Engineering an artificial zymogen by alternate frame protein folding

Abstract

Alternate frame folding (AFF) is a novel mechanism by which allostery can be introduced into a protein where none may have existed previously. We employ this technology to convert the cytotoxic ribonuclease barnase into an artificial zymogen that is activated by HIV-1 protease. The AFF modification entails partial duplication of the polypeptide chain and mutation of a key catalytic residue in one of the duplicated segments. The resulting molecule can fold in one of two "frames" to yield the wild-type structure or a circularly permuted form in which the positions of the N-and C-termini are exchanged with a surface loop. It cannot take on both structures simultaneously because each competes for a shared amino acid sequence. An HIV-1 protease recognition sequence is inserted into one of the surface loops in the nonpermuted frame, and cleavage induces a shift from the nonpermuted fold to the permuted fold. Using the AFF mechanism, we were able to suppress k cat ∕K M by 250-fold in the proenzyme relative to wild-type barnase. HIV-1 protease cleavage subsequently increases k cat ∕K M by 130-fold. AFF is significant because it is general and can in principle be used to control activity of many enzymes, including those whose functions are not regulated by any existing mechanism.mutually exclusive folding | design | HIV protease | molecular switch | barnase Z ymogens are inactive enzyme precursors that are activated by proteolytic cleavage. Natural zymogens play essential roles in apoptosis, blood coagulation, digestion, viral maturation, and other cellular processes. Artificial zymogens are of broad interest, with potential applications that include diagnostic tools and toxins that attack pathogens or diseased cells. Here we introduce a unique technique (alternate frame protein folding, or AFF) to convert the cytotoxic ribonuclease barnase (Bn) into an artificial zymogen that is activated by HIV-1 protease (PR). The significance of AFF is that it is general and can thus be applied to many enzymes, including those whose activities are not regulated by any existing mechanism.Several groups have previously engineered artificial zymogens. Raines and colleagues circularly permuted ribonuclease A (RNase A) and bridged the original N-and C-termini with a peptide linker that contained cleavage sequences for PR, the NS3 protease from hepatitis C, and plasmepsin II from Plasmodium falciparium (1-3). The linkers inhibited enzymatic activity by partially occluding the active site. Cleavage with the respective protease increased k cat ∕K M by approximately 100-fold. In a recent study we took a related approach by permuting Bn with a short linker peptide intended to force the termini together, thereby distorting the active site or unfolding the enzyme (4). Cleaving the linker with a chemical reagent relieved conformational strain and increased RNase activity. Although these strategies successfully created artificial zymogens, they rely on specific properties of the target enzyme and are therefore not general. Cyclic permutation typic...

Structural Characterization of Two Alternate Conformations in a Calbindin D9k-Based Molecular Switch

“…This order–disorder transition can be harnessed to a fluorescent 1,2 or functional output 3 to report on ligand binding or regulate the function of an enzyme, respectively. The AFF mechanism is significant because the nature of the N to N′ conformational change is expected to be similar for other proteins to which it is applied.…”

Protease-based synthetic sensing and signal amplification

“…The signal generated by protease-based signal sensors, transducers, and proximity sensors can be connected to practically any type of output, e.g., activation of natural or artificially engineered zymogens (32,33), including previously developed proteaseinducible reporter systems such as BLIP-β-lactamase fusion proteins (19,20). Because only modest affinities and specificities of protein or peptide binders are required for the construction of artificial zymogens, their development is likely to be straightforward, and neither depends on specific structural features (32) or on significant structural rearrangements (33). In vivo artificial zymogens or incorporation of appropriate cleavage sites into cellular proteins could enable actuation of such artificial signaling circuits through potentially any protein-mediated activity.…”

Synthetic protein switches: design principles and applications

“…AFF was also used to engineer allosterically regulated actuators including a zymogen of barnase [35] and a protease-sensitive ratiometric GFP sensor [36]. The latter was constructed by duplicating the tenth C-terminal strand of GFP at the N terminus while separating it with a thrombin cleavage site; in addition, a point mutation was introduced in the native tenth C-terminal strand shifting the emission spectrum from green to yellow ( Figure 2E).…”