Insights into rate‐limiting substrate dynamics during proteolysis of Kunitz‐BPTI family canonical serine protease inhibitors

Proteases are ubiquitous enzymes that catalyze the hydrolysis of peptide bonds within protein substrates; they have served as key model enzymes for studying the molecular basis for catalytic power and specificity. Protease substrate specificity is most often defined in terms of linear sequence motifs that flank the cleavage site; however, the natural substrates of proteases are proteins with 3‐dimensional shapes and complex conformational dynamics that are not well represented by 1‐dimensional sequence alone. These structural and dynamical properties can impact recognition and binding of substrates by proteases, as well as the efficiency of catalysis itself. In this study, we explore the importance of substrate structure and dynamics for proteolysis using as our model the cleavage of the Kunitz‐BPTI family of canonical serine protease inhibitors by mesotrypsin. Bovine pancreatic trypsin inhibitor (BPTI), an archetypal serine protease inhibitor of the Kunitz family, has a high affinity substrate‐like interaction with trypsin, yet is cleaved many orders of magnitude slower than other peptide substrates. Mesotrypsin, a trypsin variant, has been shown to hydrolyze Kunitz family inhibitors at accelerated rates; this is especially true of human Kunitz domain inhibitors. Amyloid precursor protein inhibitor (APPI) and amyloid precursor like protein‐2 Kunitz domain (APLP2‐KD), two human Kunitz domain family members, are hydrolyzed by mesotrypsin several hundred times faster than BPTI. Here, we present a new crystal structure of a cleavage intermediate of APLP2‐KD bound to mesotrypsin, refined to 1.4 Å resolution. This newly solved structure reveals a dramatic substrate conformational change that we hypothesize is required during Kunitz domain cleavage. Using this structure along with published APPI and BPTI complexes, we have modeled acyl‐enzyme intermediates of mesotrypsin and we have carried out molecular dynamic simulations to explore the conformational transformation that allows the progression of the hydrolysis reaction. We identify dynamic features of the inhibitor scaffolds, located far from the enzyme binding site, that correlate with observed inhibitor cleavage rates and plausibly explain their differential propensity to undergo both conformational transformation and cleavage. Our findings implicate global scaffold flexibility as a determinant of proteolytic rate, as rigid substrates possessing stabilizing features that render them highly resistant to conformational change are proteolyzed more slowly than more flexible substrates of similar structure. Support or Funding Information NIH R01 CA154387 Mayo Graduate school