7JH6

De novo designed two-domain di-Zn(II) and porphyrin-binding protein

Summary for 7JH6

• Entry DOI: 10.2210/pdb7jh6/pdb
• Descriptor: Two-domain di-Zn(II) and porphyrin-binding protein, ZINC ION, [5,10,15,20-tetrakis(trifluoromethyl)porphyrinato(2-)-kappa~4~N~21~,N~22~,N~23~,N~24~]zinc, ... (5 entities in total)
• Functional Keywords: metalloprotein, inter-domain coupling and cooperation, de novo protein design, allostery, synthetic porphyrin, de novo protein
• Biological source: synthetic construct
• Total number of polymer chains: 4
• Total formula weight: 86931.44

Authors

Schmidt, N.,Liu, L.,DeGrado, W.F. (deposition date: 2020-07-20, release date: 2020-12-09, Last modification date: 2024-04-03)

Primary citation

Pirro, F.,Schmidt, N.,Lincoff, J.,Widel, Z.X.,Polizzi, N.F.,Liu, L.,Therien, M.J.,Grabe, M.,Chino, M.,Lombardi, A.,DeGrado, W.F.
Allosteric cooperation in a de novo-designed two-domain protein.
Proc.Natl.Acad.Sci.USA, 117:33246-33253, 2020

PubMed Abstract: 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 and a 7-fold decrease in These studies establish the feasibility of designing allosterically regulated catalytic proteins, entirely from scratch.

PubMed: 33318174
DOI: 10.1073/pnas.2017062117

Experimental method

X-RAY DIFFRACTION (3.5 Å)