Structural studies of malarial aspartic proteinase activation

Malaria is a complex and devastating disease that is a major public health problem in many areas of the world. During the blood stage of the malaria infection, the malaria parasite, Plasmodium, lives in the human host's erythrocytes, where it digests large quantities of hemoglobin as a source of nutrients. Hemoglobin digestion is an ordered and highly efficient process that is carried out in a specialized acidic compartment by several of the parasite's proteolytic enzymes. These enzymes, the hemoglobinases, include a cysteine proteinase, a metalloproteinase and at least two aspartic proteinases. The aspartic proteinases, plasmepsins, are the subject of this thesis.; Like all known eukaryotic aspartic proteinases, the plasmepsins are produced as inactive precursors, or zymogens, called proplasmepsins. Proteolytic removal of the amino-terminal extension, or prosegment, at acidic pH converts the inactive zymogen into the active proteinase. We determined the x-ray crystal structure of proplasmepsin II from the most dangerous human malaria parasite Plasmodium falciparum. Comparison of the structure of this zymogen to the structure of the corresponding mature enzyme, plasmepsin II, revealed the basis of inactivity of proplasmepsin II. The prosegment and the first 15 amino acids of the mature sequence enforce a major domain rotation in the zymogen relative to the mature enzyme. This domain shift severely distorts the active site in proplasmepsin II relative to plasmepsin II. At this "immature" active site, the catalytic machinery is in the wrong orientation to carry out proteolysis. The crystal structure of proplasmepsin II was used to propose a possible pathway of autocatalytic activation that occurs at low pH in vitro, and to rationalize the need for acidic pH for maturase-assisted activation in vivo.; Until recently, our understanding of aspartic proteinase activation had been limited, on a structural level, to the gastric aspartic proteinase zymogens, human and porcine pepsinogen and human progastricsin. In all of these zymogens, the prosegment prevents activity at neutral pH by blocking a pre-assembled active site. Disruption of several salt bridges at acidic pH clears the prosegment out of the proteinase's active site, leading to autocatalyzed activation. In proplasmepsin II, the method of inactivation is fundamentally different. In this case, the active site is accessible, but incompletely formed, and therefore unable to catalyze peptide bond hydrolysis.; We also investigated the activation of plasmepsin in another human malaria parasite, P. vivax, by determining the crystal structures of plasmepsin and proplasmepsin. P. vivax plasmepsin has the structure of atypical aspartic proteinase. P. vivax proplasmepsin possesses many of the structural features that were observed in P. falciparum proplasmepsin II, including the same method of inactivation. Thus, the domain shift that prevents the formation of a functional active site in the zymogen is established as a common mode of inactivation for the proplasmepsins.; The hemoglobinases of Plasmodium have been recognized as targets for antimalarial drug design, since inhibition of these enzymes kills the parasites in culture. Crystal structures of the proplasmepsins suggest that their unusual active site architecture may also render these zymogens potential drug design targets.