A Single Point Mutation in Mitochondrial Hsp70 Cochaperone Mge1 Gains Thermal Stability and Resistance

作者：Adinarayana Marada, Srinivasu Karri, Swati Singh, Praveen Kumar Allu, Yerranna Boggula, T.M. Krishnamoorthy, Lalitha Guruprasad, Naresh Babu V. Sepuri · 发表于：Biochemistry · 年份：2016 · DOI：10.1021/acs.biochem.6b00232 · 被引用次数：19 · 研究领域：Heat shock proteins research、thermodynamics and calorimetric analyses、Protein Structure and Dynamics

Mge1, a yeast homologue of Escherichia coli GrpE, is an evolutionarily conserved homodimeric nucleotide exchange factor of mitochondrial Hsp70. Temperature-dependent reversible structural alteration from a dimeric to a monomeric form is critical for Mge1 to act as a thermosensor. However, very limited information about the structural component or amino acid residue(s) that contributes to thermal sensing of Mge1/GrpE is available. In this report, we have identified a single point mutation, His167 to Leu (H167L), within the hinge region of Mge1 that confers thermal resistance to yeast. Circular dichroism, cross-linking, and refolding studies with recombinant proteins show that the Mge1 H167L mutant has increased thermal stability compared to that of wild-type Mge1 and also augments Hsp70-mediated protein refolding activity. While thermal denaturation studies suggest flexibility in the mutant, ionic quenching studies and limited proteolysis analysis reveal a relatively more rigid structure compared to that of the wild type. Intriguingly, Thermus thermophilus GrpE has a leucine at the corresponding position akin to the Mge1 mutant, and thermophilus proteins are well-known for their rigidity and hydrophobicity. Taken together, our results show that the yeast Mge1 H167L mutant functionally and structurally mimics T. thermophilus GrpE.