Background: Mycobacterial cytochrome P450 families 125 and 142 initiate metabolism of host cholesterol. Results: Family 142 enzymes were able to oxidize cholesterol esters, whereas those in family 125 were not. Conclusion: Structural differences impede cholesterol ester oxidation only in the 125 family. Significance: The ability of the 142 family to oxidize cholesterol esters gives Mycobacterium tuberculosis access to additional pools of intracellular cholesterol.

The receptor tyrosine kinase MET is a major component controlling the invasive growth program in embryonic development and in invasive malignancies. The discovery of therapeutic antibodies against MET has been difficult, and antibodies that compete with hepatocyte growth factor (HGF) act as agonists. By applying phage technology and cell-based panning strategies, we discovered two fully human antibodies against MET (R13 and R28), which synergistically inhibit HGF binding to MET and elicit antibody-dependent cellular cytotoxicity. Cell-based phosphorylation assays demonstrate that R13 and R28 abrogate HGF-induced activation of MET, AKT1, ERK1/2, and HGF-induced migration and proliferation. FACS experiments suggest that the inhibitory effect is mediated by "locking" MET receptor in a state with R13, which then increases avidity of R28 for the extracellular domain of MET, thus blocking HGF binding without activating the receptor. In vivo studies demonstrate that the combination of R13/28 significantly inhibited tumor growth in various colon tumor xenograft models. Inhibition of tumor growth was associated with induction of hypoxia. Global gene expression analysis shows that inhibition of HGF/MET pathway significantly upregulated the tumor suppressors KLF6, CEACAM1, and BMP2, the negative regulator of phosphatidylinositol-3-OH-kinase PIK3IP1, and significantly suppressed SCF and SERPINE2, both enhancers of proliferation and invasiveness. Moreover, in an experimental metastasis model, R13/28 increased survival by preventing the recurrence of otherwise lethal lung metastases. Taken together, these results underscore the utility of a dual-antibody approach for targeting MET and possibly other receptor tyrosine kinases. Our approach could be expanded to drug discovery efforts against other cell surface proteins.

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The crystal structure of the P450cin substrate-bound nitric oxide complex and the substrate-free form have been determined revealing a substrate-free structure that adopts an open conformation relative to the substrate-bound structure. The region of the I helix that forms part of the O2 binding pocket shifts from α helix in the substrate-free form to a π helix in the substrate-bound form. Unique to P450cin is an active site residue, Asn242, in the I helix that H-bonds with the substrate. In most other P450s this residue is a Thr and plays an important role in O2 activation by participating in an H-bonding network required for O2 activation. The π/α I helix transition results in the carbonyl O atom of Gly238 moving in to form an H-bond with the water/hydroxide ligand in the substrate-free form. The corresponding residue, Gly248, in the substrate-free P450cam structure experiences a similar motion. Most significantly, the oxy-P450cam complex Gly248 adopts a position midway between the substrate-free and -bound states. A comparison between these P450cam and the new P450cin structures provides insights into differences in how the two P450s activate O2. The structure of P450cin complexed with nitric oxide, a close mimic of the O2 complex, shows that Gly238 is likely to form tighter interactions with ligands than the corresponding Gly248 in P450cam. Having a close interaction between an H-bond acceptor, the Gly238 carbonyl O atom, and the distal oxygen atom of O2 will promote protonation and hence further reduction of the oxy-complex to the hydroperoxy intermediate resulting in heterolytic cleavage of the peroxide O-O bond and formation of the active ferryl intermediate required for substrate hydroxylation.

DosS is a sensor in Mycobacterium tuberculosis that differentially responds to O2, NO, and CO, as well as to changes in the redox state of the prosthetic heme iron atom. The ferrous protein and its Fe(II)-NO and Fe(II)-CO complexes undergo autophosphorylation and subsequently transfer the phosphate group to DosR, a nuclear factor, to activate it. In contrast, autophosphorylation is negligible with the ferric protein and the Fe(II)-O2 complex. To clarify the basis for this differential response to gases, we have determined the crystal structures of the NO- and CO-complexes of the DosS GAF-A domain, which contains the heme to which the gases bind. Comparison of these crystal structures with those reported for the phosphorylation-inactive ferric GAF-A domain suggest that the GAF-A domain is in a dynamic equilibrium between active and inactive states, and that the position of Glu87 in the heme cavity, which depends on the which gas is bound, acts as a modulator of the equilibrium, and therefore of catalytic activity.