Computational design and molecular mechanism in oligomerization of C‐terminal binding protein 2

Abstract

IntroductionC‐terminal binding protein 2 (CtBP2) is moonlighting proteins, which has multiple functions and plays a key role in transcriptional control and in the maintenance of cellular metabolism (1). CtBP2 consist of D‐isomer specific 2‐hydroxyacid dehydrogenase (D2‐HDH) domains and C‐terminal intrinsically unstructured region. Recent studies have shown that the recruitment of a corepressor complex by CtBP2 depends on NADH/NAD ratio. Molecular mechanism of oligomerization of CtBP2 may provide a basis for the formation of a transcriptional corepressor complex.MethodsWe attempted to construct artificial designed proteins on the basis of in silico screening using the INTMSAlign program (2). Overexpression in E. coli cells, and purification of artificial designed CtBP2 (ArCtBP2) were performed. The oligomeric state and the binding affinity to NADH of ArCtBP2 were compared to human CtBP2 (hCtBP2) by size exclusion chromatography and intrinsic Trp fluorescence. To evaluate structural information, X‐ray crystallographic analysis of ArCtBP2 was performed.ResultsSize exclusion chromatography of ArCtBP2 shows 4 peaks for each corresponding oligomeric state, whereas that of hCtBP2 was observed as a single peak. The dissociation constants (Kd) value of NADH to ArCtBP2 was determined as 3.6 ± 0.9 μM by fluorescence spectroscopy. In addition, crystal structure of apo and NAD–bound form of ArCtBP2 at 2.0 and 1.7 Å resolution, respectively. Crystal structure of ArCtBP2 showed opened‐like structure in D2‐HDH domain compared to hCtBP2. These results suggested that the open–closed form of D2‐HDH domain is involved in the binding affinity for NADH and oligomeric state of CtBP2.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.