Using alternative preservation method in cryo-EM to overcome challenging specimen methionine tRNA synthetase

Abstract

Targeting the protein machinery of intracellular parasites protein machinery is a challenge for drug development because often those parasites have minimal genomes and depend on their host for basic metabolic functions. Mycoplasma penetrans is one such intracellular parasite that infects immunocompromised patients, in particular individuals who are infected with HIV. Despite its minimal genome, the M. penetrans methionine tRNA synthetase (MetRS) is a unique fusion of an N-terminal alanine glyoxylate aminotransferase (AGAT) domain, which forms a homodimer, and a C-terminal tRS domain, the two of which are presumed to be independent. The AGAT domain appears to increase the MetRS enzyme's substrate differentiation ability and its efficiency in amino-acylating tRNA. The structural basis for this added functionality is unknown. We have now used cryo-EM to determine the structure of this unique drug target to show that the dimer adopts a butterfly-shaped arrangement, in which the body is the AGAT dimer, and the wings are two independent tRS domains. Initial cryogenic preparations of the complex were unsuccessful, so we also used a systematic analysis of alternative sample preparation techniques to identify which was optimal for this particular protein. With the structure in hand, we will next use rational mutagenesis to test the hypothesis that substrate channeling occurs amongst the unrelated active sites to impact MetRS activity, further developing this enzyme as a possible drug target.