Towards crystal structures of antibodies and transcription factors
J. Písačková1, 2, 3, K. Procházková1,
V. Král2, M. Fábry2, P. Řezáčová1, 2
1Institute of Organic Chemistry and Biochemistry, ASCR, Flemingovo nam. 2, 166 10 Prague 6, Czech Republic
2Institute
of Molecular Genetics, ASCR, Videnska 1083, 142 20
Prague 4, Czech Republic
3Faculty of Science, Charles University in Prague, Albertov 6, 128 43 Prague 2, Czech Republic
jana.pisackova@img.cas.cz
Introduction
The knowledge of protein three-dimensional structure is important for understanding protein function. The three-dimensional structure can, among other things, provide information on regulation of protein function by interaction with ligands that can also be utilized in rational design of inhibitors or modulators. Structural information can also provide a basis for protein engineering approaches.
X-ray crystallography is the principal method of protein structure determination, close to 90% of all the protein structures deposited in the Protein Data Bank to date have been solved by this method. Crystallization of protein represents a common bottleneck in the process of structure determination as having sufficiently large monocrystal is the essential requirement for diffraction experiments. The crystallization process is influenced by large number of factors from which the protein sample properties are the most important variable. Therefore, pre-crystallization biochemical and biophysical characterization of the protein sample can help in successful crystallization. This analysis can also be beneficial for other methods of structure determination such as NMR.
The use of pre-crystallization analysis will be presented on an example of two case studies: antibody fragments and bacterial transcriptional regulator.
Recombinant
antibody fragments
Monoclonal antibody MEM-57 recognizes CD3 antigen, which is expressed on the surface of T-lymphocytes in complex with the T-cell receptor. CD3 plays role in the transduction of activation signal after the antigen is recognized by T-cell receptor. Antibody MEM-57 shows similar properties to the therapeutic anti-CD3 antibody OKT3 used as an immunosuppressant in organ transplantation. Antibody MEM-57 could be used in diagnostics of autoimmune diseases, for T-cell lymphoma classification, or as an immunosuppressant in transplantation. Antibody MEM-57 could be also used in cancer therapy in therapeutic antibody format of Bispecific T-cell Engager (BiTE). BiTE molecule consists of an anti-CD3 antibody single chain variable fragment (scFv) linked to an anti-tumor antigen scFv. BiTE induces polyclonal activation of cytotoxic T-lymphocytes exclusively in the tumor site [1]. The structural information on scFv MEM‑57 would enable humanization of the antibody for therapeutic applications.
Monoclonal antibody MEM-85 recognizes CD44 antigen, which is a cell surface receptor for hyaluronate and plays an important role in the immune system [2]. Some tumors exhibit CD44 overexpression and this is associated with bad prognosis. Antibody MEM-85 could be used in cancer diagnostics and classification or in cancer immunotherapy. MEM-85 blocks hyaluronate binding to CD44 and has been shown to induce CD44 shedding from the cell surface, similar to the shedding induced by hyaluronate binding. Thus, MEM-85 could also be used as a tool for analysis of the structural effects of hyaluronate – CD44 interaction on cellular events. Structural information on the antibody – receptor complex would allow rational design of potential hyaluronate binding inhibitors.
Deoxyribonucleoside regulator DeoR
In bacteria, transcription of metabolic genes is regulated by various catabolic repressors [3]. Bacillus subtilis can utilize deoxyribonucleosides and deoxyribose as a source of carbon and energy. The genes encoding the proteins required for their catabolism are grouped in the dra-nupC-pdp operon. Expression of this operon is repressed by binding of deoxyribonucleoside regulator protein DeoR. Expression of metabolic genes is induced by binding of small molecular effector to DeoR. The preferred effector molecule is deoxyribose-5-phosphate, but deoxyribose-1-phosphate was also described to act as an inducer. The DeoR repressor protein from Bacillus subtilis is homologous to bacterial regulator proteins of the SorC family. There is no sequence similarity between the DeoR regulators of Bacillus subtilis and Escherichia coli, even though they possess similar regulatory function. Structural information on DeoR free and ligand bound forms would elicit the regulation of DeoR function by a small molecular effector.
Experimental
section
Proteins were prepared by heterologous expression in E. coli and purified by combinations of nickel chelation chromatography and ion-exchange chromatography. Protein pre-crystallization analysis employed size-exclusion chromatography, flow cytometry, dynamic light scattering, and thermofluor assay (also known as differential scanning fluorimetry, DSF). Thermofluor assay was used for the optimization of protein stability for crystallization, for optimization of protein oligomeric homogeneity, and for characterization of protein – ligand interactions. Crystallization screening was performed by the sitting drop vapor diffusion method; crystallization optimization employed a wide range of approaches using hanging drop vapor diffusion and counter-diffusion techniques. Diffraction data were collected at beamline MX14.2 at BESSY, Berlin and processed using the HKL-3000 package. Structures of DeoR were solved by molecular replacement using MolRep program; model building was performed automatically by Buccaneer and manually by Coot programs. Structures were refined using Refmac program.
Results and
discussion
Recombinant
antibody fragments
Single-chain variable fragments (scFv) of the antibodies MEM-57 and MEM-85 were constructed: variable domains of the heavy and light chains were joined by a flexible Gly-Ser linker and further equipped with the N-terminal pelB leader sequence, and C-terminal c‑myc tag and His5 tag. The recombinant fragments were targeted into the periplasmic space of E. coli where they accumulated in a soluble form. Proteins were isolated from the host by osmotic shock. Two-step purification protocol employing nickel chelation chromatography and ion‑exchange chromatography was developed to produce high yield of pure protein: 3 mg of scFv MEM-57 and 1.5 mg of scFv MEM-85 per 1l of bacterial culture. Antigen binding activity of both antibody fragments was confirmed by flow cytometry.
The ratio between monomeric and multimeric forms of the individual scFv fragments was determined by analytical size-exclusion chromatography. In case of scFv MEM-57, equilibrium established between monomer, dimer and higher oligomers with the majority of the protein being in the monomeric form. On the contrary, the monomeric and dimeric forms of scFv MEM-85 could be separated during the purification process.
Dynamic light scattering was used to evaluate the dispersity of the individual protein preparations at high concentrations used in crystallization experiments. Protein preparations of scFv MEM-57 which showed monomodal particle size distribution yielded crystals of better quality, unlike polydisperse protein preparations. All protein preparations of scFv MEM-85 were monomodal and monodisperse.
Thermofluor assay was used to screen for the composition of the storage buffer optimal for protein stability and to evaluate protein oligomeric homogeneity. For scFv MEM-57, the positive effect of the storage buffer composition (100 mM sodium phosphate pH 7.5, 200 mM NaCl) was confirmed by the results of initial crystallization trials. Crystallization screening in the original storage buffer did not yield any crystals of scFv MEM-57. After the buffer was changed to composition which showed to be optimal for protein stability in the thermofluor assay (100 mM sodium phosphate pH 7.5, 200 mM NaCl), protein crystals were obtained in 42 out of 96 screened conditions. Optimization of crystallization conditions by a wide range of approaches using hanging drop vapor diffusion and counter-diffusion techniques is now in progress.
For scFv MEM-85, thermofluor analysis showed high thermal stability of the antibody fragment in the majority of tested buffer systems (melting temperature Tm of 340 – 342 K). Crystallization trials were unsuccessful so far owing probably to the high stability of the protein.
Deoxyribonucleoside regulator DeoR
The C-terminal effector-binding domain of DeoR from B. subtilis (C-DeoR) equipped with N-terminal His6 tag cleavable by tobacco etch virus protease was expressed in E. coli and purified using nickel chelation chromatography. Pre-crystallization analysis performed by size-exclusion chromatography and dynamic light scattering confirmed protein sample homogeneity and showed that a dimer is the biological unit of C-DeoR. The ligand binding activity of the recombinant C-DeoR was confirmed by thermofluor assay for deoxyribose-5-phosphate, but not for deoxyribose-1-phosphate. These results confirmed the role of deoxyribose-5-phosphate as a preferred inducer.
Crystallization screening trials in the presence of 50 mM deoxyribose-5-phosphate yielded needle-shaped crystals. Extensive crystallization optimization by a wide range of approaches using hanging drop vapor diffusion and counter-diffusion techniques together with protein re-purification by ion-exchange chromatography only yielded plate-like crystals of poor diffraction quality. Thermofluor assay was used to screen for the composition of the storage buffer optimal for protein stability and revealed specific thermal stabilization of C-DeoR by trisodium citrate The crystallization screening procedure was repeated in the optimized storage buffer (20 mM trisodium citrate pH 7.0, 150 mM NaCl, 0.02% (v/v) β-mercaptoethanol) both in the presence and absence of 50 mM deoxyribose-5-phosphate, which yielded three-dimensional crystals. By optimizing the protein and precipitant concentrations large three-dimensional crystals with a good diffraction quality were obtained and diffraction data sets from three different crystal forms were collected at high resolution [4]. The structures were solved by molecular replacement using putative sugar-binding transcriptional regulator from Arthrobacter aurescens TC1 as a model. Structure refinement is currently in progress.
Conclusions
Analysis of proteins by combination of biochemical and biophysical methods such as size-exclusion chromatography, dynamic light scattering, and thermofluor assay can be successfully used to help protein crystallization. We have prepared scFv fragments of antibodies MEM-57 and MEM-85. We have performed protein pre-crystallization analysis and we have optimized protein stability and homogeneity for both NMR (MEM-85) and crystallization (MEM-57). Optimization of crystallization conditions is currently in progress. In order to gain information on the structure of the deoxyribonucleoside regulator from Bacillus subtilis in the ligand free form and in the complex with deoxyribose-5-phosphate, we have prepared the C-terminal effector-binding domain of DeoR (C-DeoR). We have performed protein pre-crystallization analysis and developed crystallization protocols which yielded monocrystals of three crystal forms suitable for diffraction data collection. Three complete high-resolution data sets were collected and processed, the structures were solved using molecular replacement; structure refinement is in progress.
Acknowledgements
This work was supported by research project GA UK 567012 awarded by the Charles University in Prague, by research projects. ME08016, and 1M0505 awarded by the Ministry of Education of the Czech Republic, research project GA203/09/0820 awarded by the Grant Agency of the Czech Republic, and research projects AV0Z40550506, and AV0Z50520514 awarded by the Academy of Sciences of the Czech Republic.
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