Influenza A virus is a major human and animal pathogen with the potential to cause catastrophic loss of life. The virus reproduces rapidly, mutates frequently and occasionally crosses species barriers. The recent emergence in Asia of avian influenza related to highly pathogenic forms of the human virus has highlighted the urgent need for new effective treatments. Here we demonstrate the importance to viral replication of a subunit interface in the viral RNA polymerase, thereby providing a new set of potential drug binding sites entirely independent of surface antigen type. No current medication targets this heterotrimeric polymerase complex. All three subunits, PB1, PB2 and PA, are required for both transcription and replication. PB1 carries the polymerase active site, PB2 includes the capped-RNA recognition domain, and PA is involved in assembly of the functional complex, but so far very little structural information has been reported for any of them. We describe the crystal structure of a large fragment of one subunit (PA) of influenza A RNA polymerase bound to a fragment of another subunit (PB1). The carboxy-terminal domain of PA forms a novel fold, and forms a deep, highly hydrophobic groove into which the amino-terminal residues of PB1 can fit by forming a 3(10) helix.

Influenza virus RNA-dependent RNA polymerase is a multi-functional heterotrimer, which uses a 'cap-snatching' mechanism to produce viral mRNA. Host cell mRNA is cleaved to yield a cap-bearing oligonucleotide, which can be extended using viral genomic RNA as a template. The cap-binding and endonuclease activities are only activated once viral genomic RNA is bound. This requires signalling from the RNA-binding PB1 subunit to the cap-binding PB2 subunit, and the interface between these two subunits is essential for the polymerase activity. We have defined this interaction surface by protein crystallography and tested the effects of mutating contact residues on the function of the holo-enzyme. This novel interface is surprisingly small, yet, it has a crucial function in regulating the 250 kDa polymerase complex and is completely conserved among avian and human influenza viruses.

Structures of nitric oxide reductase (NOR) in the ferric resting and the ferrous CO states have been solved at 2.0 A resolution. These structures provide significant new insights into how NO is reduced in biological systems. The haem distal pocket is open to solvent, implicating this region as a possible NADH binding site. In combination with mutagenesis results, a hydrogen-bonding network from the water molecule adjacent to the iron ligand to the protein surface of the distal pocket through the hydroxyl group of Ser 286 and the carboxyl group of Asp 393 can be assigned to a pathway for proton delivery during the NO reduction reaction.

Nitric oxide reductase from the denitrifying fungus Fusarium oxysporum catalyzes the reduction of NO to N 2 O [Nakahara, K., et al. J. Biol. Chem. 1993, 268, 8350-8355]. Since this enzyme belongs to the cytochrome P450 superfamily [Kizawa, H., et al. J. Biol. Chem. 1991, 266, 10632-10637], it is called cytochrome P450nor (P450nor), but does not exhibit monooxygenation activity. In the present study, we examine the coordination structure of the heme iron in P450nor in the ferric-NO form by using infrared, resonance Raman, and X-ray absorption (EXAFS ) extended X-ray absorption fine structure) spectroscopies, since the ferric-NO complex is a first intermediate in the NO reduction cycle by P450nor [Shiro, Y, et al J. Biol. Chem. 1995, 270, 1617-1623. We compared the data obtained with those for the d-camphor-bound form of Pseudomonas putida camphor hydroxylase cytochrome P450cam (P450cam), a typical model of the monooxygenase. From the vibrational spectroscopic measurements, we found that the Fe-bound N-O stretching frequency (ν(N-O)) occurred at 1851 cm -1 and the Fe-NO stretching frequency (ν(Fe-NO)) at 530 cm -1 for P450nor, while those at 1806 and 522 cm -1 were observed for P450cam, respectively. The assignments were confirmed by the 15 NO substituting effect on these vibrational frequencies. These results indicated that NO binds to the ferric iron in P450nor stronger than in P450cam. Support for this was provided from the EXAFS study, which gave an Fe-N NO bond distance of 1.66 ( 0.02 Å for P450nor and 1.76 ( 0.02 Å for P450cam. These spectroscopic results suggest that, compared with P450cam, the lower steric hindrance and/or the difference in the electrostatic interactions of the ligand NO with its surroundings facilitates the donation of the NO 2pπ* electron to the iron 3dπ orbital, resulting in the strengthening of the Fe-NO and the N-O bonds of P450nor. The vibrational spectral observation of the ferrous-CO complex of P450nor supported this suggestion. This configuration can reduce the electron density on the NO ligand in P450nor, which is seemingly relevant to the NO reduction reactivity of P450nor.