ZMPSTE24 is a unique membrane bound metalloenzyme responsible for processing of lamin A proteins in humans. It does so by carrying out two proteolytic cleavage steps at two distinct sites within lamin A sequence after it is post-transitionally modified by a farnesyl chain: a C-terminal cleavage of three amino acids post a prenylation site, and an upstream N-terminal cleavage. Errors in this processing lead to age related progeroid diseases. ZMPESTE24 has also been shown to act as a translocon declogger, and off target inhibition by HIV protease inhibitors lead to accelerated aging side effects. Many mysteries still surround ZMPSTE24, including how it can recognize two distinct cleavage sites. Due to difficulty in purification of ZMPSTE24, the yeast homologue, Ste24, is being investigated. Both enzymes share significant sequence homology and can process each other’s substrates interchangeably. a-Factor, the only bona fide substrate of Ste24 in yeast, is historically used a model system to study prenylation. a-Factor is initially produced as a 36-mer peptide, and after several proteolytic cleavage and post-transitional modification steps the final processed peptide is exported as a 12-mer peptide with a prenylated cysteine and carboxymethylated C-terminus. In this work, several peptide probes based on the sequence of a-Factor are synthesized and used to discern the intricacies of Ste24. The first probe is a mimetic of a-Factor 15-mer precursor, but with a none-hydrolyzable CH3NH y isostere linkage at the cleavage site and a Cy5 fluorophore on the N-terminus. This probe was used to accurately measure the KD of binding to Ste24 using microscale thermophoresis. Several mutant enzymes were also investigated in this manner. The second probe is also based on the sequence of a-Factor’s 15-mer precursor but includes a benzophenone photolabeling moiety on the prenyl chain, and a biotin on the N-terminus. This peptide was used to map the active site within Ste24’s by photo-crosslinking it to the enzyme, enrichment using streptavidin beads, and finally visualizing using either western blots or LC-MS-MS analysis after trypsin digestion. Both WT and several mutant enzymes were photolabeled and visualized in this manner. These experiments in conjunction with activity assays are potentiating a model describing substrate binding and proteolysis by the enzyme. Overall, the work described here offers key insights into the unique structure and molecular mechanism of Ste24.

Mapping the structure and activity of the novel metalloenzyme Ste24 using peptide probes

ACS Spring 2022

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