Gary S. Laco

Structural studies of FIV and HIV-1 proteases complexed with an efficient inhibitor of FIV protease

Three forms of feline immunodeficiency virus protease (FIV PR), the wild type (wt) and two single point mutants, V59I and Q99V, as well as human immunodeficiency virus type 1 protease (HIV‐1 PR), were cocrystallized with the C2‐symmetric inhibitor, TL‐3. The mutants of FIV PR were designed to replace residues involved in enzyme‐ligand interactions by the corresponding HIV‐1 PR residues at the structurally equivalent position. TL‐3 shows decreased (improved) inhibition constants with these FIV PR mutants relative to wt FIV PR. Despite similar modes of binding of the inhibitor to all PRs (from P3 to P3′), small differences are evident in the conformation of the Phe side chains of TL‐3 at the P1 and P1′ positions in the complexes with the mutated FIV PRs. The differences mimick the observed binding of TL‐3 in HIV‐1 PR and correlate with a significant improvement in the inhibition constants of TL‐3 with the two mutant FIV PRs. Large differences between the HIV‐1 and FIV PR complexes are evident in the binding modes of the carboxybenzyl groups of TL‐3 at P4 and P4′. In HIV‐1 PR:TL‐3, these groups bind over the flap region, whereas in the FIV PR complexes, the rings are located along the major axis of the active site. A significant difference in the location of the flaps in this region of the HIV‐1 and FIV PRs correlates with the observed conformational changes in the binding mode of the peptidomimetic inhibitor at the P4 and P4′ positions. These findings provide a structural explanation of the observed Ki values for TL‐3 with the different PRs and will further assist in the development of improved inhibitors. Proteins 2000;38:29–40. Published 2000 Wiley‐Liss, Inc.

Molecular replacement with pseudosymmetry and model dissimilarity: a case study.

Crystals of human T-cell leukemia virus protease (HTLV-1 PR) have been very difficult to prepare and only native data extending to 2.6 angstroms resolution could be collected. Initial attempts to solve the structure with a variety of low-sequence-identity models utilizing proteases from other retroviruses and using a number of molecular-replacement programs were unsuccessful. The structure was finally solved using Phaser, revealing extensive pseudosymmetry and significant deviations from the starting models, features that were likely to be responsible for the initial failures. The steps taken to solve this structure and some of its intriguing crystallographic aspects are discussed.

Bay-Region Diol Epoxides of Benzo[a]Pyrene are Stealth Poisons of Topoisomerase I

Abstract Topoisomerase I transiently cleaves one strand of duplex DNA to relieve torsional strain during enzymatic processing events such as transcription and replication. Thus, topoisomerase I is an important target for anti-tumor drugs. We have examined the effects of cis- and trans-opened (+)-7R,8S,9S,10R)- and (−)-(7S,8R,9R,10S)-benzo[a]pyrene 7,8-diol 9,10-epoxide adducts at the exocyclic amino groups of the purine bases at or near a known cleavage site in a 22-mer DNA duplex. The dG adducts lie in the minor groove either upstream or downstream from the modified base, and the dA adducts are intercalated at either side of the modified base. Thus four distinct structural motifs were available for study. The dG adducts inhibit cleavage at the normal site with resultant remote cleavages being observed. In contrast, three of the four dA adducts examined appear to be initially invisible to the enzyme since they allow the normal cleavage to occur, but they prevent subsequent religation and thus act as topoisomerase “stealth poisons”.

Structure of the D30N active site mutant of FIV proteinase complexed with a statine-based inhibitor

Publisher Summary It is believed that a substrate bound to the protease would form interactions similar to peptidomimetic inhibitors. However, as a substrate would be processed rapidly, no structure of a protease/substrate complex is available. As a first step in studying such interactions, a mutant of feline immunodeficiency virus protease (FIV PR) is expressed in which the catalytic Asp30 was mutated into an Asn, leading to inactive protease—designated as FIV PR(D30N) in the chapter. A complex between this mutant and a substrate should therefore be stable. To investigate the extent of perturbation of the active site of FIV PR caused by this mutation, the crystal structure of FIV PR(D30N) is determined at 2.0 A resolution in a complex with LP-149, a statine-based inhibitor. This structure is then compared with the structure of the wild type enzyme FIV PR(wt) complexed with the same inhibitor. The study described reveals that the mode of binding of LP-149 to FIV PR(D30N) is similar to the mode of binding of LP-149 to FIV PR(wt), making the mutant a valuable model to study the interactions of substrates with FIV PR, and retroviral proteases in general. The results described show that the crystal structure of the FIV PR(D30N)/LP-149 complex is very similar to that of the FIV PR(wt)/LP-149 complex. The position and orientation of the inhibitor molecule LP-149 in both proteins is nearly identical. Some hydrogen bond distances between the inhibitor and the active site pockets of FIV PR(D30N) have been found to be slightly different from those observed in the FIV PR(wt)/LP-149 complex. These differences are however, too small to affect the binding mode of the inhibitor in the mutated active site.