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Elucidation of the Substrate Binding Site of the Yeast Zinc Metalloprotease, Ste24
Author(s) -
Theisen Chelsea,
Hsu ErhTing,
Wiley Patty,
Vervacke Jeffrey,
Distefano Mark,
Hrycyna Christine
Publication year - 2019
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2019.33.1_supplement.631.40
Subject(s) - protease , metalloproteinase , saccharomyces cerevisiae , yeast , biochemistry , chemistry , proteases , binding site , transmembrane domain , active site , serine protease , mutagenesis , substrate (aquarium) , mutant , biophysics , microbiology and biotechnology , biology , membrane , enzyme , gene , ecology
The Ste24 zinc metalloprotease from S. cerevisiae is an intriguing integral membrane protease whose precise mechanism of action has yet to be explored fully at a molecular level. This protease is unique in that it recognizes only isoprenylated substrates and performs two distinct cleavage reactions sequentially at structurally unrelated sites in the same substrate molecule, notably the mating pheromone a ‐factor in yeast. The recent X‐ray structure of yeast Ste24 revealed unique features that define a novel class of membrane protease that does not resemble any other class of intramembrane proteases described to date. The 7 transmembrane helices of Ste24 form a voluminous water‐filled intramembrane barrel‐shaped chamber that is capped at both ends. Notably, the metalloprotease active site faces the chamber interior so that substrate entry and exit is restricted and must occur through one of four side portals in the protein. Since the structure provided only minimal mechanistic information, we use it to guide our studies to define how the farnesylated yeast mating pheromone a ‐factor enters and exits the chamber, how it is recognized, and how Ste24 mediates catalysis. Herein, we aim to identify the residues that comprise the substrate binding site for isoprenylated a ‐factor in Ste24. Mutant forms of Ste24 generated by site‐directed mutagenesis were characterized for protease activity by a radioactive coupled methyltransferase‐protease assay and assayed for substrate binding using a photoactivatable analog a ‐factor containing a biotin moiety. Substrate binding was assessed by immunoblot analysis using Neutravidin‐HRP. Tandem MS/MS methods are being developed to accurately identify the labeled amino acids in or near the binding site of the labeled proteins. Preliminary results suggest that a region surrounding the active site, lying between two portal sites within the chamber, appear to be important for a ‐factor binding for the first C‐terminal cleavage reaction. Further studies will aim to determine residues important for binding for the second proteolytic cleavage reaction. Support or Funding Information National Institutes of Health This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .