title: Crystal engineering of HIV-1 reverse transcriptase for structure-based drug design.
abstract: HIV-1 reverse transcriptase (RT) is a primary target for anti-AIDS drugs. Structures of HIV-1 RT, usually determined at approximately 2.5-3.0 A resolution, are important for understanding enzyme function and mechanisms of drug resistance in addition to being helpful in the design of RT inhibitors. Despite hundreds of attempts, it was not possible to obtain the structure of a complex of HIV-1 RT with TMC278, a nonnucleoside RT inhibitor (NNRTI) in advanced clinical trials. A systematic and iterative protein crystal engineering approach was developed to optimize RT for obtaining crystals in complexes with TMC278 and other NNRTIs that diffract X-rays to 1.8 A resolution. Another form of engineered RT was optimized to produce a high-resolution apo-RT crystal form, reported here at 1.85 A resolution, with a distinct RT conformation. Engineered RTs were mutagenized using a new, flexible and cost effective method called methylated overlap-extension ligation independent cloning. Our analysis suggests that reducing the solvent content, increasing lattice contacts, and stabilizing the internal low-energy conformations of RT are critical for the growth of crystals that diffract to high resolution. The new RTs enable rapid crystallization and yield high-resolution structures that are useful in designing/developing new anti-AIDS drugs.
authors.affiliation: Center for Advanced Biotechnology and Medicine, Rutgers University, Piscataway, NJ, USA.
authors.forename: Joseph D
authors.initials: JD
authors.lastname: Bauman
authors.forename: Kalyan
authors.initials: K
authors.lastname: Das
authors.forename: William C
authors.initials: WC
authors.lastname: Ho
authors.forename: Mukta
authors.initials: M
authors.lastname: Baweja
authors.forename: Daniel M
authors.initials: DM
authors.lastname: Himmel
authors.forename: Arthur D
authors.initials: AD
authors.lastname: Clark
authors.forename: Deena A
authors.initials: DA
authors.lastname: Oren
authors.forename: Paul L
authors.initials: PL
authors.lastname: Boyer
authors.forename: Stephen H
authors.initials: SH
authors.lastname: Hughes
authors.forename: Aaron J
authors.initials: AJ
authors.lastname: Shatkin
authors.forename: Eddy
authors.initials: E
authors.lastname: Arnold
mesh_terms: D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D015195:Drug Design; D054303:HIV Reverse Transcriptase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D016296:Mutagenesis; D009570:Nitriles / Q000737:chemistry*; D015202:Protein Engineering / Q000379:methods*; D011743:Pyrimidines / Q000737:chemistry*; D018894:Reverse Transcriptase Inhibitors / Q000737:chemistry*; D000068696:Rilpivirine
chemical_list: D009570:Nitriles; D011743:Pyrimidines; D018894:Reverse Transcriptase Inhibitors; C514824:reverse transcriptase, Human immunodeficiency virus 1; D054303:HIV Reverse Transcriptase; D000068696:Rilpivirine