Microsomal P450s use specific proline-rich sequences for efficient folding, but not for maintenance of the folded structure

The amino-terminal region of microsomal P450s contains three distinct seque nce moths, the signal-anchor sequence (SA), the basic sequence (BS), and th e proline-rich sequence (PR). Studies with two P450s of the CYP2C subfamily , P4502C11 (CYP2C11) and P4502C2 (CYP2C2), have indicated that upon express ion in eukaryotic cells (yeast, COS cells, and insect cells), specific prol ine residues in PR are important for proper folding. In the present study, we have established that the PR region in a very different CYP gene family, P450c17 (CYP17), is also important for efficient folding. These studies ha ve been carried out using expression in Escherichia coli. Using P4502C11, w e have established that the folding requirements for P450s in bacteria are very similar to those in eukaryotic cells. Interestingly, when the PR from P450c17 is swapped for that of P4502C11 and visa versa, complete misfolding is observed. However, both the BS and SA can be swapped between these P450 s without affecting folding. After proper folding of P450c17, removal of th e PR by factor Xa protease has no effect on the maintenance of the P450 str ucture. Inspection of the sequences of many different CYP gene families ind icates that the PR sequence is conserved within a gene family but varies co nsiderably between families. We conclude that PR is important for directing the folding pathway leading to the functional P450, but not for maintainin g the functional form.