Structure-based Interaction Of HPCAF Protein Domains

The protein post-translational modifications are important to regulate protein functions,activities and their locations in eukaryotic cells.The protein acetylation,one of the most frequent post-translational modifications,regulates transcriptions,protein degradation,cellular metabolism and cell cycle.It plays a key role in many diseases,e.g.Cancer,Parkinson diease and Alzheimer’s.PCAF(p300/CBP-associated factor)is one of histone acetyltransferases.It is also a transcription cofactor,which mainly locate in the nucleus.Human PCAF(hPCAF)has a catalytic HAT(Histone AcetylTransferase)domain,which can acetylate histones and also non-histone proteins,a Bromo domain in its C-termus and the N-terminal undefined region,which has a ubiquitin ligase activity.The structures of HAT and C-Terminal Bromo domain have already been solved.But structures about the N-terminal region are still unknown.A region 415-492 of hPCAF has several auto-acetylation sites and a nuclear localization signal,whereas auto-acetylation can regulate nuclear localization of hPCAF and improves PCAF HAT activity.However an exact molecular mechanism is still unknown.Herein,we have explored the relationship between the N-terminal region 415-492 and the HAT domain 493-658.Firstly,we analyzed the acetylation activity of hPCAF 415-658(415-492 and 493-658 region)and the HAT domain 493-658 by enzymatic kinetic assay,where we found that the HAT activity could be inhibited by the region 415-492.We then collected and analyzed the HSQC spectra of hPCAF415-658 and also the HAT domain,which suggested that the region 415-492 could maintain a rigid conformation in solution when together with the HAT domain,possibly resulted from an interaction between the region 415-492 and the HAT domain.In order to examine the effect of autoacetylation on their interaction,we muted PCAF catalytic activity by mutating E570 to histidine in the HAT domain and inserting a P3C site inbetween.We were able to purify the region 415-492 in the acetylated and non-acetylated states.However,the interaction between HAT domain and acetylated or non-acetylated 415-492 was not observed in GST pull-down assay.We could not detect any acetylation by the HAT domain in vitro,suggesting the autoacetylation can not happen in trans.All the results indicate that the HAT domain may have a weak interaction with the 415-492 region intramolecularly.Moreover,we also examined the effect of the auto-acetyaltion of hPCAF on nuclear receptor importin α in the GST pull-down assay and did not find any obvious difference.In order to further illustrate the interaction between HAT domain and the 415-492 region,we crystallize the acetylated and non-acetylated hPCAF 415-658 respectively.Although the crystals diffracted to a high resolution,the region 415-492 could not be observed in electron density map.