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The transcription factor for immunoglobulin heavy-chain enhancer 3 (TFE3) is a member of the microphthalmia (MiT/TFE) transcription factor family. Dysregulation of TFE3 due to chromosomal abnormalities is associated with a subset of human renal cell carcinoma. Little structural information of this key transcription factor has been reported. In this study, we determined the crystal structure of the helix-loop-helix leucine zipper (HLH-Lz) domain of human TFE3 to a resolution of 2.6 Å. The HLH-Lz domain is critical for the dimerization and function of TFE3. Our structure showed that the conserved HLH region formed a four-helix bundle structure with a predominantly hydrophobic core, and the leucine zipper region contributed to the function of TFE3 by promoting dimer interaction and providing partner selectivity. Together, our results elucidated the dimerization mechanism of this important transcription factor, providing the structural basis for the development of inhibiting strategies for treating TFE3 dysregulated diseases. • TFE3 is a key transcription factor associated with a subset of human kidney cancer. • The crystal structure of the HLH-Lz domain of human TFE3 is determined. • The dimerization mechanism within HLH-Lz domain is analyzed. • We propose a possible strategy for inhibiting TFE3 activity. [ABSTRACT FROM AUTHOR]

Aminoacyl-tRNA synthetases (AARSs) catalyze the ligation of amino acids to their cognate tRNAs and therefore play an essential role in protein biosynthesis in all living cells. The KARS gene in human encodes both cytosolic and mitochondrial lysyl-tRNA synthetase (LysRS). A recent study identified a missense mutation in KARS gene (c.517T > C) that caused autosomal recessive nonsyndromic hearing loss. This mutation led to a tyrosine to histidine (YH) substitution in both cytosolic and mitochondrial LysRS proteins, and decreased their aminoacylation activity to different levels. Here, we report the crystal structure of LysRS YH mutant at a resolution of 2.5 Å. We found that the mutation did not interfere with the active center, nor did it cause any significant conformational changes in the protein. The loops involved in tetramer interface and tRNA anticodon binding site showed relatively bigger variations between the mutant and wild type proteins. Considering the differences between the cytosolic and mitochondrial tRNAlyss, we suggest that the mutation triggered subtle changes in the tRNA anticodon binding region, and the interferences were further amplified by the different D and T loops in mitochondrial tRNAlys, and led to a complete loss of the aminoacylation of mitochondrial tRNAlys. • The structure of a LysRS mutant associated with hearing loss disease was determined. • The YH mutant structure exhibited subtle differences with that of WT LysRS. • The tRNA differences may amplify the effect of YH mutation in charging mitochondrial tRNA. [ABSTRACT FROM AUTHOR]

General control nonderepressible 2 (GCN2) is a serine/threonine protein kinase, detecting a variety of stresses including amino acid starvation, reactive oxygen species, etc. in eukaryotic cells. Activation of GCN2 requires the interaction of the N-terminal RWD domain with the upstream GCN1 protein and the dimerization by the kinase domain. In this study, we determined two crystal structures of the RWD domain of human GCN2 in two different crystal packing modes. These two different crystal structures reveal a same dimer of the RWD domain, which has not been reported in previous studies. We further confirmed this novel dimer interaction in solution using gel filtration experiments, and in human embryonic kidney (HEK) 293 cells using bimolecular fluorescence complementation (BiFC) and co-immunoprecipitation (co-IP) assays. Together, this study discovers a potential protein-protein interface on the RWD domain of human GCN2, and suggests a possible regulation between the interaction of GCN1 and the formation of GCN2 dimer. • The structure of human GCN2 RWD domain is determined with two crystal packing modes. • Crystal structures indicate that human GCN2 RWD domain formed a novel dimer. • The novel dimer of human GCN2 RWD domain is experimentally confirmed in human cells. • The novel dimer interface implies protein interaction surface during cell signaling. [ABSTRACT FROM AUTHOR]