Protein conformational switches or allosteric proteins play a key role in the regulation of many essential biological pathways. Nonetheless, the implementation of protein conformational switches in protein design applications has… Click to show full abstract
Protein conformational switches or allosteric proteins play a key role in the regulation of many essential biological pathways. Nonetheless, the implementation of protein conformational switches in protein design applications has proven challenging, with only a few known examples that are not derivatives of naturally occurring allosteric systems. We have discovered that the domain swapped (DS) dimer of hCRBPII undergoes a large and robust conformational change upon retinal binding, making it a potentially powerful template for the design of protein conformational switches. Atomic resolution structures of the apo- and holo- forms illuminate a simple, mechanical mechanism involving sterically driven torsion angle flipping of two residues that drive the motion. We further demonstrate that the con-formational "readout" can be altered by addition of cross-domain disulfide bonds, also visualized at atomic resolution. Finally, as a proof of principle, we have created an allosteric metal binding site in the DS dimer, where ligand binding results in a reversible five-fold loss of metal binding affinity. The high resolution structure of the metal-bound variant illustrates a well-formed metal binding site at the inter-face of the two domains of the DS dimer, and confirms the design strategy for allosteric regulation.
               
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