10390 Biochemistry, Vol. 48, No. 43, 2009 commercial version of this purification system is the Profinity eXact Purification System (Bio-Rad Laboratories), and similar components are used for measurement of azide-dependent cleavage rates in this paper. Here we report anion-dependent cleavage kinetics for subtilisin S189 and describe the 1.8 A˚ resolution crystal structure of complexes of an inactive (S221A) form of S189 and a prodomain substrate. The crystallized prodomain construct, called pG16, includes a four-residue C-terminal tail with a SATS sequence and thus provides structural information about substrate interactions spanning the active site. Although S189, like wild-type subtilisin, has Ser as the catalytic nucleophile at residue 221, these crystal structures have Ala 221 to prevent cleavage of the substrate. We report the structure of the subtilisin-substrate complex without triggering anion, with fluoride, and with the activator azide. The resulting structures and corresponding kinetic analysis provide detailed views of protease-substrate interactions and insight into the mechanisms of catalysis and activation by anions. METHODS Definition of Mutants. (i) Subtilisin. S189 is our basic processing subtilisin whose engineering has been described ear- lier. S189 denotes subtilisin from B. amyloliquefaciens with the following mutations: Q2K, S3C, P5S, S9A, I31L, D32A, K43N, M50F, A73L, Δ75-83, Y104A, G128S, E156S, G166S, G169A, S188P, Q206C, N212G, Y217L, N218S, T254A, and Q271E (21, 23-26). We have constructed two new variants of S189 for this study. The first variant (denoted pS164) is the S221A mutant of S189. As noted above, mutation of the active site serine inactivates the enzyme and allows structural characterization of substrate interactions that span the active site. The second variant, denoted pS169, is the A1C mutant of S189. This mutation does not change the catalytic properties of the enzyme but allows the introduction of a fluorescent group at its N-terminus. The site specific label is useful for kinetic analysis as described below. (ii) Prodomain. pG60 is a stabilized version of the prodomain used in previous studies (19). pG60 denotes the prodomain from B. amyloliquefaciens with the following mutations: substitution of amino acids 17-21 (TMSTM) with GFK and substitutions A23C, K27E, V37L, Q40C, H72K, A74Y, H75R, and Y77L. pG60 is independently stable and binds to subtilisin with ∼100- fold higher affinity than the wild-type prodomain. We have constructed two new variants of pG60 for this study. The first variant, denoted pG16, constructed for the crystallographic studies, has four amino acids added to its C-terminus: S78, A79, T80, and S81. The second variant, denoted pG12, con- structed for the kinetic measurements, is pG16 followed by the 56-amino acid B1 domain of protein G (GB) (27, 28), whose tryptophan fluorescence facilitates monitoring of proteolytic release (see below). Expression and Purification. Subtilisin variants pS164 and pS169 were expressed in Escherichia coli as described for similar variants in ref 29, except that the peptide affinity purification column used an immobilized peptide of the sequence LFRAL rather than ALAL. Prodomain variants pG12 and pG16 were expressed in E. coli as described for similar variants in ref 21, except they were released from the S189 subtilisin column by elution in 0.1 M H3PO4 (pH 2.1) rather than by anion-triggered cleavage. Purity was assessed by SDS-PAGE and estimated to be >95%. Gallagher et al. FIGURE 1: (A) Cartoon representation of the pG12 fusion protein (green and yellow) in complex with pS169 subtilisin (blue). The position of the fluorescent reporter is colored red on the pS169 N-terminus. (B) Cartoon representation of GB and the pG12-pS169 complex after cleavage. Measurement of Activity of Subtilisin pS169 as a Func- tion of Anion. The N-terminal amino acid is the only free cysteine in pS169 and enables specific labeling with maleimide reagents. pS169 for kinetic measurements was labeled with Alexafluor 350 maleimide. E. coli extract containing 20 mM DTT was washed over a 1 mL LFRAL column to allow affinity binding of pS169. The column was washed with 20 mL of 10 mM HEPES (pH 6.8), 100 mM NaCl, and 1 mM EDTA (HNE). A 1.4 mL volume of a 10 mM solution of Alexafluor 350 maleimide in HNE and 20% DMSO was injected on the column and incubated for 2 h at 22 C. Excess label was then washed away in HNE. The labeled protein was eluted in 10 mM NH4OH and immediately neutralized with 1/10 volume of 1 M potassium phosphate (pH 7.2). Attached to the N-terminus of pS169, Alexafluor 350 becomes a fluorescent reporter group with an absorption maximum at 350 nm and an emission maximum at 450 nm. The substrate for kinetic measurements was pG12 (Figure 1). pG12 forms a highly stable complex with labeled pS169. In this complex, the single tryptophan of GB can be excited at 300 nm so that some energy is transferred to Alexafluor 350, the emission of which at 450 nm can then be monitored. Enzymatic cleavage releases GB from the complex and results in a decrease in the level of energy transfer, enabling time-dependent quantitation of proteolysis. The total fluorescence signal at 450 nm decreases by ∼8% upon complete cleavage of the substrate complex into the product complex and GB.