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 approximately 20 A. 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 A 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.

Fig. 1.

Details of the interactions that stabilize the loop at the C terminus of the duplicated helix. (a) L20 (the design template). (b) L20/R63A in the presence of guanidinium. Distances (black) are shown in Å; in green are the corresponding distances in the WT structure. The superimposed F_o – F_c difference map contoured at 3.3 σ (red) defines the position of the ligand.

Fig. 2.

(a) Superposition of liganded (red) on the unliganded (cyan) forms of L20/R63A. As representative examples, the alternative positions of Ser-44 are labeled. On the lower left and right are simulated-annealing omit maps (contoured at 1.1 σ) with backbone representations of the helix extended in both directions. (b) Detailed sketch showing the structures of the liganded (Upper) and the unliganded (Lower) forms. The “inserted” residues (Asn-40-Ile-50) are colored orange, and the “parent” residues (Asn-51-Ile-61, renumbered because of the 11-residue insert) are colored blue. The vertical bars connecting the two structures show the location of helix B in WT. In the presence of the guanidinium ion (Upper), the inserted helix (in orange) extends at its N terminus. In the absence of the ion (Lower), the inserted sequence occupies the position of helix B and the parent sequence extends the helix at its C terminus.

Fig. 3.

(a) Thermal factor profiles for the liganded (red) and the unliganded form (black). Some of the differences shown are presumably due to the different resolutions and different crystal packing. The most dramatic differences, however, are in the vicinity of the duplicated helix. The orange and blue bars indicate the duplicated sequence. (b) Comparison of the residue-accessible surface-area profiles of the liganded (red) and unliganded (black) structures. The orange and blue bars indicate the duplicated sequence. For parts of the protein away from the region of duplication, the two profiles are essentially identical. Within the region of duplication there are major differences highlighted in Inset (see text for discussion).