Unusual Role of Tryptophan residues in Structural and Functional properties of Mimi Virus TyrRS (TyrRS_apm)

Abstract

In crystal structure, the homodimeric (α2) mimi-virus Tyrosyl-tRNA Synthetases (TyrRSapm) showed significant variation in dimer orientation as compared to other solved structures of TyrRSs. Previous report showed that Bacillus stearothermophilus N-terminal TyrRS exists as dimer under native condition and unfolds through a monomeric intermediate. In our previous studies, we have reported that the C-terminal anticodon binding domain of TyrRSapm might have an indirect role in noncanonical dimer formation. To study the implication of the noncanonical dimer interface on the structural and functional organization of TyrRSapm, we replaced all three W residues (both of dimer interface and C-terminal domain) systematically with F residues (using site directed mutagenesis) with four different combinations (W80F/W120F, W278, W80F/W278F, W120F/W278F). We took advantage of Trp (W) fluorescence as the biophysical reporter for probing dimer interface study. The circular dichroism (CD) spectra of Trp mutants are dramatically different from wild-type indicating a huge conformation reorientation due to mutations. Another unusual feature of this enzyme is that it contains 10 cysteine residues per monomer and none of them involved in disulphide bridge formation. Using DTNB reaction as a probe again it was observed that mutants behave differently indicating a major perturbation. Fluorescence anisotropy supported by analytical ultracentrifugation data showed that TyrRSapm and its mutants exist as dimer. The unfolding pattern of N-terminal and C-terminal domain shows significant difference from each other indicating differential melting of domains. Aminoacylation reaction showed that two of the four Trp. mutants retain enzyme activities though different as compared to WT, while the other two mutants lost their activities. Hence it can be concluded that, the Trp residues play a crucial role in the structural as well as functional organization of TyrRSapm that may be reflected as the non canonical orientation of dimer conformation in crystal structure.