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CHARACTERIZATION OF THE BINDING SITE OF STE24 DURING THE -AAXING CLEAVAGE
ZMPSTE24 is a seven transmembrane domain zinc metalloprotease that resides in the ER and inner nuclear membranes of mammalian cells. The crystal structures of both the mammalian and yeast homologs, ZMPSTE24 and Ste24, respectively, were solved recently and revealed a common novel structure. Both structures contain a large chamber of mixed hydrophobicity that is capped on both sides. The canonical catalytic HExxH zinc-binding motif lies inside the chamber. Defects in the enzymatic function of human ZMPSTE24 have been shown to cause premature aging disorders. In addition to the well-defined role ZMPSTE24 and Ste24 play in the maturation of prelamin A in mammals and a-factor in yeast, both proteins have been proposed to play protective roles in Type 2 diabetes and viral infections by interactions with the cellular translocon. ZMPSTE24 can also be inhibited by several common HIV aspartyl protease inhibitors, possibly causing the frequent and common side-effects of these prescribed drugs. As of now, no precise location for substrate binding has been identified in either ZMPSTE24 or Ste24. Thus, the goal of this project is to localize residues in the enzyme that are important for substrate binding. The yeast homolog Ste24 was used as a model system as it functionally complements the mammalian enzyme and can be reliably cloned, overexpressed, and purified in an active form.
Three approaches were taken to directly determine the KD values for substrates of Ste24. The ability to perform a direct analysis of KD values of Ste24 mutations was successfully optimized using microscale thermophoresis. Through KD analysis, the Ste24 mutation G255A, while completely inactive, does not prevent substrate binding. Alternatively, L441A and L410A mutations showed both an increase in thermal stability and a decrease in binding affinity, that could explain their lower activity levels. A photoaffinity labeling-based proteomics experiment was utilized to precisely locate the site of the prenyl group to a hydrophobic patch lying just under a side portal of Ste24, near K234, during the -aaXing cleavage of a-factor maturation. To assess the method of inhibition of HIV protease inhibitors on Ste24 the conserved aspartate mutants were explored. All mutations of these aspartate residues resulted in a severe loss of Ste24 function and instability of the protein.