Proenzyme Structure and Activation of Astacin Metallopeptidase

Abstract

Proteolysis is regulated by inactive (latent) zymogens, with a prosegment preventing access of substrates to the activesite cleft of the enzyme. How latency is maintained often depends on the catalytic mechanism of the protease. For example, in several families of the metzincin metallopeptidases, a "cysteine switch" mechanism involves a conserved prosegment motif with a cysteine residue that coordinates the catalytic zinc ion. Another family of metzincins, the astacins, do not possess a cysteine switch, so latency is maintained by other means. We have solved the high resolution crystal structure of proastacin from the European crayfish, Astacus astacus. Its prosegment is the shortest structurally reported for a metallopeptidase, and it has a unique structure. It runs through the active-site cleft in reverse orientation to a genuine substrate. Moreover, a conserved aspartate, projected by a wide loop of the prosegment, coordinates the zinc ion instead of the catalytic solvent molecule found in the mature enzyme. Activation occurs through two-step limited proteolysis and entails major rearrangement of a flexible activation domain, which becomes rigid and creates the base of the substrate-binding cleft. Maturation also requires the newly formed N terminus to be precisely trimmed so that it can participate in a buried solvent-mediated hydrogen-bonding network, which includes an invariant active-site residue. We describe a novel mechanism for latency and activation, which shares some common features both with other metallopeptidases and with serine peptidases.The proteolytic activity of most metallopeptidases (MPs) 3 is regulated, and it is only exerted where and when required (1). Such control may occur through modulation of gene expression, compartmentalization, allostery, or inhibition by protein inhibitors. Another regulatory mechanism is zymogenic latency, which is provided by mostly N-terminal prosegments. These block access of substrates to the active-site cleft, and they are removed by limited proteolysis during maturation (2, 3). Such prosegments often fold independently and guide on their part the folding process of the cognate protease domains. They may also act as intramolecular chaperones or inhibitors of the mature enzymes in trans and in intracellular sorting of the zymogen (2). Therefore, the study of the molecular mechanisms by which MPs maintain latency is indispensable to an understanding of their basic mode of action. It also paves the way for the design of inhibitors that mimic the latent state so as to modulate MP activity as part of therapeutic approaches. Detailed three-dimensional structural information can contribute much to this understanding (4). However, among the MPs, only funnelins, lysostaphins, thermolysins, and matrix metalloproteinases (MMPs) have been structurally analyzed for their zymogens. Results reveal that the mature enzyme moieties are already in a competent conformation. Notwithstanding, each group displays a distinct mechanism of latency maintenance (5-11).The metzincins...

Structure, function and latency regulation of a bacterial enterotoxin potentially derived from a mammalian adamalysin/ADAM xenolog

“…The name was based on a cysteine Sγ atom replacing the catalytic solvent molecule in the zymogen (25,26). Such an aspartate-switch mechanism has been described for proastacin (27). In this zymogen, however, the PD is much shorter (34 residues), the zinc-binding aspartate is provided by a wide loop immediately downstream of a prodomain helix that occupies the primed side of the cleft, and no interactions are observed between the PD and the upper-rim strand.…”

A Novel Mechanism of Latency in Matrix Metalloproteinases

“…To date, aspartate-switch zymogenic mechanisms have been described only for astacins (7,88) and fragilysins (26), which are only distantly related MPs grouped with MMPs within the metzincins. To verify the function of Asp 25 in latency in pKly18, we used mutant pKly18-Y35A (from pKAR8), as the wild-type form (pKAR7) was insoluble.…”

A Drosophila Protease Cascade Member, Seminal Metalloprotease-1, Is Activated Stepwise by Male Factors and Requires Female Factors for Full Activity

“…To our surprise, ovulin and Acp36DE were processed in these mates ( Figure S4, C and D, respectively), albeit slowly and in the case of ovulin, not to completion, suggesting that Semp1 has a small amount of activity in the absence of processing by seminase. To parallel the activation mechanism of the canonical Astacus astacus astacin protease (Guevara et al 2010), we will refer to the removal of Semp1's propeptide region by seminase as "activational cleavage." Semp1 mutated at its predicted activational cleavage site fails to cleave its substrates Despite the fact that Semp1 cleaves ovulin and Acp36DE, and that the protease and its substrates are made in the same tissue, cleavage does not occur until these proteins are within the mated female's RT.…”

“…I46A mutation blocked the second (and final) cleavage event, but incompletely blocked the first. This type of activation mechanism exists for the canonical A. astacus astacin protease (Guevara et al 2010), which is first cleaved by a trypsin protease a few amino acids N-terminal to its propeptide cleavage site. Following this tryptic cleavage event, the remaining residues are removed from the propeptide by an intramolecular cleavage by astacin itself, producing the active protease (Guevara et al 2010).…”