Trypsin inhibition by macrocyclic and open-chain variants of the squash inhibitor MCoTI-II

MCoTl-I and MCoTl-II from the seeds of Momordica cochinchinensis are inhibitors of trypsin-like proteases and the only known members of the large family of squash inhibitors that are cyclic and contain an additional loop connecting the amino- and the carboxy-terminus. To investigate the contribution of macrocycle formation to biological activity, we synthesized a set of open-chain variants of MCoTl-II that lack the cyclization loop and contain various natural and non-natural amino acid substitutions in the reactive-site loop. Upon replacement of P1 lysine residue \#10 within the open-chain variant of MCoTl-II by the non-natural isosteric nucleo amino acid AlaG{[}beta-(guanin-9-yl)-L-alanine], a conformationally restricted arginine mimetic, residual inhibitory activity was detected, albeit reduced by four orders of magnitude. While the cyclic inhibitors MCoTl-I and MCoTl-II were found to be very potent trypsin inhibitors, with picomolar inhibition constants, the open-chain variants displayed an approximately 10-fold lower affinity. These data suggest that the formation of a circular backbone in the MCoTI squash inhibitors results in enhanced affinity and therefore is a determinant of biological activity

Electrostatic interactions of domain III stabilize the inactive conformation of μ-calpain

The ubiquitous μ- and m-calpains are Ca(2+)-dependent cysteine proteases. They are activated via rearrangement of the catalytic domain II induced by cooperative binding of Ca(2+) to several sites of the molecule. Based on the crystallographic structures, a cluster of acidic residues in domain III, the acidic loop, has been proposed to function as part of an electrostatic switch in the activation process. Experimental support for this hypothesis was obtained by site-directed mutagenesis of recombinant human μ-calpain expressed with the baculovirus system in insect cells. Replacing the acidic residues of the loop individually with alanine resulted in an up to 7-fold reduction of the half-maximal Ca(2+) concentration required for conformational changes (probed with 2-p-toluidinylnapthalene-6-sulphonate fluorescence) and for enzymic activity. Along with structural information, the contribution of individual acidic residues to the Ca(2+) requirement for activation revealed that interactions of the acidic loop with basic residues in the catalytic subdomain IIb and in the pre-transducer region of domain III stabilize the structure of inactive μ-calpain. Disruption of these electrostatic interactions makes the molecule more flexible and increases its Ca(2+) sensitivity. It is proposed that the acidic loop and the opposing basic loop of domain III constitute a double-headed electrostatic switch controlling the assembly of the catalytic domain

Primed-site probing of papain-like cysteine proteases

Pfizer JM, Assfalg-Machleidt I, Machleidt W, Moroder L, Schaschke N. Primed-site probing of papain-like cysteine proteases. INTERNATIONAL JOURNAL OF PEPTIDE RESEARCH AND THERAPEUTICS. 2007;13(1-2):93-104