A proline switch controls folding and domain interactions in the gene-3-protein of the filamentous phage fd.

The amino-terminal domains N1 and N2 of the gene-3-protein of phage fd form a bilobal structural and functional entity that protrudes from the phage tip. Domain N2 initiates the infection of Escherichia coli by binding to the F pilus. This binding results in the dissociation of the two domains and allows N1 to interact with the TolA receptor at the cell surface. The refolding of the N1-N2 fragment begins with the folding of domain N1, which takes a few milliseconds, followed by the folding of domain N2, which is complete within five minutes. The subsequent domain assembly is unusually slow and shows a time-constant of 6200 s at 25 degrees C. We found that the rate of this reaction is controlled by the trans to cis isomerization of the Gln212-Pro213 bond in the hinge subdomain of N2, a region that provides many interactions between N1 and N2 in the gene-3-protein. The substitution of Pro213 by Gly accelerated domain association 30-fold and revealed that the folding of the two individual domains and their assembly are indeed sequential steps in the refolding of the gene-3-protein. In the course of infection, the domains must separate to expose the binding site for TolA on domain N1. The kinetic block of domain reassembly caused by Pro213 isomerization could ensure that after the initial binding of N2 to the F pilus the open state persists until N1 and TolA are close enough for their mutual interaction. Pro213 isomerization might thus serve as a slow conformational switch in the function of the gene-3-protein.