Structural insight into protein recognition by RNA aptamers
Ribonuclease and SHAPE mapping were used to determine the correct predicted secondary structure of a high affinity aptamer (Lys1) selected against hen egg white lysozyme (KD ~ 30 nM). A deletion variant, Lysl .2minE (KD~ 20 nM), was engineered to delete a long, apparently unstructured region. The lysozyme-Lysl .2minE complex was solved by X-ray crystallography at a 2.0 A resolution, yielding a seventh RNA aptamerprotein structure. Solution hydroxyl-radical footprinting confirms the small binding interface observed in the crystal. Although the Lysl .2minE aptamer interacts with a positively charged face of lysozyme, the electrostatic contribution to the binding free energy is minimal. The Lysl .2minE aptamer was found to inhibit the function of lysozyme in a standard cell-wall hydrolysis assay -- a surprising result since the aptamer binding site is quite far from the catalytic site, and no structural differences between free lysozyme and that in complex with the aptamer could be detected. We determined that Lysl .2minE is able to modulate lysozyme function by inhibiting the binding of large natural substrates.;We hypothesize that an RNA aptamer selected against the myristoylated matrix domain (myrMA) of the HIV-1 Gag polyprotein may preclude Gag from trafficking to the membrane, thus preventing viral assembly. Twelve rounds of selection resulted in six high affinity RNA aptamers that bind myrMA (KD = 3 - 108 nM). Five deletion variants of the highest affinity parent aptamer (MA2) were engineered and determined to retain high affinity towards myrMA. The smallest aptamer, MA2D (43 nts), binds near the highly basic region of myrMA, although the electrostatic component contributes a modest 23% to the total free binding energy. The protein binds primarily to loop L2 of MA2D, which shares a high sequence identity with the MA-binding domain of other RNA aptamer sequences selected by three independent groups (Lochrie 1997, Purohit 2001, Ramalingam 2011). In addition, we show that MA2D disrupts myrMA-membrane interactions.;The goal of this study is to probe the mechanisms by which RNA aptamers bind their targets and regulate catalytic activity and/or cellular function. RNA aptamer structure elucidation will provide valuable insight on aptamer specificity and may reveal novel RNA protein-binding motifs.
Source: Dissertation Abstracts International, Volume: 76-05(E), Section: B.;Advisors: Mark E. Girvin.