Functional characterization of the propeptide of Plasmodium falciparum subtilisin-like protease-1.
Erythrocyte invasion by the malaria merozoite is prevented by serine protease inhibitors. Various aspects of the biology of Plasmodium falciparum subtilisin-like protease-1 (PfSUB-1), including the timing of its expression and its apical location in the merozoite, suggest that this enzyme is involved in invasion. Recombinant PfSUB-1 expressed in a baculovirus system is secreted in the p54 form, noncovalently bound to its cognate propeptide, p31. To understand the role of p31 in PfSUB-1 maturation, we examined interactions between p31 and both recombinant and native enzymes. CD analyses revealed that recombinant p31 (rp31) possesses significant secondary structure on its own, comparable with that of folded propeptides of some bacterial subtilisins. Kinetic studies demonstrated that rp31 is a fast binding, high affinity inhibitor of PfSUB-1. Inhibition of two bacterial subtilisins by rp31 was much less effective, with inhibition constants 49-60-fold higher than that for PfSUB-1. Single (at the P4 or P1 position) or double (at P4 and P1 positions) point mutations of residues within the C-terminal region of rp31 had little effect on its inhibitory activity, and truncation of 11 residues from the rp31 C terminus substantially reduced, but did not abolish, inhibition. None of these modifications prevented binding to the PfSUB-1 catalytic domain or rendered the propeptide susceptible to proteolytic digestion by PfSUB-1. These studies provide new insights into the function of the propeptide in PfSUB-1 activation and shed light on the structural requirements for interaction with the catalytic domain.
Jean
Letitia
L
Parasitology and Physical Biochemistry, National Institute for Medical Research, Mill Hill, London NW7 1AA, United Kingdom.
Hackett
Fiona
F
Martin
Stephen R
SR
Blackman
Michael J
MJ
D016367:Baculoviridae / Q000235:genetics; D001665:Binding Sites; D002942:Circular Dichroism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011498:Protein Precursors / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D017433:Protein Structure, Secondary; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D013329:Structure-Activity Relationship; D013381:Subtilisins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D014162:Transfection
D004791:Enzyme Inhibitors; D011498:Protein Precursors; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D013381:Subtilisins; C403176:subtilisin-like protease 1, Plasmodium falciparum