An Acrobatic Substrate Metamorphosis Reveals a Requirement for Substrate Conformational Dynamics in Trypsin Proteolysis

Abstract

Edited by George DeMartinoThe molecular basis of enzyme catalytic power and specificity derives from dynamic interactions between enzyme and substrate during catalysis. Although considerable effort has been devoted to understanding how conformational dynamics within enzymes affect catalysis, the role of conformational dynamics within protein substrates has not been addressed. Here, we examine the importance of substrate dynamics in the cleavage of Kunitz-bovine pancreatic trypsin inhibitor protease inhibitors by mesotrypsin, finding that the varied conformational dynamics of structurally similar substrates can profoundly impact the rate of catalysis. A 1.4-Å crystal structure of a mesotrypsinproduct complex formed with a rapidly cleaved substrate reveals a dramatic conformational change in the substrate upon proteolysis. By using long all-atom molecular dynamics simulations of acyl-enzyme intermediates with proteolysis rates spanning 3 orders of magnitude, we identify global and local dynamic features of substrates on the nanosecond-microsecond time scale that correlate with enzymatic rates and explain differential susceptibility to proteolysis. By integrating multiple enhanced sampling methods for molecular dynamics, we model a viable conformational pathway between substrate-like and productlike states, linking substrate dynamics on the nanosecond-microsecond time scale with large collective substrate motions on the much slower time scale of catalysis. Our findings implicate substrate flexibility as a critical determinant of catalysis.Protein function is determined by macromolecular geometry conferred by the folded state, as noted by Anfinsen nearly 40 years ago (1); however, recent years have brought fresh meaning to this paradigm with an increasing appreciation of the temporal dependence of protein structure. Proteins constantly sample varied conformational fluctuations about the time-averaged structures that we observe crystallographically or spectroscopically, and these conformational dynamics are in many cases closely coupled to protein function (2-4). Nowhere is this clearer than for enzymes, proteins evolved to accelerate biological chemical reactions. Varied examples have revealed that enzyme conformational dynamics can facilitate substrate binding, progression along the catalytic reaction coordinate, and product release (5-10). Most studies of protein dynamics in enzyme catalysis have naturally focused on conformational changes within the enzyme. However, for the many enzymes that catalyze reactions of protein substrates, an overlooked source of potentially relevant dynamics lies within the substrate.Trypsins are serine proteases, a class of proteolytic enzymes that have been well characterized and used to dissect and understand catalysis, most often using short oligopeptide model substrates as proxies for natural protein substrates. Following formation of a noncovalent Michaelis complex, the catalytic mechanism proceeds through two sequential steps (Scheme 1) (11). In the first step, the enzyme serine...

GASP‐1 and GASP‐2, two closely structurally related proteins with a functional duality in antitrypsin inhibition specificity: a mechanistic point of view

“…This is likely correlated to a weaker binding to trypsin. Such a result has already been reported in the binding between b Trypsin and APPI, showing greater RMSD variation than with BPTI .…”

“…The structures are made of two main domains, namely the scaffold domain and the canonical loop (Fig. B) . The latter domain (canonical loop) is also known as the binding loop to trypsin.…”

“…Several studies have thoroughly described the interactions between trypsin and their inhibitors, which have highlighted the key role of P 1 and P 2 0 [30][31][32][33]. These two residues differ in both mKu 2-1 and mKu 2-2 from BPTI, which is a Lys/Arg-Xaa inhibitor.…”

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Small molecule inhibitors of mesotrypsin from a structure-based docking screen

“…Mesotrypsin displays unique active site features and conformational dynamics [34] which can in turn influence substrate motions [39]. Diminazene, a bis-benzamidine analog with mesotrypsin inhibitory activity, presents an attractive starting point for the development of more selective mesotrypsin inhibitors.…”