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...

Metabolic deactivation of 1,25(OH) 2 D3 is initiated by modification of the vitamin-D side chain, as carried out by the mitochondrial cytochrome P450 24A1 (CYP24A1). In addition to its role in vitamin-D metabolism, CYP24A1 is involved in catabolism of vitamin-D analogs, thereby reducing their efficacy. CYP24A1 function relies on electron transfer from the soluble ferredoxin protein adrenodoxin (Adx). Recent structural evidence suggests that regioselectivity of the CYP24A1 reaction may correlate with distinct modes of Adx recognition. Here we used nuclear magnetic resonance (NMR) spectroscopy to monitor the structure of 15 N-labeled full-length Adx from rat while forming the complex with rat CYP24A1 in the ligand-free state or bound to either 1,25(OH) 2 D3 or the vitamin-D supplement 1a(OH)D3. Although both vitamin-D ligands were found to induce a reduction in overall NMR peak broadening, thereby suggesting ligand-induced disruption of the complex, a crosslinking analysis suggested that ligand does not have a significant effect on the relative association affinities of the redox complexes. However, a key finding is that, whereas the presence of primary CYP24A1 substrate was found to induce NMR peak broadening focused on the putative recognition site a-helix 3 of rat adrenodoxin, the interaction in the presence of 1a(OH)D3, which is lacking the carbon-25 hydroxyl, results in disruption of the NMR peak broadening pattern, thus indicating a ligand-induced nonspecific protein interaction. These findings provide a structural basis for the poor substrate turnover of side-chain-modified vitamin-D analogs, while also confirming that specificity of the CYP24A1-ligand interaction influences specificity of CYP24A1-Adx recognition. SIGNIFICANCE STATEMENTMitochondrial cytochrome P450 enzymes, such as CYP24A1 responsible for catabolizing vitamin-D and its analogs, rely on a protein-protein interaction with a ferredoxin in order to receive delivery of the electrons required for catalysis. In this study, we demonstrate that this protein interaction is influenced by the enzyme-ligand interaction that precedes it. Specifically, vitamin-D missing carbon-25 hydroxylation binds the enzyme active site with high affinity but results in a loss of P450-ferredoxin binding specificity.

Tuberculosis is caused by the pathogenic bacterium Mycobacterium tuberculosis (Mtb) and remains the leading cause of death by infection world-wide. The Mtb genome encodes a disproportionate number of twenty cytochrome P450 enzymes, of which the essential enzyme cytochrome P450 121A1 (CYP121A1) remains a target of drug design efforts. CYP121A1 mediates a phenol coupling reaction of the tyrosine dipeptide cyclo-L-Tyr-L-Tyr (cYY). In this work, a structure and function investigation of dimerization was performed as an overlooked feature of CYP121A1 function. This investigation showed that CYP121A1 dimers form via intermolecular contacts on the distal surface and are mediated by a network of solvent-exposed hydrophobic residues. Disruption of CYP121A1 dimers by site-directed mutagenesis leads to a partial loss of specificity for cYY, resulting in an approximate 75% decrease in catalysis. 19F labeling and nuclear magnetic resonance of the enzyme FG-loop was also combined with protein docking to develop a working model of a functional CYP121A1 dimer. The results obtained suggest that participation of a homodimer interface in substrate selectivity represents a novel paradigm of substrate binding in CYPs, while also providing important mechanistic insight regarding a relevant drug target in the development of novel anti-tuberculosis agents.

This report summarizes a symposium sponsored by the American Society for Pharmacology and Experimental Therapeutics at Experimental Biology held April 20-24 in Boston, MA. Presentations discussed the status of cytochrome P450 (P450) knowledge, emphasizing advances and challenges in relating structure with function and in applying this information to drug design. First, at least one structure of most major human drugmetabolizing P450 enzymes is known. However, the flexibility of these active sites can limit the predictive value of one structure for other ligands. A second limitation is our coarse-grain understanding of P450 interactions with membranes, other P450 enzymes, NADPH-cytochrome P450 reductase, and cytochrome b 5 . Recent work has examined differential P450 interactions with reductase in mixed P450 systems and P450:P450 complexes in reconstituted systems and cells, suggesting another level of functional control. In addition, protein nuclear magnetic resonance is a new approach to probe these protein/protein interactions, identifying interacting b 5 and P450 surfaces, showing that b 5 and reductase binding are mutually exclusive, and demonstrating ligand modulation of CYP17A1/b 5 interactions. One desired outcome is the application of such information to control drug metabolism and/or design selective P450 inhibitors. A final presentation highlighted development of a CYP3A4 inhibitor that slows clearance of human immunodeficiency virus drugs otherwise rapidly metabolized by CYP3A4. Although understanding P450 structure/function relationships is an ongoing challenge, translational advances will benefit from continued integration of existing and new biophysical approaches.