S. Shane Taremi

Molecular views of viral polyprotein processing revealed by the crystal structure of the hepatitis C virus bifunctional protease-helicase

BACKGROUND Hepatitis C virus (HCV) currently infects approximately 3% of the worlds population. HCV RNA is translated into a polyprotein that during maturation is cleaved into functional components. One component, nonstructural protein 3 (NS3), is a 631-residue bifunctional enzyme with protease and helicase activities. The NS3 serine protease processes the HCV polyprotein by both cis and trans mechanisms. The structural aspects of cis processing, the autoproteolysis step whereby the protease releases itself from the polyprotein, have not been characterized. The structural basis for inclusion of protease and helicase activities in a single polypeptide is also unknown. RESULTS We report here the 2.5 A resolution structure of an engineered molecule containing the complete NS3 sequence and the protease activation domain of nonstructural protein 4A (NS4A) in a single polypeptide chain (single chain or scNS3-NS4A). In the molecule, the helicase and protease domains are segregated and connected by a single strand. The helicase necleoside triphosphate and RNA interaction sites are exposed to solvent. The protease active site of scNS3-NS4A is occupied by the NS3 C terminus, which is part of the helicase domain. Thus, the intramolecular complex shows one product of NS3-mediated cleavage at the NS3-NS4A junction of the HCV polyprotein bound at the protease active site. CONCLUSIONS The scNS3-NS4A structure provides the first atomic view of polyprotein cis processing. Both local and global structural rearrangements follow the cis cleavage reaction, and large segments of the polyprotein can be folded prior to proteolytic processing. That the product complex of the cis cleavage reaction exists in a stable molecular conformation suggests autoinhibition and substrate-induced activation mechanisms for regulation of NS3 protease activity.

Construction and characterization of a fully active PXR/SRC-1 tethered protein with increased stability

The nuclear xenobiotic receptor PXR is a ligand-inducible transcription factor regulating drug-metabolizing enzymes and transporters and a master switch mediating potentially adverse drug-drug interactions. In addition to binding a coactivator protein such as SRC-1, the C-terminal ligand-binding domain (LBD) is solely responsible for ligand recognition and thus the ligand-dependent downstream effects. In an effort to facilitate structural studies of PXR to understand and abolish the interactions between PXR and its ligands, several recombinant PXR/SRC-1 constructs were designed and evaluated for expression, stability and activity. Expression strategies employing either dual expression or translationally coupled bicistronic expression were found to be unsuitable for producing stable PXR in a stochiometric complex with a peptide derived from SRC-1 (SRC-1p). A single polypeptide chain encompassing PXR and SRC-1p tethered with a peptidyl linker was designed to promote intramolecular complex formation. This tethered protein was overexpressed as a soluble protein and required no additional SRC-1p for further stabilization. X-ray crystal structures in the presence and absence of the known PXR agonist SR-12813 were determined to high resolution. In addition, a circular dichroism-based binding assay was developed to allow rapid evaluation of PXR ligand affinity, making this tethered protein a convenient and effective reagent for the rational attenuation of drug-induced PXR-mediated metabolism.

Key steps in the structure-based optimization of the hepatitis C virus NS3/4A protease inhibitor SCH503034

Crystal structures of protease/inhibitor complexes guided optimization of the buried nonpolar surface area thereby maximizing hydrophobic binding. The resulting potent tripeptide inhibitor is in clinical trials.

Inactivation of the human cytomegalovirus protease by diisopropylfluorophosphate

Publisher Summary Cytomegalovirus (CMV) protease is a serine protease, and labeling with diisopropylfluorophosphate (DFP) has identified Ser132 as the active site serine. The structure of the CMV protease containing the diisopropylphosphorylserine at residue 132 (DIP-CMV protease) is likely to resemble that of the tetrahedral transition-state intermediate. As the structure of the DFP-treated serine proteases resembles that of the tetrahedral transition-state intermediate, and the inactivated enzyme would not be susceptible to autoproteolysis, production of DIP-CMV protease would be useful for structure based drug design. The concentrations of DFP sufficient to yield stoichiometric incorporation of inhibitor at the active site of CMV protease, also resulted in substantial incorporation of DIP at a second site or sites. This heterogeneous incorporation would preclude crystallographic studies. For this reason, this chapter has attempted to optimize the conditions for inactivation of CMV protease with DFP, to produce pure DIP-CMV protease with minimum second site incorporation. Initial studies indicated that there was no loss of protease activity in the control samples, even when incubated for 23 hours at 22°C. A 3.8 hour incubation was sufficient to completely inactivate 180 μM protease in the presence of 4.3 mM DFP, while a 2.4 fold excess of the inhibitor inactivated only 50% of the enzyme. This indicates that CMV protease is less reactive with DFP than trypsin or chymotrypsin, and requires an order of magnitude excess of the organophosphate to achieve complete inactivation.