Bicyclic Engineered Sortase A Performs Transpeptidation under Denaturing Conditions

Abstract

Enzymes are of central importance to many biotechnological and biomedical applications. However, for many potential applications, the required conditions impede enzyme folding and therefore function. The enzyme Sortase A is a transpeptidase that is widely used to perform bioconjugation reactions with peptides and proteins. Thermal and chemical stress impairs Sortase A activity and prevents its application under harsh conditions, thereby limiting the scope for bioconjugation reactions. Here, we report the stabilization of a previously reported, activity-enhanced Sortase A, which suffered from particularly low thermal stability, using the in situ cyclization of proteins (INCYPRO) approach. After introduction of three spatially aligned solvent-exposed cysteines, a triselectrophilic cross-linker was attached. The resulting bicyclic INCYPRO Sortase A demonstrated activity both at elevated temperature and in the presence of chemical denaturants, conditions under which both wild-type Sortase A and the activity-enhanced version are inactive.

Figure 1

(A) Amino acid sequence of SrtA variant S11 including homology model (atomic coordinates available as Supporting Information). (B) Reaction of S11 with Ae2 resulting in desired cross-linked product xS11 and overalkylated species oS11. Calculated molecular weights are stated. (C) MS of product mixture resulting from reaction of S11 with Ae2 in the presence of 5 mM CaCl₂. Deconvoluted found masses are stated (for details see Supplementary Figure S3). (D) MS of reaction of S11 with Ae2 in the absence of CaCl₂. Deconvoluted found masses are stated (for details see Supplementary Figure S7).

Figure 2

(A) Strategy I: The residual chloroacetamide groups on an overalkylated oS11 are reacted with a biotinylated thiol and removed using streptavidin resin. MS spectra before (top) and after (bottom) purification are shown. Deconvoluted found masses are stated (for details see Supplementary Figures S8 and S9). (B) Strategy II: Reaction of S11 with Ae2 is not allowed to complete, and a mixture of S11 and xS11 is subjected to denaturing size exclusion chromatography for separation. MS spectra before (top) and after (bottom) purification are shown. Deconvoluted found masses are stated (for details see Supplementary Figures S10 and S12).

Figure 3

(A) Normalized first derivative of fluorescence ratio (λ = 350 nm/330 nm). Maxima-derived melting temperatures (T_i) are given (for details see Supplementary Figure S16). (B) Principle of transpeptidation activity assay, involving the cleavage of a dual-labeled substrate with fluorophore: fluorescein isothiocyanate (FITC), and quencher: 4-((4-(dimethylamino)phenyl)azo)benzoic acid (Dabcyl). (C) HPLC analysis of transpeptidation reactions performed at 60 °C after 30 min. Signal (a) corresponds to the substrate, while signals (b) and (c) correspond to the products of transpeptidation (for MS spectra see Supplementary Figure S18). (D) Fluorescence intensity measurement of transpeptidation reaction performed at different concentrations of urea. Relative initial rates (rel. v.) are stated. (E) Fluorescence intensity measurement of transpeptidase reaction performed at different concentrations of GuHCl. Relative initial rates are stated.

Figure 4

MD simulations: (A) Root mean square fluctuation (RMSF) of backbone Cα atoms (gray: 8M, red: xS11) calculated from a total of 2.5 μs simulation time. Most flexible loops are highlighted (yellow) including cross-linking positions in xS11 (111, 149, and 177). The region between residues 93–111 shows the most significant increase in flexibility upon cross-linking (gray). (B) Aligned snapshots at 50 ns intervals taken from the five, 500 ns replicates demonstrate the conformational flexibility (left: 8M, right: xS11).