UC San Diego

Metal ions are indispensable to biological function, as they are utilized for a myriad of processes encompassing signaling, electron transfer, and catalysis. These functions are made possible by exploiting intrinsic properties of the metal ions and the ligands to which they bind, represented by small molecules and metalloproteins. From the perspective of a synthetic chemist, proteins can be conceived as “macromolecular ligands”. Like those of small molecule ligands, the properties of “macromolecular ligands” can be parameterized at two discrete coordination spheres. The primary sphere is composed of the amino acid residues and exogenous ligands that coordinate the metal ion, and thus has the most direct impact on metal-based function. The secondary sphere is composed of residues that form mainly non-covalent interactions—hydrogen bonding, hydrophobic, and van der Waals (vDW) interactions—with the primary sphere. Although secondary sphere features do not directly bind the metal ion, they play an indispensable role in controlling metal-based reactivity. For example, the oxygen-binding affinity of myoglobin is influenced significantly by hydrogen bonding interactions between a histidine residue in its secondary sphere and the oxygen ligand that binds the heme co-factor. The large functional scope of metalloproteins is a testament to the wide diversity of primary and secondary spheres, and the efficiency with which metalloproteins execute these functions is a testament to the intricate interplay between the spheres. The functional potential of natural metalloproteins has long inspired protein design efforts. Two major metalloprotein design approaches are rational and de novo design. In rational design, primary and/or secondary sphere features of a natural protein are repurposed to mediate non-native functions. This approach has culminated in the engineering of new-to-nature functions spanning metal-hydride mediated ketone reduction and carbon-silicon bond coupling. While rational design represents an effective path to engineer metal-based functions, such functions are achieved within rigid and highly evolved protein folds/interfaces. Therefore, important questions remain unanswered: how does metal-based function emerge from an initially nonfunctional metalloprotein, and what are the minimum primary/secondary sphere coordination requirements to achieve metal-based functions? The work described in this dissertation stems from a de novo design approach in which both the quaternary structure and metal coordination site(s) of a metalloprotein are designed from scratch. The building block of our designed metalloprotein structures is cytochrome cb562, a natively monomeric, four-helix bundle protein. With cytochrome cb562 as our starting point, we obtain a diverse array of metalloprotein assemblies that serve as platforms for pursuing complex metal-based functions encompassing redox-based signaling and oxygen activation. In Chapter 2, we describe the sequential design of three metalloprotein trimers, TriCyt1, TriCyt2, and TriCyt3. TriCyt1, our initial trimeric construct, is obtained through a single hydrophobic mutation (G70W) at the interface of a cytochrome cb562 variant. Solution experiments indicated that TriCyt1 assembled as a trimer in the presence of all mid-to-late first row transition metals (MnII-ZnII), with yields ranging from 12% (+MnII) to 89% (+CoII). Crystal structures of NiII and CuII-supplemented TriCyt1 revealed a biologically rare His6 coordination motif. The only known natural His6 motif is present in calprotectin, a metal sequestering protein that coordinates MnII with nanomolar affinity. Motivated by the prospect of obtaining from scratch a MnII:His6 site of such high affinity, which had not yet been achieved in protein design, we sought to increase the preorganization of this metal coordination site through redesign of the C2 and C3 interfaces of TriCyt1. Computationally prescribed, mostly hydrophobic mutations at the C3 interface led to TriCyt2, a construct that trimerized with near-quantitative yield in the presence of MnII. A second round of redesign, in which salt bridges were installed at the C2 interface, led to a metal-independent, pH-switchable trimer which bound MnII with ~ 50 nM affinity. While a His6 primary sphere is suitable for achieving high affinity metal binding sites, our ability to design metalloproteins with broad functional potential hinges on our ability to engineer multiple types of metal binding sites. In Chapter 3, we describe our efforts to diversify the primary and/or secondary spheres of the TriCyt scaffold in pursuit of metalloprotein constructs which could coordinate lanthanides with high affinity and selectivity, mediate metal-dependent catalysis, and stabilize multinuclear metal coordination sites. The TriCyt series illustrates metal-ion-identity-independent assembly: the assembly path converges on the same architecture with the same metal coordination motif regardless of the metal ion added. In the TriCyt series there is low cooperativity between metal-ligand and non-covalent interactions, as metal coordination preferences play a minimal role in directing assembly. In Chapter 4, we describe our design and characterization of a variant of Rosetta interface design cytochrome 1 (A74/C96RIDC1) in which the cooperative interplay between metal-ligand and non-covalent interactions could be tuned by the redox state of the protein. In the oxidized state, C96-C96 disulfide bonds rigidify a tetrameric architecture (A74/C96RIDC14ox) whose assembly is independent of metal ion identity. Chemical reduction of the disulfide bonds gives rise to A74/C96RIDC1red, whose assembly is governed in larger part by the metal coordination preferences of exogenous metal ions. Whereas A74/C96RIDC14ox can only access two structural states (apo and metal-bound), A74/C96RIDC1red can access three distinct, metal-ion-identity dependent structural states with unique coordination environments. With five structural states whose accessibility is dependent on solution redox potential and metal ion identity, A74/C96RIDC1 represents a rare protein construct whose oligomerization path is varied through two different types of stimuli. We characterize the oligomeric and conformational states of A74/C96RIDC1 under disulfide-oxidized and disulfide-reduced states and in the presence of different mid-to-late first row transition metal ions, both in solution and via X-ray crystallography. We also employ density functional theory (DFT) and Rosetta interface calculations to attempt to quantify the energetic contributions of metal-ligand and non-covalent interactions and rationalize how differences in metal ion identity result in divergent metal-directed assembly paths.