Published online 1 August 2008 Nucleic Acids Research, 2008, Vol. 36, No. 15 5083–5092 doi:10.1093/nar/gkn464 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. Hughes 3 , 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 Received May 1, 2008; Revised July 2, 2008; Accepted July 3, 2008 ABSTRACT 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 A˚ resolution, are important for understanding enzyme function and mecha- nisms 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 non- nucleoside 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, increas- ing 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 design- ing/developing new anti-AIDS drugs. INTRODUCTION HIV-1 reverse transcriptase (RT) is the enzyme responsi- ble for generating a double-stranded linear DNA from the single-stranded RNAs packaged in HIV-1 virions. Twelve of the 25 approved anti-AIDS drugs target RT (hivinsite.ucsf.edu, 2008) and are classiﬁed as either nucleoside/nucleotide RT inhibitors (NRTIs) or non- nucleoside RT inhibitors (NNRTIs). A high rate of viral replication combined with lack of eﬃcient proofreading activities in both RT and human RNA polymerase II results in the rapid generation of mutant viruses (1). The generation of HIV-1 mutants in infected patients allows the virus to develop resistance to all of the available anti-AIDS drugs, sometimes within days to a few months of treatment (2). New anti-AIDS drugs should be designed to be eﬀective against viruses that carry known resistance mutations. Structural studies have been instrumental in developing the diarylpyrimidine (DAPY) class of NNRTIs, including TMC278/rilpivirine and TMC125/etravirine/Intelence, which eﬀectively inhibit wild-type and drug-resistant HIV-1 viruses (3,4). The DAPY NNRTIs have strategic ﬂexibility, allowing them to inhibit NNRTI-resistant viruses (5,6). Early attempts to crystallize the RT/ TMC278 complex yielded crystals that failed to diﬀract beyond 6 A˚ resolution. The conformational ﬂexibility of TMC278 may have introduced heterogeneity into the RT molecules in the crystal lattice (7), which might have been the primary cause of the persistently low resolution diﬀraction obtained in the many trials over a 5-year period. In an eﬀort to restrict the conformations of RT in the crystal lattice and improve the diﬀraction quality, a systematic protein crystal engineering approach was taken to produce an RT that could give high-resolution crystal structures of the RT/TMC278 complex. Three fundamental types of protein engineering approaches that are useful for crystallography include: (i) alterations that aﬀect the suitability of the protein for biochemical study, including mutagenesis and the addi- tion of tags for expression, solubility and puriﬁcation; (ii) changes that increase the conformational homogeneity of the protein sample and (iii) modiﬁcations of the protein that directly alter interactions at crystal contact *To whom correspondence should be addressed. Tel: +732 235 5323; Fax: +732 235 5788; Email: arnold@cabm.rutgers.edu Present address: Deena A. Oren, Structural Biology Resource Center, Rockefeller University, New York, NY, USA 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.