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.
journal_name
Nucleic Acids Resjournal_title
Nucleic acids researchauthors
Bauman JD,Das K,Ho WC,Baweja M,Himmel DM,Clark AD Jr,Oren DA,Boyer PL,Hughes SH,Shatkin AJ,Arnold Edoi
10.1093/nar/gkn464subject
Has Abstractpub_date
2008-09-01 00:00:00pages
5083-92issue
15eissn
0305-1048issn
1362-4962pii
gkn464journal_volume
36pub_type
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