De novo design of drug-binding proteins with predictable binding energy and specificity.

The de novo design of small molecule-binding proteins has seen exciting recent progress; however, high-affinity binding and tunable specificity typically require laborious screening and optimization after computational design. We developed a computational procedure to design a protein that recognizes a common pharmacophore in a series of poly(ADP-ribose) polymerase-1 inhibitors. One of three designed proteins bound different inhibitors with affinities ranging from <5 nM to low micromolar. X-ray crystal structures confirmed the accuracy of the designed protein-drug interactions. Molecular dynamics simulations informed the role of water in binding. Binding free energy calculations performed directly on the designed models were in excellent agreement with the experimentally measured affinities. We conclude that de novo design of high-affinity small molecule-binding proteins with tuned interaction energies is feasible entirely from computation.

Copper assisted sequence-specific chemical protein conjugation at a single backbone amide.

Direct, site-specific methods of protein functionalization are highly desirable for biotechnology. However, such methods are challenging due to the difficulty of chemically differentiating a single site within a large protein. Herein, we propose "metal binding targeting" strategy and develop a Copper Assisted Sequence-specific conjugation Tag (CAST) method to achieve rapid (second order rate 8.1 M-1 s-1), site-specific protein backbone chemical modification with pinpoint accuracy. We demonstrate the versatility of CAST conjugation by preparing various on-demand modified recombinant proteins, including a homogeneous antibody-drug conjugate with high plasma stability and potent efficacy in vitro and in vivo. Thus, CAST provides an efficient and quantitative method to site-specifically attach payloads on large, native proteins.

De novo design of drug-binding proteins with predictable binding energy and specificity.

The de novo design of small-molecule-binding proteins has seen exciting recent progress; however, the ability to achieve exquisite affinity for binding small molecules while tuning specificity has not yet been demonstrated directly from computation. Here, we develop a computational procedure that results in the highest affinity binders to date with predetermined relative affinities, targeting a series of PARP1 inhibitors. Two of four designed proteins bound with affinities ranging from < 5 nM to low µM, in a predictable manner. X-ray crystal structures confirmed the accuracy of the designed protein-drug interactions. Molecular dynamics simulations informed the role of water in binding. Binding free-energy calculations performed directly on the designed models are in excellent agreement with the experimentally measured affinities, suggesting that the de novo design of small-molecule-binding proteins with tuned interaction energies is now feasible entirely from computation. We expect these methods to open many opportunities in biomedicine, including rapid sensor development, antidote design, and drug delivery vehicles.

De novo metalloprotein design.

Natural metalloproteins perform many functions - ranging from sensing to electron transfer and catalysis - in which the position and property of each ligand and metal, is dictated by protein structure. De novo protein design aims to define an amino acid sequence that encodes a specific structure and function, providing a critical test of the hypothetical inner workings of (metallo)proteins. To date, de novo metalloproteins have used simple, symmetric tertiary structures - uncomplicated by the large size and evolutionary marks of natural proteins - to interrogate structure-function hypotheses. In this Review, we discuss de novo design applications, such as proteins that induce complex, increasingly asymmetric ligand geometries to achieve function, as well as the use of more canonical ligand geometries to achieve stability. De novo design has been used to explore how proteins fine-tune redox potentials and catalyse both oxidative and hydrolytic reactions. With an increased understanding of structure-function relationships, functional proteins including O2-dependent oxidases, fast hydrolases, and multi-proton/multi-electron reductases, have been created. In addition, proteins can now be designed using xeno-biological metals or cofactors and principles from inorganic chemistry to derive new-to-nature functions. These results and the advances in computational protein design suggest a bright future for the de novo design of diverse, functional metalloproteins.

De Novo Design, Solution Characterization, and Crystallographic Structure of an Abiological Mn-Porphyrin-Binding Protein Capable of Stabilizing a Mn(V) Species.

De novo protein design offers the opportunity to test our understanding of how metalloproteins perform difficult transformations. Attaining high-resolution structural information is critical to understanding how such designs function. There have been many successes in the design of porphyrin-binding proteins; however, crystallographic characterization has been elusive, limiting what can be learned from such studies as well as the extension to new functions. Moreover, formation of highly oxidizing high-valent intermediates poses design challenges that have not been previously implemented: (1) purposeful design of substrate/oxidant access to the binding site and (2) limiting deleterious oxidation of the protein scaffold. Here we report the first crystallographically characterized porphyrin-binding protein that was programmed to not only bind a synthetic Mn-porphyrin but also maintain binding site access to form high-valent oxidation states. We explicitly designed a binding site with accessibility to dioxygen units in the open coordination site of the Mn center. In solution, the protein is capable of accessing a high-valent Mn(V)-oxo species which can transfer an O atom to a thioether substrate. The crystallographic structure is within 0.6 Å of the design and indeed contained an aquo ligand with a second water molecule stabilized by hydrogen bonding to a Gln side chain in the active site, offering a structural explanation for the observed reactivity.

Coordination chemistry within a protein host: regulation of the secondary coordination sphere.

Secondary coordination spheres of metal complexes are instrumental in controlling properties that are linked to function. To study these effects in aqueous solutions artificial Cu proteins have been developed using biotin-streptavidin (Sav) technology and their binding of external azide ions investigated. Parallel binding studies were done in crystallo on single crystals of the artificial Cu proteins. Spectroscopic changes in solution are consistent with azide binding to the Cu centers. Structural studies corroborate that a Cu-N3 unit is present in each Sav subunit and reveal the composition of hydrogen bonding (H-bonding) networks that include the coordinated azido ligand. The networks involve amino acid residues and water molecules within the secondary coordination sphere. Mutation of these residues to ones that cannot form H-bonds caused a measurble change in the equilibrium binding constants that were measured in solution. These findings further demonstrate the utility of biotin-Sav technology to prepare water-stable inorganic complexes whose structures can be controlled within both primary and secondary coordination spheres.

Peroxide Activation Regulated by Hydrogen Bonds within Artificial Cu Proteins.

Copper-hydroperoxido species (CuII-OOH) have been proposed to be key intermediates in biological and synthetic oxidations. Using biotin-streptavidin (Sav) technology, artificial copper proteins have been developed to stabilize a CuII-OOH complex in solution and in crystallo. Stability is achieved because the Sav host provides a local environment around the Cu-OOH that includes a network of hydrogen bonds to the hydroperoxido ligand. Systematic deletions of individual hydrogen bonds to the Cu-OOH complex were accomplished using different Sav variants and demonstrated that stability is achieved with a single hydrogen bond to the proximal O-atom of the hydroperoxido ligand: changing this interaction to only include the distal O-atom produced a reactive variant that oxidized an external substrate.

Modular Artificial Cupredoxins.

Cupredoxins are electron-transfer proteins that have active sites containing a mononuclear Cu center with an unusual trigonal monopyramidal structure (Type 1 Cu). A single Cu-Scys bond is present within the trigonal plane that is responsible for its unique physical properties. We demonstrate that a cysteine-containing variant of streptavidin (Sav) can serve as a protein host to model the structure and properties of Type 1 Cu sites. A series of artificial Cu proteins are described that rely on Sav and a series of biotinylated synthetic Cu complexes. Optical and EPR measurements highlight the presence of a Cu-Scys bond, and XRD analysis provides structural evidence. We further provide evidence that changes in the linker between the biotin and Cu complex within the synthetic constructs allows for small changes in the placement of Cu centers within Sav that have dramatic effects on the structural and physical properties of the resulting artificial metalloproteins. These findings highlight the utility of the biotin-Sav technology as an approach for simulating active sites of metalloproteins.