Structural Determinants in Adenovirus 12 E1A Involved in the Interaction with C-Terminal Binding Protein 1


AbstractThe interaction between the C-terminal binding protein 1 (CtBP-1) and purified Ad12 E1A protein has been examined through the use of a combination of biophysical techniques. A fragment equivalent to the 77 C-terminal amino acids of Ad12 E1A (Ad12 77-a.a. E1A) was generated by limited proteolysis of Ad12 266-a.a. E1A at Phe187 and/or Tyr189 using chymotrypsin. The impact of deletion of the 189 N-terminal amino acids from E1A on the equilibrium dissociation constant Kd for binding to CtBP was assessed using ELISA in vitro binding assays and intrinsic fluorescence spectroscopy. Values of Kd of 4.0 and 38 nM were determined for full-length and truncated forms of E1A, respectively. Circular dichroism spectroscopic studies revealed that the conformation adopted by these polypeptides is dependent on the surrounding environment, which is predominately randomly folded when free in solution, but adopting a more ordered α-helical secondary structure in the presence of trifluoroethanol. Using nuclear magnetic resonance (NMR) spectroscopy to examine the interaction between Ad E1A and CtBP it was observed that the chemical shift positions of individual backbone amide nitrogen atoms were well resolved in 15N-1H-HSQC NMR spectra performed on samples of isotopically 15N-labeled Ad12 77-a.a. E1A. In the presence of CtBP, signals of backbone amide nitrogen atoms displayed increased linewidth consistent with an increase in molecular mass upon binding CtBP. In addition, some signals that have been attributed to Val254/256 and Leu259, and reside within the binding site for CtBP on E1A, are shifted in the 15N- and/or 1H-dimensions, defining specific contacts between E1A and CtBP. These data suggest that structural determinants in the C-terminal PXDLS binding motif in the rest of exon 2 and in exon 1 all contribute to optimizing the conformation of the binding site on Ad12 E1A for CtBP. However, no interaction was observed between CtBP and truncated Ad12 E1A, which no longer contained the C-terminal binding motif

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Last time updated on 6/5/2019

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