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Characterization of the binding interaction between the oncoprotein gankyrin and a grafted S6 ATPase.

Chapman AM, Rogers BE, McNaughton BR - Biochemistry (2014)

Bottom Line: A complex with the C-terminal portion of the proteosomal subunit S6 ATPase is the only available structure of a protein-protein interaction involving the oncoprotein gankyrin.However, difficulties associated with recombinant expression of S6 ATPase alone, or truncations thereof, have limited our understanding of this assembly.We replaced the C-terminal portion of FtsH from Escherichia coli with the structurally homologous C-terminal portion of S6 ATPase and used this grafted protein to characterize the gankyrin-S6 ATPase binding interaction by isothermal titration calorimetry.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and ‡Department of Biochemistry & Molecular Biology, Colorado State University , Fort Collins, Colorado 80523, United States.

ABSTRACT
A complex with the C-terminal portion of the proteosomal subunit S6 ATPase is the only available structure of a protein-protein interaction involving the oncoprotein gankyrin. However, difficulties associated with recombinant expression of S6 ATPase alone, or truncations thereof, have limited our understanding of this assembly. We replaced the C-terminal portion of FtsH from Escherichia coli with the structurally homologous C-terminal portion of S6 ATPase and used this grafted protein to characterize the gankyrin-S6 ATPase binding interaction by isothermal titration calorimetry.

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(a) Circular dichroismspectra of wild-type FtsH (wt-FtsH, top)and FtsH-S6 ATPase (bottom). (b) Co-purification of wild-type gankyrin-His6x and FtsH-S6 ATPase mutants: wt-S6 FtsH-S6 ATPase (lane 1),R342A (lane 2), R338A/R342A (lane 3), R338A/R339A/R342A (lane 4),E356A/E357A (lane 5), D359A/D362A (lane 6), and K397E (lane 7). (c)Co-purification of wild-type S6 ATPase and gankyrin-His6x mutants: wt-gankyrin (lane 1), R41A (lane 2), K116A (lane 3), R41A/K116A(lane 4), D39A/D71A (lane 5), and E182A (lane 6).
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fig2: (a) Circular dichroismspectra of wild-type FtsH (wt-FtsH, top)and FtsH-S6 ATPase (bottom). (b) Co-purification of wild-type gankyrin-His6x and FtsH-S6 ATPase mutants: wt-S6 FtsH-S6 ATPase (lane 1),R342A (lane 2), R338A/R342A (lane 3), R338A/R339A/R342A (lane 4),E356A/E357A (lane 5), D359A/D362A (lane 6), and K397E (lane 7). (c)Co-purification of wild-type S6 ATPase and gankyrin-His6x mutants: wt-gankyrin (lane 1), R41A (lane 2), K116A (lane 3), R41A/K116A(lane 4), D39A/D71A (lane 5), and E182A (lane 6).

Mentions: GraftedFtsH-S6 ATPase and wild-type FtSH (wt-FtsH) were expressedas His6x-tagged proteins in E. coli assoluble proteins (Supporting Information, Figure S1). Circular dichroism spectra of the two proteins arevirtually identical (Figure 2a), suggestingno appreciable structural change to the FtsH scaffold or grafted S6ATPase domain. The affinity of this grafted protein for gankyrin wasfirst assessed using a pull-down assay in E. coli. Binding face residues on gankyrin or FtsH-S6 ATPase were mutatedto alanine, on the basis of the findings of Yokoyama and co-workers,and their effect on complex stability was qualitatively assessed bymeasuring the amount of untagged FtsH-S6 ATPase co-purified with gankyrin-His6x.


Characterization of the binding interaction between the oncoprotein gankyrin and a grafted S6 ATPase.

Chapman AM, Rogers BE, McNaughton BR - Biochemistry (2014)

(a) Circular dichroismspectra of wild-type FtsH (wt-FtsH, top)and FtsH-S6 ATPase (bottom). (b) Co-purification of wild-type gankyrin-His6x and FtsH-S6 ATPase mutants: wt-S6 FtsH-S6 ATPase (lane 1),R342A (lane 2), R338A/R342A (lane 3), R338A/R339A/R342A (lane 4),E356A/E357A (lane 5), D359A/D362A (lane 6), and K397E (lane 7). (c)Co-purification of wild-type S6 ATPase and gankyrin-His6x mutants: wt-gankyrin (lane 1), R41A (lane 2), K116A (lane 3), R41A/K116A(lane 4), D39A/D71A (lane 5), and E182A (lane 6).
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fig2: (a) Circular dichroismspectra of wild-type FtsH (wt-FtsH, top)and FtsH-S6 ATPase (bottom). (b) Co-purification of wild-type gankyrin-His6x and FtsH-S6 ATPase mutants: wt-S6 FtsH-S6 ATPase (lane 1),R342A (lane 2), R338A/R342A (lane 3), R338A/R339A/R342A (lane 4),E356A/E357A (lane 5), D359A/D362A (lane 6), and K397E (lane 7). (c)Co-purification of wild-type S6 ATPase and gankyrin-His6x mutants: wt-gankyrin (lane 1), R41A (lane 2), K116A (lane 3), R41A/K116A(lane 4), D39A/D71A (lane 5), and E182A (lane 6).
Mentions: GraftedFtsH-S6 ATPase and wild-type FtSH (wt-FtsH) were expressedas His6x-tagged proteins in E. coli assoluble proteins (Supporting Information, Figure S1). Circular dichroism spectra of the two proteins arevirtually identical (Figure 2a), suggestingno appreciable structural change to the FtsH scaffold or grafted S6ATPase domain. The affinity of this grafted protein for gankyrin wasfirst assessed using a pull-down assay in E. coli. Binding face residues on gankyrin or FtsH-S6 ATPase were mutatedto alanine, on the basis of the findings of Yokoyama and co-workers,and their effect on complex stability was qualitatively assessed bymeasuring the amount of untagged FtsH-S6 ATPase co-purified with gankyrin-His6x.

Bottom Line: A complex with the C-terminal portion of the proteosomal subunit S6 ATPase is the only available structure of a protein-protein interaction involving the oncoprotein gankyrin.However, difficulties associated with recombinant expression of S6 ATPase alone, or truncations thereof, have limited our understanding of this assembly.We replaced the C-terminal portion of FtsH from Escherichia coli with the structurally homologous C-terminal portion of S6 ATPase and used this grafted protein to characterize the gankyrin-S6 ATPase binding interaction by isothermal titration calorimetry.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and ‡Department of Biochemistry & Molecular Biology, Colorado State University , Fort Collins, Colorado 80523, United States.

ABSTRACT
A complex with the C-terminal portion of the proteosomal subunit S6 ATPase is the only available structure of a protein-protein interaction involving the oncoprotein gankyrin. However, difficulties associated with recombinant expression of S6 ATPase alone, or truncations thereof, have limited our understanding of this assembly. We replaced the C-terminal portion of FtsH from Escherichia coli with the structurally homologous C-terminal portion of S6 ATPase and used this grafted protein to characterize the gankyrin-S6 ATPase binding interaction by isothermal titration calorimetry.

Show MeSH
Related in: MedlinePlus