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A Phenylnorstatine Inhibitor Binding to HIV-1 Protease: Geometry, Protonation and Subsite-Pocket Interactions Analyzed at Atomic Resolution
Brynda, Jiří ; Řezáčová, Pavlína ; Fábry, Milan ; Hořejší, Magdalena ; Štouračová, Renata ; Sedláček, Juraj ; Souček, M. ; Hradílek, M. ; Lepšík, M. ; Konvalinka, J.
The x-ray structure of a complex of HIV-1 protease (PR) with a phenylnorstatine inhibitor Z-Pns-Phe-Glu-Glu-NH2 has been determined at 1.03 A, the highest resolution so far reported for any HIV PR complex. The inhibiot shows subnanomolar Ki values for both the wild-type PR and the variant representing one of the most common mutations linked to resistance developoment. The structure displays a unique pattern of hydrogen bonding to the two catalytic aspartate residues. The high resolution permits to assess the donor/acceptor realtions of this hydrogen bonding and to indicate a proton shared by the two catalytic residues. Structural mechanism for the unimpaired ihnibition of the protease Val82Ala mutant is also suggested, based on energy calculations and analyses.
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Molecular cloning, E.coli expression and purification of SCFV antibody fragments of diagnostic/therapeutic interest
Král, Vlastimil ; Fábry, Milan ; Hořejší, Magdalena ; Závada, Jan ; Sedláček, Juraj
We describe molecular cloning, expression, purification and properties of two single chain antibody variable fragments (scFv) of potential diagnostic use, namely scFv M75 and scFv Tu-20. The former scFv is derived from a monoclonal antibody M75 specific for a cell surface protein MN/CA IX, strongly associated with many types of human carcinomas. The latter scFv is derived from a monoclonal antibody TU-20 specific for neuronal beta-III-tubulin.
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Crystallographic study of an anti=carbonic anhydrase IX monoclonal antibody M75
Štouračová, Renata ; Závada, Jan ; Závadová, Zuzana ; Pastoreková, S. ; Brynda, Jiří ; Fábry, Milan ; Král, Vlastimil ; Hořejší, Magdalena ; Sedláček, Juraj
Carbonic anhydrase IX (CA IX) is a cell surface protein, strongly associated with certain types of human carcinomas. Structural study of a CA IX-binding monoclonal antibody (mAb) M75, complexed with its epitope peptide may contribute toward elucidation of the role of CA IX. Monoclonal antibody M75 was obtained and proved to react excellently with native and denaturated CA IX. Using synthetic oligopeptides, the epitope of mAb M75 was localized in the proteoglycan domain of CA IX, in the region of a tandem repeat and identified as amino acids PGEEDLP. The Fab fragment was obtained by papain cleavage. We obtained crystals of free Fab M75 and Fab M75 complexed with two different epitope peptides. The data set for Fab M75 was collected and the structure solving is underway.
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Anti-HIV Proteinase Monoclonal Antibody F11.2.32 That Inhibits Enzyme Activity
Štouračová, Renata ; Lescar, J. ; Brynda, Jiří ; RIottot, M. ; Chitarra, V. ; Fábry, Milan ; Hořejší, Magdalena ; Řezáčová, Pavlína ; Bentley, G. ; Sedláček, Juraj
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Vazba fenylnorstatinového inhibitoru na proteázu HIV-1: geometrie, protonace a interakce kapes podřadných míst analyzované při atomovém rozlišení
Brynda, Jiří ; Řezáčová, Pavlína ; Fábry, Milan ; Hořejší, Magdalena ; Štouračová, Renata ; Sedláček, Juraj ; Souček, Milan ; Hradilek, Martin ; Lepšík, Martin ; Konvalinka, Jan
The x-ray structure of a complex of HIV-1 protease (PR) with a phenylnorstatine inhibitor Z-Pns-Phe-Glu-Glu-NH2 has been determined at 1.03 A, the highest resolution so far reported for any HIV PR complex. The inhibiot shows subnanomolar Ki values for both the wild-type PR and the variant representing one of the most common mutations linked to resistance development. The structure displays a unique pattern of hydrogen bonding to the two catalytic aspartate residues. The high resolution permints to assess the donor/acceptor relations of this hydrogen bonding and to indicate a proton shared by the two catalytic residues. Structural mechanism for the unimpaired inhibition of the protease Val82Ala mutant is also suggested, based on energy calculations and analyses
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