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Nucleic Acids Research Advance Access published online on August 1, 2008
Nucleic Acids Research, doi:10.1093/nar/gkn464
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© 2008 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Structural Biology

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

Joseph D. Bauman1,2, Kalyan Das1,2, William C. Ho1,2, Mukta Baweja1, Daniel M. Himmel1,2, Arthur D. Clark, Jr1,2, Deena A. Oren1,2, Paul L. Boyer3, Stephen H. Hughes3, Aaron J. Shatkin1 and Eddy Arnold1,2,*

1Center for Advanced Biotechnology and Medicine, 2Department of Chemistry and Chemical Biology, Rutgers University, Piscataway, NJ and 3NCI-Frederick Cancer Research and Development Center, Frederick, MD, USA

*To whom correspondence should be addressed. Tel: +732 235 5323; Fax: +732 235 5788; Email: arnold{at}cabm.rutgers.edu

Received May 1, 2008. Revised July 2, 2008. Accepted July 3, 2008.

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.

Present address: Deena A. Oren, Structural Biology Resource Center, Rockefeller University, New York, NY, USA.


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