Background: Steroidogenic cytochrome P450 17A1 (CYP17A1) performs hydroxylase and lyase reactions, with only the latter facilitated by cytochrome b 5 . Results: NMR mapping confirms the CYP17A1/b 5 interface and reveals substrate modulation of the interaction. Conclusion: Allosteric communication exists between the buried CYP17A1 active site and its peripheral b 5 binding site. Significance: The CYP17A1 reaction mechanism may be governed by proximal conformational control.

Background: Crystallography provides a static structure of cytochrome P450 17A1 (CYP17A1). Results: Solution NMR reveals an ensemble of CYP17A1 conformational substates. Conclusion: Ligand, cytochrome b 5 , or temperature alters the conformational CYP17A1 substates present. Significance: Changes in conformations probably modulate human steroidogenesis by CYP17A1.

Hantaviruses are distributed worldwide and can cause a hemorrhagic fever or a cardiopulmonary syndrome in humans. Mature virions consist of RNA genome, nucleocapsid protein, RNA polymerase, and two transmembrane glycoproteins, G1 and G2. The ectodomain of G1 is surface-exposed; however, it has a 142-residue C-terminal cytoplasmic tail that plays important roles in viral assembly and host-pathogen interaction. Here we show by NMR, circular dichroism spectroscopy, and mutagenesis that a highly conserved cysteine/histidine-rich region in the G1 tail of hantaviruses forms two CCHC-type classical zinc fingers. Unlike classical zinc fingers, however, the two G1 zinc fingers are intimately joined together, forming a compact domain with a unique fold. We discuss the implication of the hantaviral G1 zinc fingers in viral assembly and host-pathogen interaction.

The RNA virus that causes the Crimean Congo Hemorrhagic Fever (CCHF) is a tick-borne pathogen of the Nairovirus genus, family Bunyaviridae. Unlike many zoonotic viruses that are only passed between animals and humans, the CCHF virus can also be transmitted from human to human with an overall mortality rate approaching 30%. Currently, there are no atomic structures for any CCHF virus proteins or for any Nairovirus proteins. A critical component of the virus is the envelope Gn glycoprotein, which contains a C-terminal cytoplasmic tail. In other Bunyaviridae viruses, the Gn tail has been implicated in host-pathogen interaction and viral assembly. Here we report the NMR structure of the CCHF virus Gn cytoplasmic tail, residues 729 -805. The structure contains a pair of tightly arranged dual ␤␤␣ zinc fingers similar to those found in the Hantavirus genus, with which it shares about 12% sequence identity. Unlike Hantavirus zinc fingers, however, the CCHF virus zinc fingers bind viral RNA and contain contiguous clusters of conserved surface electrostatics. Our results provide insight into a likely role of the CCHF virus Gn zinc fingers in Nairovirus assembly.Recent outbreaks of the Crimean Congo Hemorrhagic Fever (CCHF) 2 virus along with the reported ability of the virus to transfer between humans have raised concerns of a widespread pandemic (1). The virus is transmitted to humans by tick bite or by direct handling of infected animal meat or blood (1, 2). Infection causes a hemorrhagic fever and myalgia resulting in mortality rates approaching 30% (1-3). The virus contains an antisense RNA genome divided into three segments, and named according to lengths as the S, M, and L (for Small, Medium, and Large) segments (4). The viral proteins are the nucleocapsid protein, two membrane glycoproteins Gn and Gc (also referred to as G1 and G2 in other Bunyaviridae) (5, 6), a nonstructural protein (NSm) (7), and an RNA polymerase (4). In the mature virion, the Gn glycoprotein contains a 176 residue ectodomain followed by a 24 residue transmembrane region and terminates in a long cytoplasmic tail consisting of ϳ100 residues (5, 7).Recent results from other related Bunyaviridae viruses suggest the role of the Gn tail in viral assembly. For example, alanine mutagenesis of the cytoplasmic tails of Uukuniemi virus (genus Phlebovirus) (8) and Bunyamwera virus (genus Orthobunyavirus) (9) affect the ability of virus-like particles (VLPs) to effectively incorporate ribonucleoproteins, thus intimating a role for Gn tails in genome packaging. More recently, the Gn tail of Puumala virus (genus Hantavirus) was shown to co-immunoprecipitate with the Puumala nucleocapsid protein (10). These results suggest that the CCHF virus Gn tail plays an equally important role in viral assembly of genus Nairovirus.The sequence of the CCHF virus cytoplasmic tail is somewhat variable in Nairoviruses (ϳ24% identity) and even more so when compared with other Bunyaviruses (12% identity with Hantavirus Gn tails). However, one characteristic feature present in ...

To accomplish key physiological processes ranging from drug metabolism to steroidogenesis, human microsomal cytochrome P450 enzymes require the sequential input of two electrons delivered by the FMN domain of NADPH-cytochrome P450 reductase. Although some human microsomal P450 enzymes can instead accept the second electron from cytochrome b 5 , for human steroidogenic CYP17A1, the cytochrome P450 reductase FMN domain delivers both electrons, and b 5 is an allosteric modulator. The structural basis of these key but poorly understood protein interactions was probed by solution NMR using the catalytically competent soluble domains of each protein. Numerous microsomal cytochrome P450 enzymes play key roles in human drug metabolism and the biosynthesis, interconversions, and degradation of hormones, vitamins, fatty acids, and bile acids. These reactions are all critically dependent on a single multidomain NADPH-cytochrome P450 oxidoreductase (CPR) 3 enzyme (1). CPR is required to donate the first electron required for cytochrome P450 catalysis, but frequently it also performs a second reduction of P450 required for substrate monooxygenation (2). The N terminus of CPR forms a membrane-spanning anchor that co-localizes reductase to the endoplasmic reticulum (3, 4) alongside microsomal P450 enzymes. The remainder of CPR consists of two flavin-binding domains separated by a linker domain and a flexible hinge. One domain of CPR contains the binding sites for the NADPH-reducing agent and the flavin adenine nucleotide (FAD) cofactor that initially accepts electrons from NADPH, whereas the N-terminal domain binds the flavin mononucleotide (FMN) cofactor that accepts electrons from FAD and transfers them to the P450. In initial structures of CPR (4), the FAD and FMN were closely associated in space, an arrangement supporting efficient FAD-to-FMN electron transfer, but a conformation that precludes FMN-to-P450 electron transfer. Later structures of a CPR mutant with a deletion in the linker region revealed several different conformations of CPR in which the FMN domain was exposed and would be available for electron delivery to P450 (5). It has been proposed that CPR oscillates between these states during its electron delivery cycle (5-9). Although interactions between CPR and a given P450 enzyme may involve some hydrophobic contributions (10), the primary factors are thought to consist of charge pairing interactions between the anionic surface of the FMN domain and cationic surface residues on the proximal side (nearest the axial heme coordination) of P450 enzymes (11-13).Such electrostatically mediated protein interactions and subsequent electron transfer from the CPR FMN domain to human cytochrome P450 17A1 (CYP17A1) (14, 15) support two distinct steroidogenic reactions as follows: 1) hydroxylation of pregnenolone or progesterone at the carbon 17 position; and 2) a 17,20-lyase reaction in which 17␣-hydroxypregnenolone is converted into the initial androgen, dehydroepiandrosterone (16). The former reaction is necessary for hu...