Use of sequence duplication to engineer a ligand-triggered, long-distance molecular switch in T4 lysozyme.
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.
Institute of Molecular Biology, Howard Hughes Medical Institute, and Department of Physics, University of Oregon, Eugene, OR 97403-1229, USA. brain@uoxray.uoregon.edu
Mohammad S
MS
Yousef
Walter A
WA
Baase
Brian W
BW
Matthews
D017122:Bacteriophage T4 / Q000201:enzymology; D001665:Binding Sites; D018360:Crystallography, X-Ray; D019020:Directed Molecular Evolution; D019791:Guanidine / Q000494:pharmacology; D008024:Ligands; D009113:Muramidase / Q000737:chemistry*; D044367:Phase Transition / Q000187:drug effects; D017354:Point Mutation; D011487:Protein Conformation / Q000187:drug effects; D015202:Protein Engineering / Q000379:methods*; D012091:Repetitive Sequences, Nucleic Acid
D008024:Ligands; D009113:Muramidase; D019791:Guanidine