Caught after the Act: a human A-type metallocarboxypeptidase in a product complex with a cleaved hexapeptide.

A/B-type metallocarboxypeptidases (MCPs) are among the most thoroughly studied proteolytic enzymes, and their catalytic mechanisms have been considered as prototypes even for several unrelated metalloprote(in)ase families. It has long been postulated that the nature of the side chains of at least five substrate residues, i.e., P4-P1', influence Km and kcat and that once the peptide or protein substrate is cleaved, both products remain in the first instance bound to the active-site cleft of the enzyme in a double-product complex. Structural details of binding of substrate to the nonprimed side of the cleft have largely relied on complexes with protein inhibitors and peptidomimetic small-molecule inhibitors that do not span the entire groove. In the former, the presence of N-terminal globular protein domains participating in large-scale interactions with the surface of the cognate catalytic domain outside the active-site cleft mostly conditions the way their C-terminal tails bind to the cleft. Accordingly, they may not be accurate models for a product complex. We hereby provide the structural details of a true cleaved double-product complex with a hexapeptide of an MCP engaged in prostate cancer, human carboxypeptidase A4, employing diffraction data to 1.6 A resolution (Rcryst and Rfree = 0.159 and 0.176, respectively). These studies provide detailed information about subsites S5-S1' and contribute to our knowledge of the cleavage mechanism, which is revisited in light of these new structural insights.

Papaya glutamine cyclotransferase shows a singular five-fold beta-propeller architecture that suggests a novel reaction mechanism.

Cyclisation of N-terminal glutamine and/or glutamate to yield pyroglutamate is an essential posttranslational event affecting a plethora of bioactive peptides and proteins. It is directly linked with pathologies ranging from neurodegenerative diseases to inflammation and several types of cancers. The reaction is catalysed by ubiquitous glutaminyl cyclotransferases (QCs), which present two distinct prototypes. Mammalian QCs are zinc-dependent enzymes with an alpha/beta-hydrolase fold. Here we present the 1.6-A-resolution structure of the other prototype, the plant analogue from Carica papaya (PQC). The hatbox-shaped molecule consists of an unusual five-fold beta-propeller traversed by a central channel, a topology that has hitherto been described only for some sugar-binding proteins and an extracellular nucleotidase. The high resistance of the enzyme to denaturation and proteolytic degradation is explained by its architecture, which is uniquely stabilised by a series of tethering elements that confer rigidity. Strikingly, the N-terminus of PQC specifically interacts with residues around the entrance to the central channel of a symmetry-related molecule, suggesting that this location is the putative active site. Cyclisation would follow a novel general-acid/base working mechanism, pivoting around a strictly conserved glutamate. This study provides a lead structure not only for plant QC orthologues, but also for bacteria, including potential human pathogens causing diphtheria, plague and malaria.

Staphylococcal methicillin resistance: fine focus on folds and functions.

Globalisation has entailed a massive increase in trade and human mobility facilitating the rapid spread of infectious agents, including those that are drug resistant. A particularly serious threat to human health is posed by methicillin-resistant staphylococcal strains which have acquired molecular mechanisms to evade the action of beta-lactam antibiotics (BLAs). Full expression of high-level methicillin resistance involves a complex network of molecules and depends primarily on sufficient expression of a penicillin-binding protein with low sensitivity towards BLAs. Other factors include the fine-tuned regulation of autolytic activity of cell-wall components, as well as an optimal rate of peptidoglycan precursor formation and a highly specific peptidoglycan precursor structure. Three-dimensional structural data are available on several of the pieces involved in the jigsaw puzzle and provide a molecular basis for the understanding of methicillin resistance and for the design of new therapeutic strategies.