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Title
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Crystal engineering of HIV-1 reverse transcriptase for structure-based drug design.
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Authors
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J.D.Bauman,
K.Das,
W.C.Ho,
M.Baweja,
D.M.Himmel,
A.D.Clark,
D.A.Oren,
P.L.Boyer,
S.H.Hughes,
A.J.Shatkin,
E.Arnold.
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Ref.
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Nucleic Acids Res, 2008,
36,
5083-5092.
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PubMed id
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Abstract
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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.
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