ABSTRACT
Blood-feeding exoparasites are rich sources of protease inhibitors, and the mosquito Aedes aegypti, which is a vector of Dengue virus, Yellow fever virus, Chikungunya virus and Zika virus, is no exception. AaTI is a single-domain, noncanonical Kazal-type serine proteinase inhibitor from A. aegypti that recognizes both digestive trypsin-like serine proteinases and the central protease in blood clotting, thrombin, albeit with an affinity that is three orders of magnitude lower. Here, the 1.4â Å resolution crystal structure of AaTI is reported from extremely tightly packed crystals (â¼22% solvent content), revealing the structural determinants for the observed inhibitory profile of this molecule.
Subject(s)
Aedes/chemistry , Insect Proteins/chemistry , Insect Vectors/chemistry , Serine Peptidase Inhibitors, Kazal Type/chemistry , Thrombin/chemistry , Aedes/metabolism , Amino Acid Sequence , Animals , Binding Sites , Cloning, Molecular , Crystallography, X-Ray , Gene Expression , Genetic Vectors/chemistry , Genetic Vectors/metabolism , Insect Proteins/genetics , Insect Proteins/metabolism , Insect Vectors/metabolism , Molecular Docking Simulation , Pichia/genetics , Pichia/metabolism , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Protein Multimerization , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Alignment , Sequence Homology, Amino Acid , Serine Peptidase Inhibitors, Kazal Type/genetics , Serine Peptidase Inhibitors, Kazal Type/metabolism , Thrombin/antagonists & inhibitors , Thrombin/genetics , Thrombin/metabolismABSTRACT
Purine nucleoside phosphorylase (PNP; EC 2.4.2.1) is a key enzyme of the purine-salvage pathway. Its ability to transfer glycosyl residues to acceptor bases is of great biotechnological interest owing to its potential application in the synthesis of nucleoside analogues used in the treatment of antiviral infections and in anticancer chemotherapy. Although hexameric PNPs are prevalent in prokaryotes, some microorganisms, such as Bacillus subtilis, present both hexameric and trimeric PNPs. The hexameric PNP from B. subtilis strain 168, named BsPNP233, was cloned, expressed and crystallized. Crystals belonging to different space groups (P32(1), P2(1)2(1)2(1), P6(3)22 and H32) were grown in distinct conditions with pH values ranging from 4.2 to 10.5. The crystals diffracted to maximum resolutions ranging from 2.65 to 1.70 Å.
Subject(s)
Bacillus subtilis/enzymology , Purine-Nucleoside Phosphorylase/chemistry , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/isolation & purification , Cloning, Molecular , Crystallization , Crystallography, X-Ray , Gene Expression , Models, Molecular , Protein Structure, Quaternary , Purine-Nucleoside Phosphorylase/genetics , Purine-Nucleoside Phosphorylase/isolation & purificationABSTRACT
HIV polymorphism is responsible for the selection of variant viruses resistant to inhibitors used in AIDS treatment. Knowledge of the mechanism of resistance of those viruses is determinant to the development of new inhibitors able to stop, or at least slow down, the disease's progress caused by new mutations. In this paper, the crystallization and preliminary crystallographic structure solution for two multi-resistant 99 amino acid HIV proteases, both isolated from Brazilian patients failing intensive anti-AIDS therapy are presented, viz. the subtype B mutant, with mutations Q7K, S37N, R41K, K45R, I54V, L63P, A71V, V82A and L90M, and the subtype F (wild type), naturally carrying mutations Q7K, I15V, E35D, M36I, S37N, R41K, R57K, D60E, Q61N, I62V, L63S, I64L and L89M, with respect to the B consensus sequence. Both proteins crystallized as a complex with the inhibitor TL-3 in space group P6(1)22. X-ray diffraction data were collected from these crystals to resolutions of 2.1 and 2.6 A for the subtype B mutant and subtype F wild type, respectively, and the enzyme structures were solved by molecular replacement. The crystals of subtype F HIV protease are, to the best of the authors' knowledge, the first protein crystals obtained for a non-B HIV protease.