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.

Fig. 1.

X-ray crystal structure of WT Bn. The dark gray region corresponds to the C-terminal segment of Bn-AFF that is duplicated and fused to the N-terminus. Amino acid positions 1 (N-terminus), 93 (site of circular permutation), 102 (catalytic acid; alpha carbon shown), and 110 (C-terminus) are indicated.

Fig. 2.

Amino acid sequences of Bn variants and mechanism of AFF switching. (A) Amino acid sequences of enzymes created in this study. Numbering is according to the WT Bn sequence; prime superscripts indicate amino acids in the F2 segment of Bn-AFF. Duplicate segments are shown in red and blue. Open and closed circles denote the presence of His102 or Ala102, respectively. Dashed lines represent the PR cleavage site (IFLETS). Solid lines symbolize the flexible linker used to bridge the original N- and C-termini in Bn-F2 and Bn-AFF. (B) Schematic illustrating the topological change that underlies the F1⇆F2 fold shift. F1 and F2 analogs are shown. Dashed line and open/closed circles are the same as in panel A. (C) Schematic of AFF switching mechanism. Wavy lines indicate unfolded regions; colors, dashed line, and open/closed circles are the same as in panel A.

Fig. 3.

CD spectra of Bn-AFF variants and their F1/F2 analogs. Lines are as follows: Bn-F1 (Solid Gray), Bn-F2 (Dashed Gray), Bn-F2(P94) (Dotted Black), Bn-AFF(P94 + A96) (Dashed Black), Bn-AFF(P94′) (Solid Black).

Fig. 4.

Urea-induced denaturation of Bn-AFF before stability optimization (A), and after stability optimization (B). Symbols in panel A are: Bn-F1 (Open Circles), Bn-AFF (Closed Circles), Bn-F2 (Triangles). Symbols in panel B are: Bn-F1 (Open Circles), Bn-AFF(P94′) (Closed Squares), Bn-F2(P94) (Inverted Triangles). Lines are best fits of the data to the linear extrapolation equation.

Fig. 5.

Limited trypsin digestion of Bn-AFF(P94′) characterized by mass spectrometry. Peaks marked with an asterisk or +2 indicate an impurity or the +2 charge species of a parent peak, respectively. Samples are as follows: Bn-AFF(P94′) (A), Bn-AFF(P94^′) + trypsin (B), PR-digested Bn-AFF(P94′) (C), PR-digested Bn-AFF(P94^′) + trypsin (D).

Fig. 6.

Hydrolysis of the fluorogenic RNA substrate 5′ 6-FAM-ArGAA-3′TAMRA by Bn-AFF(P94′). Lines are best fits of the data to equations in ref. .