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3643099485
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note
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Disulfide cross-linking was chosen for the following reasons: (i) thiol-disulfide interchanges proceed readily under physiological conditions; (ii) disulfide bond formation is reversible and selective, and as such unlikely to yield an irrelevant cross-linked species through kinetic trapping, especially in the presence of a thiol-reducing agent; and (iii) efficient methods exist for site-specific attachment of tethered thiols to DNA (14) and for mutating other residues in a protein to cysteine.
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3643050419
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note
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HIV-1 RT was expressed in Escherichia coli strain BL21(DE3) as follows. An expression vector containing both p66 and p51 coding sequences was constructed, with independent promoters and attenuating sequences and opposite directions of transcription. The COOH-terminus of p51 also contained a six-histidine tag. Cysteine mutations were introduced into helix H of p66 to create tethering sites for a modified template:primer. In addition to a helix H mutation (Q258C, G262C, or W266C) [in which the first residue at the indicated position is mutated to the second residue (16)], each of the variant proteins used in this study had (i) the C280S mutation, introduced in prior crystallographic studies on RT (6), (ii) the E478Q mutation, introduced to eliminate RNase H catalytic activity (42) for purposes of future studies on RNA:DNA hybrids, and (iii) a P1K mutation to enhance expression levels. The three variant RT proteins retain one of the two cysteine residues present in wild-type p66 (C38) and both wild-type cysteine residues in p51 (C38 and C280). Cell pellets were lysed with a French press, and the enzyme was purified by Ni-nitrilotriacetic acid affinity chromatography, followed by cation exchange chromatography (sulfopropyl-Sepharose fast flow) and a final step of size-exclusion chromatography (Superdex 200).
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20
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3643054644
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note
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Single-letter abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.
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We introduced the thiol tether into the template: primer through postsynthetic modification using the convertible nucleoside approach (74). Cystamine deprotection gave the C2 tether protected as the mixed 2-aminoethyl disulfide, which was used for biochemical studies; 3-aminopropane disulfide deprotection gave the C3 tether, which was used for crystallization. Template:primers containing the C2 and C3 tethers show virtually identical cross-linking specificity but exhibit different kinetics, with C3 being faster than C2 (43).
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3643139110
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Each RT mutant (4 μM) was mixed with the thioltethered template:primer (10 μM) and a dNTP and ddNTP cocktail (100 μM each, refer to Fig. 2B) in the presence of 2 mM β-mercaptoethanol, which was added to increase the specificity of cross-linking. After a 2-hour incubation at 25°C, the cross-linking reaction was quenched by the addition of a thiolcapping reagent, methyl methanethiolsulfonate (20 mM), and the products were analyzed by SDS - polyacrylamide gel electrophoresis (SDS-PAGE) under nonreducing conditions.
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2, 0.1 mM EDTA]. Nonreducing PAGE analysis of the crystals clearly indicated that RT(Q258C) and the template strand were quantitatively cross-linked (43), thus confirming the presence of the disulfide linker in the structure. Crystals were stabilized in a solution containing all the reservoir components, but with the concentration of PEG 6000 elevated to 16%, with 1 mM dTTP added. The crystals were briefly soaked (3 to 5 min) in a cryoprotecting solution containing all components of the stabilizing solution plus 25% glycerol, then mounted in a nylon loop and flash-frozen in liquid nitrogen. Diffraction data were collected at the X25 beamline at National Synchrotron Light Source (NSLS) and the F1 beamline at Cornell High-Energy Synchrotron Source (CHESS) Laboratory (Table 1). Data were integrated, scanned, and reduced with the HKL software (44) and the CCP4 suite of crystallographic programs (45). The high-resolution structure of HIV-1 RT with a bound NNRTI (8) was used for molecular replacement calculations with programs from the CCP4 package and AMoRe (46). The search model was modified by removing the fingers, palm, and thumb of p66. With data from 12 to 3.5 Å, two solutions were evident, one significantly stronger than the other. The RNase H domains of the two molecules in the asymmetric unit form a continuous β sheet, as in previous RT crystal forms. Early stages of rebuilding [with the program O (47)] and adding back missing domains and nucleic acids exploited the two-fold noncrystallographic symmetry by iterative real-space averaging [program RAVE (48)]. Subsequent grouped-B refinement with X-PLOR (49) showed that the second molecule is less well ordered than the first, and in later stages we carried out all rebuilding in the density for the first molecule, using the coordinates thus obtained to generate both molecules for the next refinement round. Density for the A-form segment of the DNA duplex was evident in early stages, and the position of the DNA was confirmed by obtaining diffraction data from a complex with an iodinated primer (iodines at positions n-10 and n-17). Additional base pairs and the dNTP were added as the refinement proceeded. Phases were improved by many rounds of rebuilding and refinement, with noncrystallographic symmetry (NCS) restraints in all refinement protocols. NCS was defined by structural domains. Once all the atoms in the final model had been built, the thermal parameters were refined individually, with tight restraints. This procedure was necessary because of the significant differences in ordering of the two molecules in the asymmetric unit. The final structure (Table 1) has good geometry (root mean square standard deviations of bond lengths and bond angles are 0.014 Å and 2.7°, respectively).
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2-chain in the extended conformation, with a disulfide torsion angle of 90°. A tether with a distance of ∼5 Å between the terminal carbons can be modeled by allowing one gauche torsional interaction in the tether, which requires little investment of energy.
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note
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We especially acknowledge B. Harris, who helped initiate this project; we thank the staffs of beamlines X25 at NSLS and F1 at CHESS; M. Lawrence, M. Jacobs, K. Leong, D. Erlanson, and R. Butcher and other members of the Harrison-Wiley and Verdine research groups; S. Doublie, T. Ellenberger, R. D'Aquila, and R. Colgrove, for discussion and advice; and E. Arnold, for coordinates of a refined complex before publication. The work was supported by NIH grants GM-39589 (to S.C.H.) and GM-44853 (to G.L.V.), NIH postdoctoral award GM-18621 (to H.H.), and by the Landon T. Clay Research Endowment (Harvard University). S.C.H. is an Investigator in the Howard Hughes Medical Institute. The coordinates of the complex have been deposited in the Brookhaven Protein Databank (1RTD).
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