Molecular reconstruction of flavocytochrome P450 BM3

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Determining the structure of biological molecules is critical for understanding their physiological function. Excellent examples include Crick and Watson's DNA structure, the structure of hemoglobin solved by Max Perutz and (more recently) the structures of the complex molecular machines (ATP synthase and the respiratory complexes) that facilitate energy generation from oxidation of foodstuffs (lipids, carbohydrates etc) to enable life. The major techniques used to determine the three dimensional organization of atoms in biological (and other) molecules are X-ray crystallography (diffraction of X-rays from crystals of the molecule, e.g. a protein) and nuclear magnetic resonance (NMR) that is used on molecules in solution. However, there are limitations to application of these methods for structural determination of large biomolecules (such as complex proteins and enzymes). Typically, determining structures of molecules larger in size than 30 kDa becomes challenging and expensive for NMR. While X-ray diffraction methods are more "tolerant" to the size of the molecule in question, it is absolutely dependent on ability to obtain crystals of the target molecule. This can be difficult (or impossible), and even if crystals are obtained they may not diffract X-rays sufficiently well to enable a structure to be solved. Crystals of large, complex proteins containing several subunits or individual structural segments (domains) are often difficult to crystallize, particularly if regions of the protein are highly mobile in solution.The subject of this proposal is an important member of the cytochrome P450 (P450) class of enzymes named flavocytochrome P450 BM3 (BM3). P450s are critical in human drug/xenobiotic detoxification, and play important roles in steroid metabolism. They do this by introducing oxygen atoms into their substrates. P450s are widely studied due to their importance in drug metabolism, as well as being important drug targets in their own right, but to date there are no true structural data to describe how P450s interact with their partner enzymes -notably cytochrome P450 reductase (CPR). In this work, we will apply a new combination of tools (spectroscopic, modelling, protein engineering and analytical methods) to define the structural organization of the BM3 enzyme - which is a "model" system in the P450 enzyme family, and which is also a natural "fusion enzyme" formed by direct linkage of a fatty acid metabolizing P450 to a CPR enzyme. While mammalian P450s and CPR are membrane associated, insoluble proteins, BM3 is fully soluble and can be prepared in large amounts for characterization. The BM3 protein has several domains and we have shown recently that it is functional as a dimer. BM3 can be broken down (by genetic engineering) into its individual domains (e.g. the CPR and P450 modules) and structures of its individual domains have been solved by X-ray crystallography. However, intact BM3 has been refractory to crystallography, for reasons described above. The crux of this proposal is to provide a structural model for the BM3 dimer through synergistic application of diverse spectroscopic, modelling and other methods to enable aspects of shape, domain mobility and inter-monomer distances to be defined, allowing the structure of the BM3 dimer to be reconstructed. The model will then be validated, and tools developed for BM3 will then be applied to another important enzyme - nitric oxide synthase (NOS) which has a similar dimeric architecture to BM3, but a more complicated mode of regulation due to interactions with the calcium binding protein calmodulin. NOS has also been refractory to crystallization, but is critical for human neurotransmission, immune function and blood pressure control. Collectively, these studies on BM3/NOS will provide a new route to determining the structural layout of complex multidomain proteins, as well as insights into how these heme-binding proteins interact with their linked CPR partner.