Crystal engineering of HIV-1 reverse transcriptase for structure-based drug design

• Published in: Nucleic acids research Vol. 36; no. 15; pp. 5083 - 5092
• Main Authors: Bauman, Joseph D, Das, Kalyan, Ho, William C, Baweja, Mukta, Himmel, Daniel M, Clark, Arthur D. Jr, Oren, Deena A, Boyer, Paul L, Hughes, Stephen H, Shatkin, Aaron J, Arnold, Eddy
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 01-09-2008; Oxford Publishing Limited (England)
• Subjects: Cloning, Molecular; Crystallography, X-Ray; Drug Design; HIV Reverse Transcriptase - chemistry; HIV Reverse Transcriptase - genetics; HIV Reverse Transcriptase - metabolism; Human immunodeficiency virus 1; Models, Molecular; Mutagenesis; Nitriles - chemistry; Protein Engineering - methods; Pyrimidines - chemistry; Reverse Transcriptase Inhibitors - chemistry; Rilpivirine; Structural Biology
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/gkn464

HIV-1 reverse transcriptase (RT) is a primary target for anti-AIDS drugs. Structures of HIV-1 RT, usually determined at ~2.5-3.0 Å 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 Å resolution. Another form of engineered RT was optimized to produce a high-resolution apo-RT crystal form, reported here at 1.85 Å 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.