Abelardo M. Silva

Structural Analysis of Plasmepsin II

Malaria is one of the world’s most devastating diseases, afflicting several hundred million people a year and killing an estimated two million of them, mostly children.1 The etiologic agent is a parasitic protozoan of the genus Plasmodium. The organism causes disease in its intraerythrocytic phase. A prominent feature of its development inside red blood cells is the degradation of hemoglobin. During its short cycle, the parasite consumes nearly all of the host hemoglobin, generating amino acids for its growth and maturation.2 This is a vast catabolic process since hemoglobin constitutes 95% of the soluble red blood cell protein. Degradation occurs in an acidic digestive vacuole and involves a series of proteases. Two aspartic proteases, termed plasmepsins,3′6 and one cysteine protease, called falcipain,4 have been purified from the digestive vacuole and characterized.5 The plasmepsins appear to make an initial attack on the native hemoglobin molecule, which is followed by proteolysis of the large fragments into small peptides by falcipain.5,7 Intact hemoglobin cannot be cleaved by falcipain unless the substrate is first denatured.8 Cysteine protease inhibitors cause osmotic swelling of the digestive vacuole and ultimately death of the parasite,4 perhaps through accumulation of plasmepsin digestion products that are too large to exit the vacuole.8 The products of digestive vacuole proteolysis are small peptides, which appear to be exported for terminal degradation by cytoplasmic expeptidases.

Structures of PmSOD1 and PmSOD2, two superoxide dismutases from the protozoan parasite Perkinsus marinus.

Perkinsus marinus, a facultative intracellular parasite of the eastern oyster Crassostrea virginica, is responsible for mass mortalities of oyster populations. P. marinus trophozoites survive and proliferate within oyster hemocytes, invading most tissues and fluids, thus causing a systemic infection that eventually kills the host. The phagocytosis of P. marinus trophozoites lacks a respiratory burst, suggesting that the parasite has mechanisms that actively abrogate the hosts oxidative defense responses. One mechanism and the first line of defense against oxidative damage is the dismutation of superoxide radical to molecular oxygen and hydrogen peroxide by superoxide dismutases (SODs). P. marinus possesses two iron-cofactored SODs, PmSOD1 and PmSOD2. Here, the crystallization and X-ray structures of both PmSOD1 and PmSOD2 are presented.

Molecular Dynamics of HIV-1 Protease in Complex with a Difluoroketone-Containing Inhibitor: Implications for the Catalytic Mechanism

The structures of three aspartic proteases, penicillopepsin, endothiapepsin and rhizopuspepsin, have been determined in complexes with difluorostatone (DFS)-containing peptide-based inhibitors [1–3]. In all three structures, the hydrated core of the inhibitors display similar conformations and interactions with the active site aspartate groups. One of the gem-hydroxyls is positioned between both aspartates, the other one interacts with a single aspartate, and the fluorines do not interact with either of them. Since one of the hydroxyl groups is located near the position of the active site water observed in the native structures, these inhibitors have been proposed as analogs of transition state intermediates of peptide bond hydrolysis.

Computer simulation and analysis of the reaction pathway for the decomposition of the hydrated peptide bond in aspartic proteases.

Reaction models based on experimental structures can be used to reveal new details of a reaction, including the exploration of transition states (TS). The TSs usually described in studies of enzyme-inhibitor structures are actually reaction intermediates (RI) - short-lived but stable species that can be found during a reaction. Actual TSs are mathematically well-characterized entities, but are inherently unstable species [1]. An enzymatic reaction mechanism can be understood as a recognition process whereby the enzyme preferentially recognizes TSs. A very high affinity for substrates, RIs or products will result in a kinetically unfavorable process. On the other hand, enzymatic stabilization of the TS is a requirement for the catalytic process. Thus, a TS mimic is potentially the highest affinity inhibitor that can be designed based on the knowledge of the structure and the details of the mechanism of action of an enzyme. However, the study of reaction paths, which includes the characterization of TSs, is one of the most complex exercises in computational chemistry.