Alfredo Jacobo-Molina

Review of HIV-1 reverse transcriptase three-dimensional structure: Implications for drug design

Two recent X-ray crystallographic studies have resulted in the three-dimensional structure determination of the reverse transcriptase (RT) enzyme from the human immunodeficiency virus type 1 (HIV-1) [Kohlstaedt et al., Science, 256 (1992) 1783; Jacobo-Molina et al., Proc. Natl. Acad. Sci. USA, 90 (1993) 6320]. This report reviews the structure of the reverse transcriptase heterodimer and provides a detailed description of the folding and topology of the individual subdomains. The interactions of the enzyme with bound template- primer are highlighted. Structure-function relationships have been established and are discussed for several conserved sequence motifs located within the enzyme. Each of these motifs is found to interact significantly with template-primer during the polymerization process. This review integrates the findings of both structure determinations, in particular, to relate these structures to strategies for drug design and development. The structures of both the nucleoside and nonnucleoside inhibitor binding sites are described, and the spatial relationship between the two sites is discussed in light of some novel possibilities for drug development. The first indication of an HIV-1 RT drug-resistant mutation manifested in the p51 subunit is presented. This mutation is located in a region of p51 that is proximal to the nonnucleoside binding pocket. The mechanisms of HIV-1 RT inhibition by both nucleoside and nonnucleoside classes of inhibitors are discussed in relation to the structure of the enzyme. In addition, the implications of the structure for understanding and avoiding the development of resistance of HIV-1 reverse transcriptase to antiviral inhibitors are discussed.

[15] Crystallization of human immunodeficiency virus type 1 reverse transcriptase with and without nucleic acid substrates, inhibitors, and an antibody fab fragment

Publisher Summary This chapter details the methodology used to produce diffraction-quality crystals of a number of Reverse Transcriptase (RT) complexes. The chapter includes description of the purification of the enzyme and of a noninhibitory Fab used in some of the crystallization experiments, because the reproducible preparation of high-quality crystals of HIV-1 RT is critically dependent on the protocols used to purify each of these proteins. The protocol used for the purification of Fab 28 yields one of the isoelectric variants. Crystallization of the RT Fab complex is carried out in hanging-drop vapor diffusion experiments at 4° over reservoirs containing 0.5 ml of crystallization solution. The parent HIV-1 reverse transcriptase used in the preparation of all the crystal forms described in this chapter is a mutant RT that has serine substituted for cysteine at amino acid position 280. It is fully active in polymerization and Ribonuclease H (RNase H) activities and is resistant to oxidative inactivation of RNase H. Crystals of RT that contain one or two amino acid substitutions have been prepared in complexes with Fab28 and dsDNA.

Crystallization and preliminary X-ray diffraction analysis of nucleoside diphosphate kinase from Myxococcus xanthus

Nucleoside diphosphate (NDP) kinase catalyzes the transfer of the gamma-phosphate from a nucleoside triphosphate to a nucleoside diphosphate. Human and rodent forms of this enzyme have been shown to be suppressors of metastasis. Crystals that diffract X-rays to high resolution have been obtained for the recombinant Myxococcus xanthus NDP kinase expressed in and purified from Escherichia coli. Two crystal forms have been obtained. Both forms are orthorhombic, space group I222 (or I2(1)2(1)2(1)) with a = 267.1 A, b = 74.0 A and c = 75.1 A for form I and a = 53.5 A, b = 74.0 A and c = 75.1 A for form II. Form I appears to have five molecules in the asymmetric unit approximately related to each other by a translation of 0.2 along the a axis. Diffraction data have been recorded to 1.9 A for form I and to 2.2 A for form II.