Structure of equine infectious anemia virus proteinase complexed with an inhibitor

Abstract

Equine infectious anemia virus (EIAV), the causative agent of infectious anemia in horses, is a member of the lentiviral family. The virus-encoded proteinase (PR) processes viral polyproteins into functional molecules during replication and it also cleaves viral nucleocapsid protein during infection. The X-ray structure of a complex of the I54G mutant of EIAV PR with the inhibitor HBY-793 was solved at 1.8 A resolution and refined to a crystallographic R-factor of 0.136. The molecule is a dimer in which the monomers are related by a crystallographic twofold axis. Although both the enzyme and the inhibitor are symmetric, the interactions between the central part of the inhibitor and the active site aspartates are asymmetric, and the inhibitor and the two flaps are partially disordered. The overall fold of EIAV PR is very similar to that of other retroviral proteinases. However, a novel feature of the EIAV PR structure is the appearance of the second a-helix in the monomer in a position predicted by the structural template for the family of aspartic proteinases. The parts of the EIAV PR with the highest resemblance to human immunodeficiency virus type 1 PR include the substrate-binding sites; thus, the differences in the specificity of both enzymes have to be explained by enzyme-ligand interactions at the periphery of the active site as well.

Expression, Characterisation and Mutagenesis of the Aspartic Proteinase from Equine Infectious Anaemia Virus

“…1) and the crystal structures that have now been elucidated for both enzymes [19,21,221, the residues in EIAV proteinase which form the S2fS2' pocket for substrate1 inhibitor (Fig. l), are Ala28, Thr30, Va132, Ile53, Ile89 from one monomer and Va156' contributed by the other subunit (Fig.…”

“…The structure solved for [GlyS4]proteinase complexed with the HBY-793 inhibitor [19] reveals that the Phe side chains of PlP1' of the inhibitor nestle very closely together with the large naphthaline rings of P3P3' respectively, with the P31P3' substituents fitting very snugly into a pocket consisting of the imino ring of Pr086', the side chain of Va187' and underlined with the side chains of Arg8' and Asp29 (Fig. 4).…”

“…The symmetrical inhibitor, A-76889 (compound 20, Appendix 1) has an identical structure from P2 to P2' but differs from HBY-793 in its P3 and P3' substituents (and in lacking the tert-butyl sulfone substituents in P4 and P4'); A-76889 was unable to inhibit EIAV wildtype or [Gly54]proteinases (as discussed in detail in [19]). The inhibitor A-77003 (compound 19 in Table 4 and Appendix 1) is identical to A-76889 apart from the stereochemistry of one of the central hydroxyl groups [21] and this too was ineffective as an inhibitor of EIAV wild-type or [Gly54]proteinases.…”

“…It thus appears that even with inhibitors such as A-77003 and A-76889, which are relatively longer than the other weak inhibitors listed in Table 4 and Appendix 1, that if the substituents in P3P3' are too small or flexible, they cannot make effective enough contacts with the enzyme to anchor themselves securely into the active site. (These aspects are considered in detail in [19]. )…”

“…In this case, with the P3' side chain oriented inwards to occupy the PI' position as in FIV proteinase, the lack of the 4-carbon side chain in [Gly54]proteinase may allow the edge of this naphthylalanine ring to be more exposed to solvent. This access will be closed off by the presence of an Ile side chain, as in FIV proteinase (which does have an Ile at the equivalent position [19,201).…”