Structural And Functional Studies Of The Human Signal Recognition Particle SRP72

The co-translational targeting or insertion of secretory and membrane proteins into the endoplasmic reticulum(ER)is a key biological process mediated by the signal recognition particle(SRP).Mammalian SRP can be divided into an Alu domain comprising the terminal RNA regions and SRP9/SRP14 protein subunits,and an S domain comprising the central RNA region and the other four protein subunits.Consistent with the role in nuclear export of eukaryotic SRP,SRP68 and SRP72 are detected in cytosol as well as the nucleolus and are highly conserved in organisms ranging from yeast to human.The two proteins form a stable heterodimer and are essential for the proper functioning of eucaryotic SRP.Reconstituted SRP lacking the SRP68/72 heterodimer failed to recognize signal peptide,to direct tanslocation,or to arrest elongation.Many cryo-electron microscopy studies at low resolution have been reported for the eukaryotic SRP-RNC(ribosome-nascent chain complex)targeting complex that have helped to illuminate its biological function.However,structural information on the two largest SRP proteins,SRP68 and SRP72,is limited,especially regarding their interaction.In this research,through extensive screening of apo-SRP72 and the SRP68/72 complex truncated variants,the crystals of human apo-SRP721-163 and SRP68509-614-SRP721-163 complex were obtained.Both structures were determined by molecular replacement and single-wavelength anomalous diffraction,respectively.SRP72 is composed of nine long antiparallel a-helix(al-a9)in the complex,forming a concave surface to which SRP68 peptide binds tightly.The SRP68-binding domain of SRP72 contains four atypical tetratricopeptide repeats(TPR)and a flexible C-terminal cap.Apo-SRP72 exists predominantly as a tail-to-tail dimer in solution,with significant populations of monomer and tetramer are also present in solution.Comparing with those in the apo-SRP72,the larger size of the interface area,interaction and stability were found in the SRP68/72 complex.Furthermore,a detailed structure comparison between the SRP68/72 complex and apo-SRP72 revealed that one of the molecule in the apo-SRP72 homodimer would sterically clash with the SRP68 in the complex and hinder the binding of SRP68.To bind SRP68,the SRP72 homodimer disassociate,and the indispensable C-terminal cap undergoes a pronounced conformational change to assist formation of the SRP68/72 heterodimer.A 23-residue polypeptide of SRP68(588-610)is sufficient for tight binding to SRP72(1-163)through its unusually hydrophobic and extended surface.Thus we successfully narrowed down the binding regions involved in the SRP68/72 interaction.Structural,biophysical and mutagenesis analyses revealed that cancer-associated mutations disrupt the SRP68/72 interaction and their co-localization within ER in mammalian cells.The results highlight the essential role of the SRP68/72 interaction in SRP-mediated protein translocation,and provide a structural basis for disease diagnosis,pathophysiology and drug design.Together,we successfully determined the first structure of human apo-SRP72 and SRP68/72 complex at high resolution,providing the direct evidence and a better mechanistic insight.Structural analysis,mutagenesis and a serious of in vitro or in vivo biochemical experiments yielded insights into deeply understanding SRP68/72 and also provided the structural basis for eukaryote-specific SRP72 and SRP68/72 complex required for protein translocation.These data suggest a role for mutation of SRP72 and SRP68 in human diseases so that accelerate the study on the drug design against diseases,such as cancers.