ͉ x-ray structure analysis ͉ virostatics Freely available online through the PNAS open access option.