Use of sequence duplication to engineer a ligand-triggered, long-distance molecular switch in T4 lysozyme

Abstract

We have designed a molecular switch in a T4 lysozyme construct that controls a large-scale translation of a duplicated helix. As shown by crystal structures of the construct with the switch on and off, the conformational change is triggered by the binding of a ligand (guanidinium ion) to a site that in the wild-type protein was occupied by the guanidino head group of an Arg. In the design template, a duplicated helix is flanked by two loop regions of different stabilities. In the ''on'' state, the N-terminal loop is weakly structured, whereas the C-terminal loop has a well defined conformation that is stabilized by means of nonbonded interactions with the Arg head group. The truncation of the Arg to Ala destabilizes this loop and switches the protein to the ''off'' state, in which the duplicated helix is translocated Ϸ20 Å. Guanidinium binding restores the key interactions, restabilizes the C-terminal loop, and restores the ''on'' state. Thus, the presence of an external ligand, which is unrelated to the catalytic activity of the enzyme, triggers the inserted helix to translate 20 Å away from the binding site. The results illustrate a proposed mechanism for protein evolution in which sequence duplication followed by point mutation can lead to the establishment of new function.T he ability to create and manipulate ligand-induced conformational changes is one of the major challenges in protein engineering and biotechnology. This ability demands a detailed understanding of the interplay between binding, structure, dynamics and energetics (1, 2). Several steps have been made toward developing possible ''nanoallostery'' modules, many cases of which have used a protein template already known to change conformation upon ligand binding. The approaches involve either mutating residues so that one state is preferentially stable over others (3-7), manipulating the binding specificity so that unnatural ligands can bind (8-10), or fusing the template to another protein that can sense the signal (11-13). Such approaches are subject to the structural and functional limitations imposed on the template during evolution. In an experiment notable for the use of different templates (14), allosteric switching was observed when two proteins (ubiquitin and barnase), neither of which undergoes conformational changes in its native form, were fused in such a way that the folding of one protein unfolded the other. However, the lack of a regulatory site that bound ligand(s) limited the ability to switch between the two states.In the present report, a nanostructural module was added to T4 lysozyme. Part of the module, a duplicated secondary structure element, switches conformation upon binding a ligand, in this case guanidinium ion. The ligand is unrelated to the function of the protein, but its binding induces a large-scale conformational change.The reference protein on which the design is based is designated L20 and has been described in ref. 15. In this protein, residues 40-50, corresponding to the B helix of T4 lysozyme, are duplicated ...

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