Allosteric cooperation in a de novo-designed two-domain protein

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

The emergence of life and the evolution of the three superkingdoms required the recombination of preexisting protein domains to perform ever-increasingly complex functions (1). The majority (∼90%) of multidomain proteins are made up of end-to-end linked domains; in the remaining ∼10% of cases, a domain insertion occurs, creating a continuous and a discontinuous domain (2, 3). In enzymes and small molecule binding proteins, the bilobed architecture facilitates the formation of active sites between individual domains; redox-active proteins often combine multiple domains to orient multiple cofactors for productive electron transfer; and allosterically regulated proteins combine binding domains with catalytic or signal-transducing domains (4). Therefore, the addition of a domain to an existing protein expands, alters, or modulates its functionality (5). Protein engineers have been inspired by this modularity to generate artificial multidomain proteins with improved properties or to create nanostuctured and sensor devices (6–10). Toward this end, different methodologies have been developed to fuse the different domains by: 1) introducing designed or naturally occurring peptide linkers (11); 2) superimposing and fusing one or two turns of terminal alpha helices of connecting helical proteins (12, 13); and 3) computationally designing new structural elements to interface the different domains in a fragment based approach (14). In each case, the domain architectures of the artificial multidomain proteins fell into either end-to-end or domain insertion topology. However, the de novo design and structure determination of allosterically regulated multidomain proteins (in which both domains are designed from scratch) have not been reported.Here, we describe the design of a protein that combines domains capable of binding ZnP, the Zn5,10,15,20-tetrakis(trifluoromethyl)porphinato], and diiron cofactors into a single tightly coupled framework. While multicofactor proteins have been widely used to explore redox coupling (15–22), high-resolution structures of multicofactor proteins have not been described in the literature, limiting what can be learned and achieved in such systems. The present work differs in two fundamental manners from earlier work on the design of multicofactor proteins. First, the goal of the present study was to examine how the binding of redox-inert ZnP cofactor allosterically modulates the catalytic activity of a second diiron-binding domain. Second, the structure of the designed multicofactor protein was determined by X-ray crystallography. It is also noteworthy that the computational methods, adopted in this work, could be readily extended to the design of electronically coupled systems for light-triggered electron energy storage and utilization, particularly given the ability to design proteins that incorporate metal ion clusters (15, 21, 23–31).The diiron-binding component of our two-domain protein is based on the DF family of de novo proteins (32, 33), which have been optimized to catalyze various two and four-electron reactions, including ferroxidase, oxidase, and monooxygenase activities (34–40). The second domain is based on PS1, which binds the synthetic ZnP (41). PS1 has a well-structured hydrophobic core, which positions the porphyrin-binding domain for productive interaction with this cofactor. The structure of PS1 has been solved by NMR in both the apo-bound and ZnP-bound state. The structures are nearly identical in the hydrophobic core, but the apo-protein is more open and flexible near the opposite end of the bundle. This structural transition allows the protein to bind the cofactor in an alligator-like chomping motion. We reasoned that such transition might be used to regulate the properties of the DF diiron site in the neighboring domain. Both DF and PS1 are four-helix bundles, so we envisioned a coaxial arrangement to facilitate interdomain communication, as in bacterial signaling proteins (42). However, DF and PS1 have distinct bundle architectures with respect to their interhelical packing, helical offsets, and helical registers, which together presented challenges for structural design. To address these challenges, we extended previous fragment-based approaches (43–48) and designed artificial multidomain proteins with allosterically communicating sites.