STRUCTURAL AND CATALYTIC ROLES OF CYSTEINE RESIDUES IN HUMAN LYSYL-tRNA SYNTHETASE

Abstract

Aminoacyl-tRNA synthetases (AARS) are essential and ubiquitous enzymes responsible for attaching amino acids to their cognate tRNAs during protein biosynthesis in a process known as aminoacylation. Comparing the amino acid sequences of bacteria (prokaryotes) to human (eukaryotic) AARSs, human AARS contain substantially more cysteine (Cys) residues than their bacteria counterpart. It is not known whether these Cys residues play roles in catalyzing aminoacylation or other functions.

Human lysyl-tRNA synthetase (hLysRS), one of the most conserved synthetases, has eight Cys residues, while E. coli LysRS has only one Cys. In addition to aminoacylation, LysRS is involved in the synthesis of the signaling molecule diadenosine tetraphosphate (Ap4A). We utilized chemical modification, site-directed mutagenesis and steady-state kinetic analysis to examine the roles of cysteine residues in catalysis and structure of hLysRS. Recombinant hLysRS was expressed in E. coli and purified by affinity chromatography. The hLysRS activity was substantially reduced upon the modification using two Cys modifying reagents, iodoacetamide and N-ethylmaleimide. Titration of hLysRS using 5, 5'-dithiobis (2-nitrobenzoic acid) (DTNB) indicated that four cysteine residues in hLysRS are reactive to DTNB while the other four are not accessible to the DTNB. Site-directed mutagenesis of Cys residues in human lysyl-tRNA synthetase indicated that mutations of Cys residues at position 338, 496 and 534 eliminate both Ap4A synthesis and lysylation activities. The remaining hLysRS Cys mutants showed reduced catalytic efficiencies in terms of their kcat and Km for aminoacylation. The cysteine-null mutant hLysRS, in which all cysteine residues were substituted with alanine, lost lysylation activity. Analysis of the secondary structure of hLysRS by circular dichroism indicated the mutation did not have significant effect on the secondary structure. However, significant changes in intrinsic fluorescence of mutants suggest the effect on the tertiary structure.

The interactions and spatial orientation of the proteins in the MSC were investigated using fluorescence resonance energy transfer. Aspartyl-tRNA synthetase and p38 were fluorescently labeled using genetically encoded tetracysteine and green fluorescent protein. Association among human tRNA synthetases, p43 and p38 of the multisynthetase complex was demonstrated by fluorescence titration and resonance energy transfer.