title: A Single Point Mutation in Mitochondrial Hsp70 Cochaperone Mge1 Gains Thermal Stability and Resistance.
abstract: 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.
authors.affiliation: Department of Biochemistry, School of Life Sciences, University of Hyderabad , Hyderabad 500046, T.S., India.
authors.forename: Adinarayana
authors.initials: A
authors.lastname: Marada
authors.affiliation: Department of Biochemistry, School of Life Sciences, University of Hyderabad , Hyderabad 500046, T.S., India.
authors.forename: Srinivasu
authors.initials: S
authors.lastname: Karri
authors.affiliation: School of Chemistry, University of Hyderabad , Hyderabad 500046, T.S., India.
authors.forename: Swati
authors.initials: S
authors.lastname: Singh
authors.affiliation: Department of Biochemistry, School of Life Sciences, University of Hyderabad , Hyderabad 500046, T.S., India.
authors.forename: Praveen Kumar
authors.initials: PK
authors.lastname: Allu
authors.affiliation: Department of Biochemistry, School of Life Sciences, University of Hyderabad , Hyderabad 500046, T.S., India.
authors.forename: Yerranna
authors.initials: Y
authors.lastname: Boggula
authors.affiliation: Department of Biochemistry, School of Life Sciences, University of Hyderabad , Hyderabad 500046, T.S., India.
authors.forename: Thanuja
authors.initials: T
authors.lastname: Krishnamoorthy
authors.affiliation: School of Chemistry, University of Hyderabad , Hyderabad 500046, T.S., India.
authors.forename: Lalitha
authors.identifier: 0000-0002-1579-4611
authors.initials: L
authors.lastname: Guruprasad
authors.affiliation: Department of Biochemistry, School of Life Sciences, University of Hyderabad , Hyderabad 500046, T.S., India.
authors.forename: Naresh Babu V
authors.initials: NBV
authors.lastname: Sepuri
mesh_terms: D000595:Amino Acid Sequence; D002942:Circular Dichroism; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D006358:Hot Temperature; D033681:Mitochondrial Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D024101:Mitochondrial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008958:Models, Molecular; D018832:Molecular Chaperones / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D017354:Point Mutation; D011489:Protein Denaturation; D000072417:Protein Domains; D017510:Protein Folding; D055503:Protein Multimerization; D055550:Protein Stability; D058767:Protein Unfolding; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
chemical_list: D018840:HSP70 Heat-Shock Proteins; C086039:MGE1 protein, S cerevisiae; D033681:Mitochondrial Membrane Transport Proteins; D024101:Mitochondrial Proteins; D018832:Molecular Chaperones; D029701:Saccharomyces cerevisiae Proteins