Metalloprotein and metallo-DNA/RNAzyme design: current approaches, success measures, and future challenges

Abstract

Specific metal-binding sites have been found in not only proteins but also DNA and RNA molecules. Together these metalloenzymes consist of a major portion of the enzyme family and can catalyze some of the most difficult biological reactions. Designing these metalloenzymes can be both challenging and rewarding because it can provide deeper insights into the structure and function of proteins and cheaper and more stable alternatives for biochemical and biotechnological applications. Toward this goal, both rational and combinatorial approaches have been used. The rational approach is good for designing metalloenzymes that are well characterized, such as heme proteins, while the combinatorial approach is better at designing those whose structures are poorly understood, such as metallo-DNA/RNAzymes. Among the rational approaches, de novo design is at its best when metal-binding sites reside in a scaffold whose structure has been designed de novo (e.g., alpha-helical bundles). Otherwise, design using native scaffolds can be equally effective, allowing more choices of scaffolds whose structural stability is often more resistant to multiple mutations. In addition, computational and empirical designs have both enjoyed successes. Because of the limitation in defining structural parameters for metal-binding sites, a computational approach is restricted to mostly metal-binding sites that are well defined, such as mono- or homonuclear centers. An empirical approach, even though it is less restrictive in the metal-binding sites to be designed, depends heavily on one's knowledge and choice of templates and targets. An emerging approach is a combination of both computational and empirical approaches. The success of these approaches can be measured not only by three-dimensional structural comparison between the designed and target enzymes but also by the total amount of insight obtained from the design process and studies of the designed enzymes. One of the biggest advantages of designed metalloenzymes is the potential of placing two different metal-binding sites in the same protein framework for comparison. A final measure of success is how one can utilize the insight gained from the intellectual exercise to design new metalloenzymes, including those with unprecedented structures and functions. Future challenges include designing more complex metalloenzymes such as heteronuclear metal centers with strong nanomolar or better affinities. A key to meeting this challenge is to focus on the design of not only primary but also secondary coordination spheres using a combination of improved computer programs, experimental design, and high-resolution crystallography.

Figure 1

Examples of metal-specific DNAzymes. (A) Mg²⁺-dependent “10–23” DNAzyme with RNA nuclease activity; B) Pb²⁺-dependent “8–17” DNAzyme with RNA nuclease activity; C) Cu²⁺-dependent DNAzyme with ligase activity; and D) DNAzyme that catalyzes porphyrin metallation. The letter “N” in the sequence represents any nucleotides capable for forming Watson-Crick base pairs. R=A or G, Y=C or T.

Figure 2

Two DNAzymes with different selectivity: Zn²⁺ selective (clone 18) and Co²⁺ selective (Clone 11). Predicted secondary structures of Clone 18 (A) and Clone 11 (B), and detailed sequence alignment in the sequence where it was randomized during the selection process (C).

Figure 3

The Greek key β-barrel scaffold has been utilized by at least 600 different types of proteins with diverse active site structures and functions.

Figure 4

The similarities between CcP and MnP in the overall structure (A) and in the active site structure (B); the difference between CcP and MnP active site structure (C) and Mn(II)-binding site (D). The pink ball in (A) represents Mn(II) while the blue ball in (A) represents Ca(II).

Figure 5

Structural comparison of the Co(II) derivative of a designed metal-binding site in CcP (blue) and Mn(II) center in MnP (red).

Figure 6

Schematic illustration of engineering the Cu_A center into azurin through loopdirected mutagenesis.

Figure 7

(A) Type 1 blue copper and replacement of cysteine and methionine with unnatural amino acids. (B). Type 2 copper protein CuZnSOD.

Figure 8

(A) Overlay of the crystal structure of WTMb (thin) and a structural model of Cu_BMb based on computer modeling and energy minimization (thick); (B) overlay of the crystal structure of heme-cooper center in CcO (thin) and the same structural model of Cu_BMb (thick) as in (A).

Figure 9

Crystal structures of Cu_A site in azurin and CcO as viewed from the Cu₂(S_cys)₂ plane (A), and perpendicular to the plane (B).

Figure 10

(A) Overlay of backbone structures of type 1 blue copper azurin and designed Cu_A azurin; (B) effects of methionine mutations on the reduction potential of the metal site in Cu_A azurin and type 1 blue copper azurin.

Figure 11

Designed artificial organometalloproteins. (A) A blue copper azurin containing a covalently attached ferrocene; (B) a Phe15Cys/Cys112Gly/Met121Gly variant of the blue copper azurin containing a covalently attached ferrocene.