Determining the Structural and Energetic Basis of Allostery in a De Novo Designed Metalloprotein Assembly

Abstract

Despite significant progress in protein design, the construction of protein assemblies that display complex functions (e.g., catalysis or allostery) remains a significant challenge. We recently reported the de novo construction of an allosteric supramolecular protein assembly (Zn-R1) in which the dissociation and binding of Zn ions were coupled over a distance of 15 Å to the selective hydrolytic breakage and formation of a single disulfide bond. Zn-R1 was constructed by Zn-templated assembly of a monomeric protein (R1, a derivative of cytochrome cb) into a tetramer, followed by progressive incorporation of noncovalent and disulfide bonding interactions into the protein-protein interfaces to create a strained quaternary architecture. The interfacial strain thus built allowed mechanical coupling between the binding/dissociation of Zn and formation/hydrolysis of a single disulfide bond (C38-C38) out of a possible six. While the earlier study provided structural evidence for the two end-states of allosteric coupling, the energetic basis for allosteric coupling and the minimal structural requirements for building this allosteric system were not understood. Toward this end, we have characterized the structures and Zn-binding properties of two related protein constructs (R1 and R1) which also possess C38-C38 disulfide bonds. In addition, we have carried out extensive molecular dynamics simulations ofR1 to understand the energetic basis for the selective cleavage of the C38-C38 disulfide bond upon Zn dissociation. Our analyses reveal that the local interfacial environment around the C38-C38 bond is key to its selective cleavage, but this cleavage is only possible within the context of a stable quaternary architecture which enables structural coupling between Zn coordination and the protein-protein interfaces.

Design and Construction of Functional Supramolecular Metalloprotein Assemblies

“…To understand the energetic basis of allosteric coupling in C38/C81/C96 R1 4 , we carried out extensive molecular dynamics calculations, which revealed that allostery was governed by an entropy/enthalpy trade-off . This trade-off primarily involves the hydrophobic i 1 interfaces, which rearrange upon Zn II dissociation to form favorable packing interactions that overcome the energetic cost incurred by reduced configurational freedom of the assembly.…”

An efficient, step-economical strategy for the design of functional metalloproteins

“…As this strategy is predicated upon the use of well-folded protein building blocks and natural amino acids, its application in the laboratory evolution of enzymatically-active metalloproteins operative in oxidative or hydrolytic processes can be readily envisioned. 35…”

Engineering protein assemblies with allosteric control via monomer fold-switching

“…The main advantage is, of course, that the assembly process becomes controllable by an external cue, for example, the concentration of a bivalent drug 23 , or even multiple cues, such as metal concentration, pH and chelators 21,22 , or metal identity, redox potential, and mechanical stress 28 . The combination of two of these mechanisms leads to even more sophisticated behavior, such as the re-creation of an allosteric transition by introducing structural strain between metal binding and disulfide bond formation on a designed protein tetramer 29,30 .…”