Molecular Insight Into The Arginine-binding Specificity Of CASTOR1

The mechanistic Target Of Rapamycin Complex 1(mTORC1)can sense and integrate multiple signals fromthe external environment,and regulate a large number of life processes which are directly related to growth and development,including gene transcription and translation,synthesis and degradation of proteins and lipids,and autophagy.Among the wide range of signal inputs that impinge on the mTORC1 activity,amino acids,such as leucine and arginine,are particularly potent activators upstream of mTORC1 in the anabolism and autophagy pathways.With the availability of amino acids,mTORC1 is recruited to the lysosomal membrane through mediation of small GTPases Rag A,-B,-C and-D,where its kinase activity is stimulated by the lysosome-anchored small GTPase Rheb.The activity of the Rag GTPases is mediated by the upstem GATOR1 and GATOR2 complexes.Recently,several amino acid sensing proteins are reported to function as upstream regulators of the Rag GTPases and the mTORC1 pathway.CASTOR1 is a cytoplasm-localized arginine sensorwhich can interact with GATOR2 in the absence of arginine.The binding of arginine to CASTOR1 disrupts the CASTOR1-GATOR2 interaction and hence GATOR2 is released to form the CASTOR1-GATOR2 complex,leading to the inhibition of GATOR1 and finally activate the mTORC1 signaling.To understand the molecular mechanism of the arginine binding specificity of CASTOR1,we determined the crystal structure of CASTOR1 in complex with arginineat 2.5 ? resolution.We found that CASTOR1 comprises of four tandem ACT domains: ACT1 and ACT3 adopt the typical ?????? topology and function in dimerization via the conserved residues from helices ?1 of ACT1 and ?5 of ACT3;whereas ACT2 and ACT4,both comprising of two non-sequential regions,assume the unusual ?????? topology and contribute an arginine-binding pocket at the interface.The bound arginine makes a number of hydrogen-bonding interactions and extensive hydrophobic contacts with the surrounding residues of the binding pocket.To validate the functional roles of these residues,we performed mutagenesis and ITC analyses,and the resultsdemonstrate that Ser111 and Asp304 of CASTOR1 play a critical role in the argininebinding.Our ITC assays also show that CASTOR1 displays a high bindingaffinity for arginine,but has no measurable binding for other amino acids including leucine,lysine,and histidine,and several similar molecules including ornithine,citrullineand creatine.Using the Dali server to carry out the structural similarity search in the Protein Data Bank,we found that the overall architecture of CASTOR1 resembles the allosteric regulatory domains found in many aspartate kinases.In conclusion,our structural and functional data together reveal the molecular basis for the arginine binding specificity of CASTOR1 in the arginine-dependent activation of the mTORC1 signaling.