In the above article, we did not indicate a Sequence Read Archive (SRA) accession number for the DNA sequence data obtained to draw our conclusions. These data are available under the accession number SRP039006 at http://www.ncbi.nlm.nih.gov/sra/.

P450cam from Pseudomonas putida is the best-characterized member of the vast family of cytochrome P450s, and it has long been believed to have a more rigid and closed active site relative to other P450s. Here we report X-ray structures of P450cam crystallized in the absence of substrate and at high and low [K + ]. The camphor-free structures are observed in a distinct open conformation characterized by a water-filled channel created by the retraction of the F and G helices, disorder of the B′ helix and loss of the K + binding site. Crystallization in the presence of K + alone does not alter the open conformation, while crystallization with camphor alone is sufficient for closure of the channel. Soaking crystals of the open conformation in excess camphor does not promote camphor binding or closure, suggesting resistance to conformational change by the crystal lattice. This open conformation is remarkably similar to that seen upon binding large tethered substrates, showing that it is not the result of a perturbation by the ligand. Re-dissolved crystals of the open conformation are observed as a mixture of P420 and P450 forms, which is converted to the P450 form upon addition of camphor and K + . These data reveal that P450cam can dynamically visit an open conformation that allows access to the deeply buried active site without being induced by substrate or ligand.Cytochrome P450s are ubiquitous heme monooxygenases that activate O 2 for oxygen atom insertion into a wide variety of substrates (1). Over 10000 forms of P450s have been identified in bacteria, archaea, plants, fungi, and all higher eukaryotes (2). Examples include major drug metabolizing enzymes of the liver, and biosynthetic enzymes involved in steroid and prostaglandin pathways. These enzymes all share a common protein fold first observed for the camphor metabolizing P450cam from Pseudomonas putida (3,4).The large diversity in the specificity or promiscuity of P450s is believed to be due largely to variations in the structure of the substrate binding channel, which is defined by elements including the F and G helices, FG loop and regions near the B′ helix, which fold over and around the heme and the I helix to enclose the substrate and position it for attack by the ferryl heme center. Sequence variation of these elements result in P450s with structurally distinct substrate binding channels, and large differences in these regions have been observed in the structures of bacterial (5), microsomal (6), and mitochondrial P450s (7), supporting the view that the different conformations of the F, G and B′ helices are responsible for the large diversity in size, shape and specificity of the substrate binding cavity. † This work was supported by grant GM41049 from the NIH. ‡ The atomic coordinate and structures factors were deposited in the Protein Data Bank under the codes 3L61 (substrate-free, 200 mM P450cam is the most well-characterized of all P450s, and in many ways it has served as the archetypal model for all P450s, particularly those with high s...

Vaccine-elicited mAbs bind the HIV-1 Env CD4bs differently from broadly neutralizing infection-induced mAbs.

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