Characterization of the N-terminal targeting signal binding domain of the mitochondrial outer membrane receptor, Tom20.
hTom20 is an outer mitochondrial membrane receptor involved in protein translocation. The cytosolic domain (aa30-145) and selected truncated versions of this domain were overexpressed and purified to study the structure-function relationship of this protein. Our studies reveal that the secondary structure of the cytosolic domain is very resistant to unfolding by guanidine-HCl and urea and is stabilized mainly by hydrophobic interactions. However, the tertiary structure of the N-terminal targeting signal binding domain (aa30-90) is more flexible. The first 30 amino acids of the cytosolic domain (aa30-60) are involved in recognizing N-terminal targeting signals and in stabilizing the cytosolic domain on the lipid surface. Moreover, we show that specifically aa30-48 interact with the membrane surface; a construct containing aa48-145 will only bind to the membrane surface in the presence of an N-terminal targeting signal peptide. The C-terminal region of hTom20 (aa141-145) interacts with the N-terminal region of hTom20, helping to stabilize the proper conformation of the N-terminal targeting signal binding domain. Finally, hTom20 interacts with the N-terminal targeting signal of preornithine carbamyl transferase fused to dihydrofolate reductase very weakly (Kd = 8 microM), as would be expected if this interaction was the first in a series orchestrated by the import receptor complex to draw the targeted protein into the mitochondrion.
Schleiff
E
E
Department of Biochemistry, McGill University, Montreal, Canada. schleiff@med.mcgill.ca
Turnbull
J L
JL
D002942:Circular Dichroism; D005454:Fluorescence Polarization; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology; D026901:Membrane Transport Proteins; D008928:Mitochondria / Q000378:metabolism*; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D011498:Protein Precursors / Q000737:chemistry / Q000378:metabolism; D021382:Protein Sorting Signals / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011956:Receptors, Cell Surface
D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D010446:Peptide Fragments; D011498:Protein Precursors; D021382:Protein Sorting Signals; D011956:Receptors, Cell Surface; C084008:TOMM20 protein, human