Recombinant proteins
The vector pCPH6P-HIV1-IN used for bacterial expression of HIV-1 IN with a cleavable hexahistidine (His 6 ) tag was previously described45. To obtain pCPH6P-SIVtal-IN, the DNA region encoding HIV-1 IN was replaced with a codon-optimized fragment encoding SIVtal IN (the corresponding amino acid sequence was derived from NCBI GenBank entry CAJ57812)46. HIV-1 and SIVtal INs were produced in endonuclease-A-deficient Escherichia coli PC2 cells (BL21(DE3), endA::TetR, T1R, pLysS)47 transformed with pCPH6P-HIV1-IN and pCPH6P-SIVtal-IN, respectively. The cells were grown in LB medium containing 120 μg ml−1 ampicillin to an absorbance at 600 nm (A 600 ) of around 0.9 and supplemented with 50 μM ZnCl 2 , and protein expression was induced by addition of 0.01% (w/v) isopropyl-β-d-1-thiogalactopyranoside for 4 h at 30 °C. Bacterial cells were lysed by sonication in core buffer containing 1 M NaCl and 20 mM Tris-HCl, pH 7.5 supplemented with 1 mM phenylmethylsulfonyl fluoride and cOmplete EDTA-free protease inhibitor cocktail (Roche). To prevent aggregation of HIV-1 IN, all buffers used during purification of this protein were supplemented with 7.5 mM 3-((3-cholamidopropyl) dimethylammonio)-1-propanesulfonate (CHAPS); the detergent was not required and was avoided during purification of SIVtal IN. The supernatant was precleared by centrifugation and incubated with NiNTA agarose (Qiagen) for 1 h °C at 4 °C in the presence of 15 mM imidazole to capture His 6 -tagged proteins. The resin was washed extensively in core buffer supplemented with 15 mM imidazole, and the protein was eluted with 200 mM imidazole in core buffer. To remove the hexahistidine tag, the eluate was incubated with human rhinovirus 14 3C protease (1:50 (w/w) ratio) overnight at 4 °C in the presence of 5 mM dithiothreitol (DTT). Cleaved proteins were diluted with ice-cold 20 mM Tris-HCl, pH 7.5 to adjust NaCl concentration to around 150 mM and immediately injected into a precooled 5 ml HiTrap Heparin HP column (GE Healthcare). A linear 0.15–1 M NaCl gradient in 7.5 mM CHAPS, 20 mM Tris-HCl, pH 7.5 (for HIV-1 IN) or 30 mM HEPES-NaOH, pH 7.5 (SIVtal IN) was used to elute the proteins; peak fractions were combined, supplemented with 1 mM DTT and the NaCl concentration was adjusted to 1 M. HIV-1 and SIVtal INs were concentrated using a 10-kDa cut-off Vivaspin device (Generon) to 8–10 and 1 mg ml−1, respectively, snap-frozen in liquid nitrogen and stored at −80 °C.
BLI analysis
All of the experiments were conducted using the Octet R8 instrument (Sartorius) at 25 °C in base buffer containing 150 mM NaCl, 20 mM Tris-HCl, pH 7.5, 1 mM DTT, 0.05% (v/v) Tween-20 and 10 U ml−1 SUPERaseIn RNase inhibitor (LifeTechnologies, AM2696); sensorgrams were recorded using Octet BLI Discovery Software (Sartorius). Biotinylated RNA oligonucleotides (10 or 20 nM; Integrated DNA Technologies) were immobilized on Octet Streptavidin biosensors to reach 0.2 nm wavelength shift threshold in base buffer. The sensors were moved to wells containing 0.5, 0.25 or 0.125 μM HIV-1 or SIVtal IN in base buffer, and IN binding was recorded for 300 s. Dissociation was recorded for 300 s in the same buffer without IN. In control experiments, sensors without immobilized RNA were exposed to varying IN concentrations to test for non-specific binding.
HDX–MS
Individual proteins and protein–RNA complexes (3 µM, final) were incubated with 40 µl D 2 O buffer for 3, 30 and 180 s at room temperature in triplicate. The labelling reaction was quenched by adding chilled 2.4% (v/v) formic acid in 2 M guanidinium hydrochloride and immediately frozen in liquid nitrogen. The samples were stored at −80 °C before analysis. The quenched protein samples were rapidly thawed and processed for proteolytic cleavage by pepsin followed by reversed-phase HPLC separation of the resulting peptides. In brief, the protein was passed through an Enzymate BEH immobilized pepsin column (2.1 × 30 mm, 5 µm, Waters) at 200 µl min−1 for 2 min and the peptic peptides were trapped and desalted on a 2.1 × 5 mm C18 trap column (Acquity BEH C18 Van-guard pre-column, 1.7 µm, Waters). Trapped peptides were subsequently eluted and separated over 11 min using a 5–43% gradient of acetonitrile in 0.1% (v/v) formic acid at 40 µl min−1. Peptides were separated on a reverse-phase column (Acquity UPLC BEH C18 column 1.7 µm, 100 mm × 1 mm; Waters). Peptides were detected on the Cyclic mass spectrometer (Waters) acquiring over an m/z of 300 to 2,000, with the standard electrospray ionization source and lock mass calibration using [Glu1]-fibrino peptide B (50 fmol µl−1). The mass spectrometer was operated at a source temperature of 80 °C with a spray voltage of 3.0 kV. Spectra were collected in positive-ion mode.
Peptide identification was performed by MSe software48 using an identical gradient of increasing acetonitrile in 0.1% (v/v) formic acid over 12 min. The resulting MSe data were analysed using Protein Lynx Global Server software (Waters) with an MS tolerance of 5 ppm. Mass analysis of the peptide centroids was performed using DynamX software (Waters). Only peptides with a score >6.4 were considered. The first round of analysis and identification was performed automatically by DynamX software; however, all peptides (deuterated and non-deuterated) were manually verified at every timepoint for the correct charge state, presence of overlapping peptides and correct retention time. Deuterium incorporation was not corrected for back-exchange and represents relative, rather than absolute changes in deuterium levels. Changes in hydrogen–deuterium amide exchange in any peptide may be due to a single amide or a number of amides within that peptide. All timepoints in this study were prepared at the same time and individual timepoints were acquired on the mass spectrometer on the same day. The MS data have been deposited to the ProteomeXchange Consortium through the PRIDE49 partner repository under dataset identifier PXD070910.
SEC–MALLS analysis
For size-exclusion chromatography coupled to multiangle laser light scattering (SEC–MALLS) analysis, SIVtal IN (100 μl) in 0.5 M NaCl, 3 mM NaN 3 , 0.5 mM Tris-(2-carboxyethyl)phosphine (TCEP) and 25 mM Tris-HCl (pH 7.5) was injected onto the Superdex-200 Increase 10/300 column (Cytiva) equilibrated in the same buffer. Chromatography was performed at 25 °C, at a flow rate of 1 ml min−1 using the JASCO-4000 semimicro HPLC system. Scattered light intensities and protein concentrations in the eluate were measured using the DAWN-HELEOS II laser photometer and an OPTILAB-TrEX differential refractometer (Wyatt Technology), respectively. The data were analysed using ASTRA software v.7.3.2 (Wyatt Technology) using recordings from both detectors, assuming a specific refractive index increment (dn/dc) of 0.186 ml g−1. The weight-averaged molar mass of SIVtal IN in chromatography peaks was determined from the combined data of three independent experiments with IN diluted to 1, 0.5 and 0.25 mg ml−1.
Cryo-EM data collection on the SIVtal IN–RNATAR complex
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