Structural model for deoxycytidine deamination mechanisms of the HIV-1 inactivation enzyme APOBEC3G - PubMed (original) (raw)

Apo3G native and N-mutant F/W interactions with Alu RNAs. A and B, the apparent dissociation constant (Kd) of Apo3G native and mutants from Alu RNAs was determined by monitoring changes in rotational anisotropy of free fluorescein-labeled RNA (50 n

m

) following the addition of enzyme as a function of concentration. A, Apo3G native (●) binds to a 91-nt Alu RNA sequence derived from 7SL RNA with an apparent Kd of 446 n

m

, which is a 2.5-fold higher affinity interaction than for the N-mutant F/W (▴, Kd of 1200 n

m

). B, the Apo3G native (●) and N-mutant F/W (▴) bind a 108-nt small cytoplasmic Alu (scAlu) RNA with approximately the same affinity, having an apparent Kd of 220 and 357 n

m

, respectively. C and D, AFM was used to determine the oligomeric state of Apo3G native (C) and N-mutant F/W (D) in the presence of a 91-nt Alu RNA and 5 m

m

MgCl2. The apparent dissociation constants for native and mutant Apo3G in the AFM buffer were 374 and 742 n

m

, respectively. Volume distributions of Apo3G are plotted against a percentage of total proteins. The predicted peaks for a monomer (42 nm3), dimer (97 nm3), and tetramer (208 nm3) are denoted with a bracket. Total volume distribution is to 1000 nm3 (see

supplemental Fig. S6

). Partial volume distribution is shown here to give a focused view of the monomer, dimer, and tetramer populations. For Apo3G native and N-mutant F/W, the population that distributes from 0 to 240 nm3 is ∼50 and ∼80%, respectively. Total proteins counted were: C, 361; and D, 211. Representative AFM images of Apo3G native and N-mutant F/W are shown for each condition. Images are 300 × 300 nm with a height scale of 5.5 nm.