Molecular reconstruction of flavocytochrome P450 BM3

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.

Bacillus megaterium flavocytochrome P450 BM3 (BM3) is a model for the P450 superfamily, and is a fusion enzyme formed by covalent linkage of a fatty acid hydroxylase P450 to a eukaryotic-like cytochrome P450 reductase (CPR). BM3 has enormous catalytic advantages over its membrane-bound eukaryotic P450/CPR counterparts, and has been extensively engineered to perform diverse chemical transformations. Crystallization of BM3 has proved impossible, although structures of its individual domains are now all solved. Reasons for its failure to crystallize likely relate to its dimeric state, domain flexibility and adventitious intra- and intermolecular disulfide bond formation in its FAD binding domain. To define structure of the catalytically relevant dimer of BM3, we will combine hydrodynamic, analytical and spectroscopic methods to identify the domain interactions that lead to dimer formation. Molecular constraints (using pulsed ELDOR [PELDOR] measurements to define interflavin radical, FAD-to-heme and substrate radical-to-substrate radical distances) will be established to enable us to define how domains fit in a molecular envelope (provided by small angle X-ray scattering [SAXS], analytical ultracentrifugation [AUC] and multiangle laser light scattering [MALLS] studies of BM3). Modelling will allow us to define likely configurations of domains that facilitate BM3 catalysis in the dimeric state, and models will be validated by mutagenesis/enzymatic analysis and protein crosslinking to establish dimer structure and BM3 electron transfer mechanism. Tools developed will then be applied to characterize the dimer in eukaryotic nitric oxide synthase (NOS), which shares BM3's flavocytochrome nature and inter-monomer electron transfer mechanism. Spin-labelled calmodulin (CaM) will be used in PELDOR studies to define CaM binding mode and influence on NOS domain organization. These studies will provide novel tools to address structural properties of multidomain proteins.

This research proposal aims to develop a new approach to determining structures of complex multidomain enzymes. Specifically, this will involve the application of a combination of spectroscopic (small angle X-ray scattering [SAXS] and pulsed electron-electron double resonance [PELDOR]), hydrodynamic (multi-angle laser light scattering [MALLS] and analytical ultracentrifugation [AUC]), structural (X-ray crystallography), mutagenesis and molecular modelling studies. The initial target enzyme for these studies is the biotechnologically important cytochrome P450-P450 reductase fusion enzyme flavocytochrome P450 BM3, a model system in the P450 superfamily of enzymes. Application of these methods to wild-type and mutant forms of the intact BM3 and its component domains (specifically, mutants that prevent non-specific inter- and intradomain disulfides) will enable us to define properties such as domain molecular shape, association state and cross-domain interactions, and (using PELDOR) distance constraints between radical states of cofactors, and between EPR active substrate molecules in the active site of the P450. The fact that BM3 is a dimer means that these distance measurements will provide important distance constraints that will go hand-in-hand with the other spectroscopic/hydrodynamic data to enable production of models of the structure of the BM3 dimer. Synergistic application of these methods will provide a new route to determining structures of multidomain enzymes that are refractory to crystallization, and could be widely adopted to provide accurate models for physiologically important enzymes. With a structure for BM3 established, we will validate the model by a combination of protein engineering and targeted chemical modification. We will then apply a similar strategy to generate a model structure of human iNOS, a calmodulin regulated flavocytochrome critical to human health.

Beneficiaries will include researchers (in academia and industry) working on complex multidomain enzymes, who could adopt similar strategies to enable enhanced knowledge of structure/mechanism in these systems. The approaches are applicable to a wide range of enzymes of biotechnological/biomedical interest and thus adoption of this systematic approach to their structural elucidation can provide novel insights by using internal cofactors forming radicals, or spin-labels attached to surface cysteines by mutagenesis, to provide important distance constraints to facilitate molecular modelling. In absence of crystal structures, the methods developed have multiple applications for e.g. academic researchers wishing to understand the structural basis for enzyme mechanism, and Pharma researchers striving to obtain data to enable specific therapeutic targeting. For the 3 human NOS isoforms, there is great interest in generating isoform specific drugs - which may be feasible by using our tool set to define differences in their structures. Development of such therapeutics for NOS enzymes would have widespread benefits for public health, and one could envisage a similar scenario for other such human targets. Understanding BM3 dimer structure will be critical to rationalise the electron transfer and regulatory mechanisms that makes it the fastest and most biotechnologically applied P450. This would have great potential for biotech companies to generate similar CPR-P450 fusion enzymes to enhance production of high value pharmaceuticals e.g. steroids, lipids. Knowledge of the structural constraints that enable efficient domain interactions and electron transfer in BM3 could be mimicked to generate more industrially applicable catalysts for cost-effective oxychemical production. Such commercial applications could be achieved in 3-5 years from recognition and adoption of our approaches. The project will provide excellent training in protein engineering and several modern structural methods to equip the PDRA with key skills to enhance their future employability.