Crystal Structures of Ancestral Orthologues Reveal the Molecular Basis of Thermostability in Thermophilic EF-Tus

• The molecular basis of protein thermostability is diverse and unclear. To better understand it, we solved high-resolution crystal structures of four 0.5 – 3.5 billion year old ancestral bacterial Elongation Factor-Tus (EF-Tu). Structural comparison revealed two key interactions, unique for the thermophilic EF-Tus; i) a hydrogen bond between a G-domain tyrosine and the α- phosphate of the guanine nucleotide that stabilizes GTP/GDP; and ii) an inter-domain salt-bridge, tethering the Domains II and III via an arginine and a glutamic acid, respectively. We could reverse the thermostability profiles of the thermophilic and mesophilic EF-Tus by adding or removing these interactions, which were validated with aggregation, biophysical, and functional assays. Further, molecular dynamics simulations demonstrated that these interactions contribute to thermostability via the Mg2+ in the nucleotide binding site. Furthermore, the inter-domain interaction restricts the transition between the open and the closed conformations of the EF-Tus, thereby regulating their thermoactivity. 

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