Probability Analysis of Variational Crystallization and Its Application to gp120, The Exterior Envelope Glycoprotein of Type 1 Human Immunodeficiency Virus (HIV-1)

摘要

The extensive glycosylation and conformational mobility of gp120, the envelope glycoprotein of type 1 human immunodeficiency virus (HIV-1), pose formidable barriers for crystallization. To surmount these difficulties, we used probability analysis to determine the most effective crystallization approach and derive equations which show that a strategy, which we term variational crystallization, substantially enhances the overall probability of crystallization for gp120. Variational crystallization focuses on protein modification as opposed to crystallization screening. Multiple variants of gp120 were analyzed with an iterative cycle involving a limited set of crystallization conditions and biochemical feedback on protease sensitivity, glycosylation status, and monoclonal antibody binding. Sources of likely conformational heterogeneity such asN-linked carbohydrates, flexible or mobile N and C termini, and variable internal loops were reduced or eliminated, and ligands such as CD4 and antigen-binding fragments (Fabs) of monoclonal antibodies were used to restrict conformational mobility as well as to alter the crystallization surface. Through successive cycles of manipulation involving 18 different variants, we succeeded in growing six different types of gp120 crystals. One of these, a ternary complex composed of gp120, its receptor CD4, and the Fab of the human neutralizing monoclonal antibody 17b, diffracts to a minimum Bragg spacing of at least 2.2 Å and is suitable for structural analysis. The extensive glycosylation and conformational mobility of gp120, the envelope glycoprotein of type 1 human immunodeficiency virus (HIV-1), pose formidable barriers for crystallization. To surmount these difficulties, we used probability analysis to determine the most effective crystallization approach and derive equations which show that a strategy, which we term variational crystallization, substantially enhances the overall probability of crystallization for gp120. Variational crystallization focuses on protein modification as opposed to crystallization screening. Multiple variants of gp120 were analyzed with an iterative cycle involving a limited set of crystallization conditions and biochemical feedback on protease sensitivity, glycosylation status, and monoclonal antibody binding. Sources of likely conformational heterogeneity such asN-linked carbohydrates, flexible or mobile N and C termini, and variable internal loops were reduced or eliminated, and ligands such as CD4 and antigen-binding fragments (Fabs) of monoclonal antibodies were used to restrict conformational mobility as well as to alter the crystallization surface. Through successive cycles of manipulation involving 18 different variants, we succeeded in growing six different types of gp120 crystals. One of these, a ternary complex composed of gp120, its receptor CD4, and the Fab of the human neutralizing monoclonal antibody 17b, diffracts to a minimum Bragg spacing of at least 2.2 Å and is suitable for structural analysis. In conventional crystallizations of biological macromolecules, the protein or other macromolecular subject is treated as a fixed entity to be tested in a multitude of crystallization conditions. Despite advances such as sophisticated screening procedures (1Carter Jr., C.W. Carter C.W. J. Biol. Chem. 1979; 254: 12219-12223Abstract Full Text PDF PubMed Google Scholar, 2Jancarik J. Kim S.H. J. Appl. Crystallogr. 1991; 24: 409-411Crossref Scopus (2076) Google Scholar) and crystallization robots (3Kelders H.A. Kalk K.H. Gros P. Hol W.G. Protein Eng. 1987; 1: 301-303Crossref PubMed Scopus (13) Google Scholar, 4Morris D.W. Kim C.Y. McPherson A. BioTechniques. 1989; 7: 522-527PubMed Google Scholar), this approach often fails for components from complex biological systems. One of these, the subject of this study, is the HIV-1 1The abbreviations used are: HIV, human immunodeficiency virus; PAGE, polyacrylamide gel electrophoresis; PEG, polyethylene glycol; Fab, anti