The Structures And Catalytic Mechanism Of N-terminal Acetyltransferase NatB Complex

N-terminal acetylation is one of the most common protein modifications in eukaryotes.It is not only involved in regulating protein stability,subcellular localization,and protein-protein interactions,but also plays an important role in cell growth,differentiation,metabolism and tumorigenesis.In eukaryotic cells,six N-terminal acetyltransferases,NatA-F,have been characterized,which belong to the GNAT superfamily.Several NAT structures,including NatA,NatD and NatE,have been determined.NatB is important in maintaining the structure and function of cytoskeleton,cell proliferation and regulating flowering time and fruit development in plant.NatB complex,composed of a catalytic subunit Naa20 and an auxiliary subunit Naa25,acetylates specifically on those polypeptides with N-terminal Met-Glu-,Met-Asp-,Met-Asn-or Met-Gln-.However,the molecular mechanism of how NatB recognizes and catalyzes these specific substrates is unclear.In this thesis we solved high-resolution crystal structures of NatB+CoA+Met and NatB+CoA-MDSEVA complex.By structural analyses,we discovered several important features of NatB that are different from other N-terminal acetyltransferases.Our reciprocal co-immunoprecipitation experiments demonstrated that the interactions between the catalytic and auxiliary subunit of NatB are important to maintain the integrity of NatB holoenzyme.In NatB+CoA-MDSEVA complex,the first two amino acids of the substrate peptides,methionine and aspartic acid,form many hydrogen bonds in NatB substrate-binding pocket.Moreover,the side chain of aspartic acid forms a salt bridge with imidazole base of the conserved His74,which is different from NatD and NatE.Mutation of His74 caused a disruption in NatB kinetics,supporting its important role in the recognition and catalysis of specific substrates.Even though this site is also a conserved His in NatA,it is not involved in substrate interaction.The crystal structure of NatB complex not only deepens our understanding to the substrate-specific N-terminal acetylation but also provides a structural foundation to design small molecule inhibitors with therapeutic potential.