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. 2010 Apr 6;49(13):2821-33.
doi: 10.1021/bi902116z.

Homodimerization of the p51 subunit of HIV-1 reverse transcriptase

Affiliations

Homodimerization of the p51 subunit of HIV-1 reverse transcriptase

Xunhai Zheng et al. Biochemistry. .

Abstract

The dimerization of HIV reverse transcriptase (RT), required to obtain the active form of the enzyme, is influenced by mutations, non-nucleoside reverse transcriptase inhibitors (NNRTIs), nucleotide substrates, Mg ions, temperature, and specifically designed dimerization inhibitors. In this study, we have utilized nuclear magnetic resonance (NMR) spectroscopy of the [methyl-(13)C]methionine-labeled enzyme and small-angle X-ray scattering (SAXS) to investigate how several of these factors influence the dimerization behavior of the p51 subunit. The (1)H-(13)C HSQC spectrum of p51 obtained at micromolar concentrations indicates that a significant fraction of the p51 adopts a "p66-like" conformation. SAXS data obtained for p51 samples were used to determine the fractions of monomer and dimer in the sample and to evaluate the conformation of the fingers/thumb subdomain. All of the p51 monomer observed was found to adopt the compact, "p51C" conformation observed for the p51 subunit in the RT heterodimer. The NMR and SAXS data indicate that the p51 homodimer adopts a structure that is similar to the p66/p51 heterodimer, with one p51C subunit and a second p51 subunit in an extended, "p51E" conformation that resembles the p66 subunit of the heterodimer. The fractional dimer concentration and the fingers/thumb orientation are found to depend strongly on the experimental conditions and exhibit a qualitative dependence on nevirapine and ionic strength (KCl) that is similar to the behavior reported for the heterodimer and the p66 homodimer. The L289K mutation interferes with p51 homodimer formation as it does with formation of the heterodimer, despite its location far from the dimer interface. This effect is readily interpreted in terms of a conformational selection model, in which p51(L289K) has a much greater preference for the compact, p51C conformation. A reduced level of dimer formation then results from the reduced ratio of the p51E(L289K) to p51C(L289K) monomers.

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Figures

Figure 1
Figure 1
Structures of RT and predicted SAXS parameters. The structures in panels a–e were used to predict the SAX scattering profiles in panel g. The color coding of the SAXS curves in panel g is identical to the coloring of the structures in panels a–e. A detailed discussion of the component structures is contained in the text. Panel f shows 3DLK with p51 colored cyan and p66 colored white except for the RNase H domain that is colored black. Orange spheres indicate the position of the methyl group of the methionine residues. The side chain of L289 is shown with magenta spheres.
Figure 2
Figure 2
1H-13C HSQC spectra of a) 57 μM [methyl-13C] methionine66 RT, b) 50 μM [methyl-13C] methionine51 RT. and c) 30 μM [methyl-13C] p51 subunit. All samples were dissolved in the NMR buffer: 10 mM Tris-HCl-d11, pD 7.6, 200 mM KCl, 1.5 mM sodium azide, 4 mM MgCl2, and 100μM DSS as an internal chemical shift standard, in D2O. Spectra were obtained at 25°C. Schematic figures at the bottom indicate the subunit labeling pattern, with filled circles indicating [methyl-13C] methionine labeling. The spectra of the p66/p51 heterodimer preparations shown in panels a and b are reproduced from Figure 2a and 2b of Zheng et al. (38). Preparation of the selectively labeled RT heterodimer is described in that reference.
Figure 3
Figure 3
1H-13C HSQC spectra of [methyl-13C] methionine p51 mutants: a) M16L; b) M230L, M357L; c) L289K; d) L289K (black) and L289K + nevirapine (red). The arrows in (d) indicate nevirapine-induced shifts. Residue M16 produces two resonances, labeled M16A and M16B, with the M16A/M16B ratio considerably higher in p66 and p51E subunits than in the p51C subunit. The M357 residue in the p51E subunit also produces two resonances, one of which, M357B, overlaps M230. The intensity of the M357B resonance appears to be related to the proximity of M357 to the dimer interface. Temperature and buffer conditions were as in Figure 2.
Figure 4
Figure 4
SAXS scattering profiles analyzed with OLIGOMER. The experimental SAXS data is indicated with plus signs and the fit of a linear combination of predicted scattering profiles is drawn with a black line for a) 60 μM p51 and b) 40 μM p51. The χ2 of the fit for a) and b) is 4.8 and 3.1, respectively. All other SAXS fits are displayed in Supporting Information.
Figure 5
Figure 5
Fraction of p51 models present in solution. Based on OLIGOMER analysis of SAXS data the fraction of each p51 model present is shown in a bar graph, with the total set to 100%. Results of the analysis for each sample are summarized in a Table included as Supporting Information. Each column is a different sample indicated at the bottom of the figure. The bars are colored to be similar to the structures shown in figure 1. No p51E monomer, either open (green) or closed (red) was found in the analysis. The additives indicated at the bottom of the columns are: Mg – 4 mM MgCl2; NVP - 0.5 mM nevirapine, AC – 10 % acetonitrile. All samples were dissolved in 50 mM Tris-HCl, 200 mM KCl, pH 8.0 buffer.
Figure 6
Figure 6
Size exclusion chromatography profiles. a) p51 in the absence (black) or presence (red) of nevirapine; b) p51L289K in the absence (black) or presence (red) of nevirapine. For reference, the RT elution profile is indicated with a blue line. Maximum elution positions for the p51 monomer, dimer, and RT heterodimer are indicated by dotted lines.
Figure 7
Figure 7
Effect of KCl on p51C monomer/dimer ratio. Samples contained 50 μM p51C280S in 50 mM Tris-HCl, pH 8.0, 4 mM MgCl2, and the concentrations of KCl indicated. Fractional component concentrations were determined using the OLIGOMER analysis of the SAXS data, as in Figure 5. Only two species were observed under the conditions of the study: the cyan bars represent the compact monomer, p51C, and the yellow bars correspond to the dimer with a closed fingers/thumb conformation, p51Eclosed/p51C.
Figure 8
Figure 8
Effect of nevirapine on p51 NMR spectra. a) 1H-13C HSQC spectra of uncomplexed [methyl-13C] methionine51 RT (50 μM) in the absence (black) and presence (red) of 200 μM nevirapine; b) 1H-13C HSQC spectra of uncomplexed [methyl-13C] methionine p51 (30 μM) in the absence (black) and presence (red) of 100 μM nevirapine. The arrows indicate resonance shifts in response to the nevirapine. NMR parameters and buffer as in Figure 2. Schematic figures at the bottom indicate the subunit labeling pattern, with filled circles indicating [methyl-13C] methionine labeling; the triangle at the active site represents nevirapine. The spectrum of the p66/p51 heterodimer preparation shown in panel a is reproduced from Figure 7c of Zheng et al. (38). Preparation of the selectively labeled heterodimer is described in that reference.
Figure 9
Figure 9
Illustrative numerical calculations of molecular species concentrations based on a conformational selection dimerization model. a) The fractional p51C, p51E, and dimeric p51E/p51C concentrations are calculated as a function of KC for parameters KD = 2 μM, p51T = 50 μM. The KC values that would approximately correspond to wt and to L289K mutant p51 are indicated. b) Molecular species determined as a function of NNRTI concentration for the model shown in Scheme 1. The calculation corresponds to the parameters p51T = 100 μM, KC = 0.03, KD = KD2 =0.2 μM, KI = KI2 = 1.0 μM. Thus, for the calculation shown, the binding of the inhibitor does not contribute directly to the affinity of the monomers, but only indirectly, through the effect on the conformational distribution of p51 monomers.
Scheme 1
Scheme 1

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