1 publication

1 publication

Allosteric Cooperation in a De Novo-Designed Two-Domain Protein

DeGrado, W.F.; Lombardi, A.

Proc. Natl. Acad. Sci. U.S.A. 2020, 117, 33246-33253, 10.1073/pnas.2017062117

We describe the de novo design of an allosterically regulated protein, which comprises two tightly coupled domains. One domain is based on the DF (Due Ferri in Italian or two-iron in English) family of de novo proteins, which have a diiron cofactor that catalyzes a phenol oxidase reaction, while the second domain is based on PS1 (Porphyrin-binding Sequence), which binds a synthetic Zn-porphyrin (ZnP). The binding of ZnP to the original PS1 protein induces changes in structure and dynamics, which we expected to influence the catalytic rate of a fused DF domain when appropriately coupled. Both DF and PS1 are four-helix bundles, but they have distinct bundle architectures. To achieve tight coupling between the domains, they were connected by four helical linkers using a computational method to discover the most designable connections capable of spanning the two architectures. The resulting protein, DFP1 (Due Ferri Porphyrin), bound the two cofactors in the expected manner. The crystal structure of fully reconstituted DFP1 was also in excellent agreement with the design, and it showed the ZnP cofactor bound over 12 Å from the dimetal center. Next, a substrate-binding cleft leading to the diiron center was introduced into DFP1. The resulting protein acts as an allosterically modulated phenol oxidase. Its Michaelis–Menten parameters were strongly affected by the binding of ZnP, resulting in a fourfold tighter Km and a 7-fold decrease in kcat. These studies establish the feasibility of designing allosterically regulated catalytic proteins, entirely from scratch.

Metal: Fe; Zn
Ligand type: Amino acid
Host protein: Due Ferri
Anchoring strategy: Amino acid
Optimization: ---
Max TON: 10
ee: ---
Notes: diFe-DFP3: Km 2.9 mM, kcat 0.7 min-1, 10 turnovers for 1 mM substrate, 20 uM protein. On binding ZnP, Km decreased 4x, and kcat decreased 7x, resulting in a lower kcat/Km overall.