Biochemistry 2009, 48, 10389–10394 DOI: 10.1021/bi900577n 10389 Structure of a Switchable Subtilisin Complexed with a Substrate and with the Activator Azide† Travis Gallagher,*,‡,§ Biao Ruan,‡, )Mariya London,‡, )Molly A. Bryan,‡, )and Philip N. Bryan‡,^ ‡Center for Advanced Research in Biotechnology, 9600 Gudelsky Drive, Rockville, Maryland 20850, §National Institute of Standards and Technology, Gaithersburg, Maryland 20899, P)otomac Affinity Proteins, LLC, Gaithersburg, Maryland 20878, and ^University of Maryland Biotechnology Institute, Baltimore, Maryland 21202 Received April 3, 2009; Revised Manuscript Received August 24, 2009 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 A˚ 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. The secreted serine protease subtilisin BPN0 from Bacillus amyloliquefaciens is active against a broad range of substrate sequences, appropriate for digestion of extracellular proteins (1-3). Like all secreted proteases, subtilisin is initially synthesized as a proenzyme (4). Prosubtilisin is autoprocessed to create a transient complex of the 77-amino acid N-terminal prodomain and the 275-amino acid mature enzyme (5, 6). Substrate prefer- ences are typified by the cleavage site of prosubtilisin, which is the substrate in the enzyme’s autocatalytic maturation process. The cleavage site has the sequence AHAY.AQSV, with the AHAY portion becoming the C-terminus of the 77-residue prodomain and the AQSV portion becoming the N-terminus of the mature enzyme (7). Extensive structural and enzymatic data have shown that most substrate contacts are made with the first four residues on the acyl side of the scissile bond, which are denoted P1-P4, numbering from the scissile bond toward the N-terminus of the substrate (8). The most effective substrates include a large uncharged residue at P1, Ser or Ala at P2, and a hydrophobe at P4 (9, 10). Previously, we have engineered subtilisin into a specific, processing protease and coengineered the prodomain into an optimized recognition sequence. This involved four steps: (1) engineering of highly stable and refoldable subtilisin (11, 12), (2) modifications of binding pockets resulting in a strong preference †This work was supported in part by National Institutes of Health Grant R44 GM076786 to B.R. and a grant from the Bill and Melinda Gates Foundation to P.N.B. through the Grand Challenge Exploration Initiative. *To whom correspondence should be addressed. Telephone: (240) 314-6204. Fax: (240) 314-6225. E-mail: gallaghe@umbi.umd.edu. for sequences of the form P4-P1 = YRAL (13-17), (3) selection of an independently stable prodomain with high affinity for subtilisin (18, 19), and (4) engineering a tunable active site by means of a D32A1 mutation which renders activity dependent on the presence of certain small anions such as fluoride and azide (20-22). The resulting switchable subtilisin, whose activity against cognate sequences (e.g., YRAL-) can be accelerated more than 3000-fold by 100 mM azide, is denoted S189. The ability to modulate the rate of the cleavage reaction with anion concentration creates a processing system with a virtual on-off switch. Switchable proteases are useful biotechnologi- cally as well as in studies of mechanism. The anion-switchable subtilisin has been used to develop a one-step affinity chroma- tography system for the purification of recombinant proteins. The purification system comprises a target protein fused to the C-terminus of an engineered prodomain and an immobilized subtilisin that is virtually inactive in the absence of a triggering anion. The ability to isolate the binding and cleavage steps with a triggering mechanism allows subtilisin to be used as both the affinity ligand and processing enzyme for affinity purification and processing of proteins fused to the prodomain (21). The 1A shorthand for denoting amino acid substitutions employs the single-letter amino acid code as follows: N218S denotes the change of asparagine 218 to serine, and Δ75-83 denotes the deletion of residues 75-83 from the amino acid sequence. Abbreviations: DMSO, dimethyl sulfoxide; DTT, dithiothreitol; EDTA, ethylenediaminetetraacetic acid; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; PAGE, polyacrylamide gel electrophoresis; PEG, polyethylene glycol; RCSB, Research Collaboratory for Structural Bioinformatics; SDS, sodium dodecyl sulfate. r 2009 American Chemical Society Published on Web 09/17/2009 pubs.acs.org/Biochemistry