Rational engineering enables co-crystallization and structural determination of the HIV-1 matrix-tRNA complex

Summary Host tRNAs specifically interact with the matrix domain (MA) of HIV-1 major structural polyprotein, Gag, to control its membrane localization and virion assembly. In this protocol, we describe the purification and engineering of HIV-1 MA and tRNA, and the co-crystallization and structure determination of the complex using X-ray crystallography. Rational engineering of the tRNA surface created tRNA-tRNA packing contacts that drove the formation of diffraction-quality co-crystals. This protocol can be adapted to solve other ribonucleoprotein complex structures containing structured RNAs. For complete details on the use and execution of this protocol, please refer to Bou-Nader et al. (2021).
Graphical abstract
Highlights • Protocol to purify HIV-1 matrix domain and to transcribe human tRNALys3 in high yields • Use of limited proteolysis to engineer HIV-1 MA protein for co-crystallization • Rational tRNA engineering to promote RNA-RNA contacts for co-crystallization • Details for crystallization of MA-tRNA complex and steps for structural determination
Host tRNAs specifically interact with the matrix domain (MA) of HIV-1 major structural polyprotein, Gag, to control its membrane localization and virion assembly. In this protocol, we describe the purification and engineering of HIV-1 MA and tRNA, and the co-crystallization and structure determination of the complex using X-ray crystallography. Rational engineering of the tRNA surface created tRNA-tRNA packing contacts that drove the formation of diffraction-quality co-crystals. This protocol can be adapted to solve other ribonucleoprotein complex structures containing structured RNAs.