Structure of a Switchable Subtilisin Complexed with a Substrate and with the Activator Azide

Abstract

An engineered variant of the protease subtilisin from Bacillus amyloliquefaciens, in which the D32A mutation renders the enzyme’s activity dependent on the presence of certain small anions such as fluoride or azide, has been produced. This modified enzyme has applications as an azide or fluoride-triggered expression−purification tool. We report activity measurements showing that the enzyme is activated more than 3000-fold by azide and describe the 1.8 Å resolution structure of an inactive form (by replacing the catalytic nucleophile Ser 221 with alanine) of the protease, in complex with azide and with a substrate that spans the active site. Both enzyme and substrate have been engineered to increase their stability and the affinity of their interaction. The substrate is based on a stabilized subtilisin prodomain, extended across the active site by the addition of four residues at its C-terminus. In the crystal structure, the substrate is well-ordered across the active site, and the azide anion is observed bound adjacent to Ala 32. The structures of the substrate complex in three different crystals (anion-free, fluoride-soaked, and azide-soaked) are compared. These structures provide extensive information for understanding subtilisin’s substrate binding and catalytic mechanism, and for the development of biotechnology tools based on anion-activated proteolysis. The mechanism of anion-dependent proteolysis appears to be a slight modification of the accepted charge-relay mechanism for serine proteases.

Engineering protein-specific proteases: targeting active RAS

“…Structure of RAS with a bound GTP analog (PDB code 6Q21), (4,5), highlighting the YSAM site in Switch 2. B: RAS-specific protease based on an X-ray structure of 3BGO.pdb (6). Cognate sequence QEEYSAM-RD is modeled in the binding cleft.…”

“…The engineering process to develop specificity for the RAS sequence QEEYSAM involved extensive modification of the Bacillus protease, subtilisin BPN' (6,(17)(18)(19)(20)(21)(22)(23)(24), a canonical serine protease in which the scissile peptide bond is attacked by a nucleophilic serine (S221). The nucleophilicity of S221 results from its interactions with the catalytic histidine (H64) and aspartic acid (D32) that together form a charge relay system (25).…”

“…The design strategy was based on the hypothesis that a linkage between substrate binding and chemical rescue can be created by mutating sub-sites to optimize interactions with the desired cognate sequence and combining these with mutation of a catalytic amino acid. To test the robustness of this hypothesis we generated two different active site variations: 1) D32G variants that are activated by nitrite or azide; 2) H64G variants that are activated by imidazole (6,17). Initial design was based on the X-ray structure of an inactive (D32A, S221A) variant of subtilisin (3BGO.pdb) (6).…”

“…In many natural proteases the cognate sequence influences the chemical steps in peptide hydrolysis and not just binding steps (16). To engineer highly sequence-specific subtilisins we combined two previous observations: 1) Mutations at remote binding pockets for substrate side chains (sub-sites) can distort the subtilisin active site and (10,12) 2) Mutating a catalytic amino acid in an enzyme can allow chemical rescue with a small molecule cofactor (6,17). We leveraged these observations to create an engineered protease that requires both conformational and chemical rescue by the substrate and cofactor, respectively, in order to achieve high levels of activity.…”

Engineering Environmentally-Stable Proteases to Specifically Neutralize Protein Toxins

“…The position of the catalytic serine 221 is shown in pink as well as glycine 166 at the back of the S1 pocket. The depiction is based on 3BGO.pdb (15).…”