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Structural basis of HIV-1 and HIV-2 protease inhibition bya monoclonal antibody
Řezáčová, Pavlína ; Lescar, J. ; Brynda, Jiří ; Fábry, Milan ; Bentley, G. A. ; Sedláček, Juraj
The murine monoclonal antibody 1696, produced by immunisation with the HIV-1 protease,inhibits the catalytic activity of the enzyme of both the HIV-1 and HIV-2 isolates, with inhibitionconstants in the low nanomolar range. This antibody cross-reacts with peptides that include theN-terminus of the enzyme (residues 1-7), a region which is highly conserved in sequence amongdifferent viral strains and which, furthermore, is crucial for homodimerization to the activeenzymatic form.<?xml:namespace prefix = o ns = "urn:schemas-microsoft-com:office:office" />We report here two crystal structures of a recombinant single-chain Fv fragment of mAb 1696,expressed in E. coli, as a complex with a cross-reactive peptides from the HIV-1 PR and theHIV-2 PR at 2.7 ? resolution and 1.9 ? resolution respectively. On the basis of the interactionsseen in the complex three-dimensional structures, the cross-reactivity between mAb 1696 withthe HIV-1 and HIV-2 protease and their N-terminal peptides can be explained. In addition, acandidate mechanism of HIV PR inhibition by mAb 1696 is proposed which may help thedesign of alternative HIV protease inhibitors, aimed at dissociating the homodimeric viral enzyme.
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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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Cryocrystallography of biological Macromolecules
Řezáčová, Pavlína
Crystals of biological macromolecules at near to room temperature are sensitive to X-rays and frequently suffer from radiation damage, especially when X-ray experiments are carried out on highly intense synchrotron beamlines. Performing such experiments at cryogenic temperatures greatly reduce, or eliminate radiation damage and thus produce higher quality diffraction data.
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