The Structural And Functional Study Of BDF5 Bromodomain From Trypanosoma Brucei

Trypanosoma brucei is a kind of unicellular protozoan widely spreaded by Glossina in Sub-Saharan Africa.Trypanosoma brucei is known for causing sleeping sickness in human and nagana in animals,as a result it has led to huge healthcare threat and economic loss.There is no effective drug to cure the sleeping sickness.Typanosomatid can escape from clearance of the host immune system by switching the variant surface glycoprotein(VSG)coat on cell surface periodically.Recently,it’s found that the transcription of VSG is tightly controlled by histone modification.Althouth there are some common features shared by Trypanosoma brucei and higher eukarytes.the epigenetic modification of histones in Trypanosoma brucei is different from higher eukarytes in some respects.Bromomain is an important ε-N-acetylation histone binding domain.There are five proteins containing bromodomain in Trypanosoma brucei.BDF5 contains two tandem bromomains(BDF5-bromol and BDF5-bromo2)and a long C-terminal loop.Sequence alignment shows that BDF5-bromol of Trypanosoma brucei displays low similarity to bromodomains of higher eukarytes.We determined the solution structure of BDF5-bromol by NMR spectroscopy.BDF5-bromol consists of four a helices(αZ,αA,αB and αC)and three linking loops(ZA loop,AB loop and BC loop).The four αhelices form a barrel,the long ZA loop and the short BC loop form a hydrophobic cage for binding acetylated lysine in histone along with the surrounding αB and αC.Besides.BDF5-bromol from Trypanosoma brucei adopts similar three-dimensinal structure of left-handed up-and-down four-helix bundle,comparing to those of bromodomains from higher eukarytes.Meantime,we purified GST-BDF5-bromol recombinant protein,and GST pull-down experiment confirmed that BDF5-bromol could bind histone in Tiypanosoma brucei.Furthermore,AlphaLIS A showed that BDF5-bromol could recognize H4K12ac and H4K5K8K12K16ac from human,which suggests BDF5-bromol might play its role by recognizing the similar acetylated site on histone H4 of Trypanosome brucei.In addition,we screened synthesized acetylated histone peptides(13 peptides)from Trypanosoma brucei by far-western experiment.It’s indicated that BDF5-bromol could recognize H4K4ac and H4K10ac peptides.Furthermore,NMR chemical shifts perturbation experiments identified the surface of BDF5-bromol interacting with H4K4ac/H4K10ac peptides.