The crystal structure of pentalenene synthase at 2.6 angstrom resolution reveals critical active site features responsible for the cyclization of farnesyl diphosphate into the tricyclic hydrocarbon pentalenene. Metal-triggered substrate ionization initiates catalysis, and the alpha-barrel active site serves as a template to channel and stabilize the conformations of reactive carbocation intermediates through a complex cyclization cascade. The core active site structure of the enzyme may be preserved among the greater family of terpenoid synthases, possibly implying divergence from a common ancestral synthase to satisfy biological requirements for increasingly diverse natural products.

Pharmacological activation of the STING (stimulator of interferon genes)–controlled innate immune pathway is a promising therapeutic strategy for cancer. Here we report the identification of MSA-2, an orally available non-nucleotide human STING agonist. In syngeneic mouse tumor models, subcutaneous and oral MSA-2 regimens were well tolerated and stimulated interferon-β secretion in tumors, induced tumor regression with durable antitumor immunity, and synergized with anti–PD-1 therapy. Experimental and theoretical analyses showed that MSA-2 exists as interconverting monomers and dimers in solution, but only dimers bind and activate STING. This model was validated by using synthetic covalent MSA-2 dimers, which were potent agonists. Cellular potency of MSA-2 increased upon extracellular acidification, which mimics the tumor microenvironment. These properties appear to underpin the favorable activity and tolerability profiles of effective systemic administration of MSA-2.

Transition metal binding sites in proteins are typically comprised of 3-4 protein ligands, most of which are also embedded in hydrogen bond networks. For instance, in human carbonic anhydrase II (CAII) the carboxamide side chain of Q92 accepts a hydrogen bond from H94, the carboxylate side chain of E l 17 accepts a hydrogen bond from H119, and the backbone carbonyl oxygen of N244 accepts a hydrogen bond from H96. In order to probe the structural importance of these hydrogen bond networks, we have determined the three-dimensional structures of Q92A, Q92N, Q92E, Q92L, and E117A CAIIs by X-ray crystallographic methods. When interpreted in light of functional measurements (catalytic activity, protein-zinc affhity) made by Kiefer and colleagues (Kiefer, L. L.; Paterno, S. A,; Fierke, C. A. J. Am. Chem. SOC., preceding paper in this issue), these high-resolution structures allow for detailed structure-function correlations which illuminate the general role of hydrogen bond networks with the second shell of residues surrounding protein-metal binding sites. Due to their structural and electrostatic contributions, these second shell residues, Le., "indirect" metal ligands, fine-tune the pKa and reactivity of zinc-bound solvent;additionally, these residues may contribute a factor of up to lo4 to protein-metal affinity in a tetracoordinate metal site. It is therefore imperative that indirect metal ligands be considered in de novo designs of avid protein-metal binding sites. Additionally, indirect ligand-direct ligand-metal networks are important for protein-nucleic acid recognition, e.g., in the C2H2 class of zinc-finger transcription factors.

Nonstructural protein 5B (NS5B) of hepatitis C virus (HCV) possesses an RNA-dependent RNA polymerase activity responsible for viral genome RNA replication. Despite several reports on the characterization of this essential viral enzyme, little is known about the reaction pathway of NS5B-catalyzed nucleotide incorporation due to the lack of a kinetic system offering efficient assembly of a catalytically competent polymerase/template/ primer/nucleotide quaternary complex. In this report, specific template/primer requirements for efficient RNA synthesis by HCV NS5B were investigated. For intramolecular copy-back RNA synthesis, NS5B utilizes templates with an unstable stem-loop at the 3 terminus which exists as a single-stranded molecule in solution. A template with a stable tetraloop at the 3 terminus failed to support RNA synthesis by HCV NS5B. Based on these observations, a number of single-stranded RNA templates were synthesized and tested along with short RNA primers ranging from two to five nucleotides. It was found that HCV NS5B utilized di-or trinucleotides efficiently to initiate RNA replication. Furthermore, the polymerase, template, and primer assembled initiation-competent complexes at the 3 terminus of the template RNA where the template and primer base paired within the active site cavity of the polymerase. The minimum length of the template is five nucleotides, consistent with a structural model of the NS5B/RNA complex in which a pentanucleotide single-stranded RNA template occupies a groove located along the fingers subdomain of the polymerase. This observation suggests that the initial docking of RNA on NS5B polymerase requires a single-stranded RNA molecule. A unique ␤-hairpin loop in the thumb subdomain may play an important role in properly positioning the single-stranded template for initiation of RNA synthesis. Identification of the template/primer requirements will facilitate the mechanistic characterization of HCV NS5B and its inhibitors.Infection by hepatitis C virus (HCV) is a significant human medical problem. HCV is recognized as the causative agent for most cases of non-A and non-B hepatitis (8), with an estimated prevalence of 170 million cases (i.e., 2 to 3%) globally (36). Four million individuals may be infected in the United States alone. Upon first exposure to HCV only about 10% of infected individuals develop acute clinical hepatitis, while others appear to resolve the infection spontaneously. In the most instances, however, the virus establishes a chronic infection that persists for decades. This usually results in recurrent and progressively worsening liver inflammation, which often leads to more severe disease states such as cirrhosis and hepatocellular carcinoma (31, 32). Currently, there are no broadly effective treatments for the debilitating progression of chronic HCV.HCV is an enveloped positive-stranded RNA virus belonging to the Flaviviridae family (24). The HCV genome encodes a polyprotein of 3,010 to 3,033 amino acids (30). The nonstructural (NS) proteins and the cataly...

Various classes of nucleotidyl polymerases with different transcriptional roles contain a conserved core structure. Less is known, however, about the distinguishing features of these enzymes, particularly those of the RNA-dependent RNA polymerase class. The 1. 9 A resolution crystal structure of hepatitis C virus (HCV) nonstructural protein 5B (NS5B) presented here provides the first complete and detailed view of an RNA-dependent RNA polymerase. While canonical polymerase features exist in the structure, NS5B adopts a unique shape due to extensive interactions between the fingers and thumb polymerase subdomains that serve to encircle the enzyme active site. Several insertions in the fingers subdomain account for intersubdomain linkages that include two extended loops and a pair of antiparallel alpha-helices. The HCV NS5B apoenzyme structure reported here can accommodate a template:primer duplex without global conformational changes, supporting the hypothesis that this structure is essentially preserved during the reaction pathway. This NS5B template:primer model also allows identification of a new structural motif involved in stabilizing the nascent base pair.