Enhancing Robustness of Sortase A by Loop Engineering and Backbone Cyclization

Abstract Staphylococcus aureus sortase A (SaSrtA) is widely used for site‐specific protein modifications, but it lacks the robustness for performing bioconjugation reactions at elevated temperatures or in presence of denaturing agents. Loop engineering and subsequent head‐to‐tail backbone cyclization of SaSrtA yielded the cyclized variant CyM6 that has a 7.5 °C increased melting temperature and up to 4.6‐fold increased resistance towards denaturants when compared to the parent rM4. CyM6 gained up to 2.6‐fold (vs. parent rM4) yield of conjugate in ligation of peptide and primary amine under denaturing conditions.

Abstract: Staphylococcus aureus sortase A( SaSrtA) is widely used for site-specific protein modifications, but it lacks the robustness for performing bioconjugation reactions at elevated temperatures or in presenceo fd enaturing agents. Loop engineering and subsequent head-to-tail backbone cyclization of SaSrtA yieldedt he cyclized variant CyM6 that has a7 .5 8Ci ncreased meltingt emperature and up to 4.6-fold increased resistance towards denaturants when compared to the parent rM4. CyM6 gained up to 2.6-fold (vs. parent rM4) yield of conjugate in ligationo f peptide and primary amine under denaturing conditions. Functionalization of proteins is often performed by targeting reactive endogenousa mino acid side chains (e.g. -NH 2 in lysine or -SH in cysteine residues). [1] However,i nm any cases, cysteine is limiteda sa ccessible nucleophile of proteins. [2] In contrast, the high abundance of accessible lysine often leads to heterogeneouslym odified products (e.g. 19 lysine residues are present on the surfaceo facellulase). [3,4] Bond-forming enzymes such as sortase, [5] butelase, [6] transglutaminase, [7] lipoic acid ligase, [8] biotin ligase, [9] phosphopantetheinyl transferase, [10] SnoopLigase, [11] and SpyLigase [12] have emerged as powerful tools for site-specific protein modifications. Sortases are a family of transpeptidases that are found in Gram-positive bacteria. [13] The mechanism of transpeptidationo fStaphylococcus aureus sortase A(SaSrtA) is well studied. SaSrtA recognizes protein 1( P1) which harborsa nL PXTG (in which Xe quals any amino acid) motif and cleaves the amide bond between threo-nine and glycine. The resulting protein-sortase thioester is then attacked by the amino group of an N-terminal glycine residue of protein 2( P2), resulting in ap rotein conjugate of protein 1a nd protein 2( Scheme 1). [14] Owing to the high degree of site-specificity and the minor size ( 5A Ar esidues)o ft he sorting motif, sortase-mediated transpeptidation, also knowna ss ortagging, has become a widelyu sed methodf or protein conjugation, [15] cyclization, [16] labelling/functionalization, [17] purification [18] and/or immobilization. [19,20] Given to the comparably high transpeptidase activity amongt he sortases, [21] SaSrtA is most frequently used in sortagging applications. [22] However, the overall low activity (catalytic efficiency k cat /K M = 160 s À1 m À1 ) [9,23] andt he requirement of calcium as ac ofactor [24] of SaSrtA wild type (WT) hinders broader utilizationo fs ortagging. Protein engineering campaigns werep erformed to yield SaSrtA variants with increasedc atalytic efficiencies in aqueous solutions( up to 105fold), [9,23] organic co-solvents (up to 6.3-fold), [25] or in absence of calcium cofactor( up to 114-fold). [26] By employing engineered SaSrtA variants,e xpanded applications such as reversible surface functionalization, [27] in vivo protein modifications, [28] as well as continuous and flow-based protein immobilization and labellingw ere achieved. [29,30] Notably,t he advantages such as low nucleophile concentration and immediate product release in continuous and flow-based systems highlight the potential of sortagging in industrial applications. [29] The latter further requires robustS aSrtA variants with high process stabilities (e.g. thermal/storage stability, resistance to denaturants) over al ong period of time. Af ew studies reported the engineering of SaSrtA WT for increased stabilityt owards thermal and chemical denaturation (e.g. 11.2 8Ci mprovement in melting temperature (T m )a nd 4.5-fold resistance in presence of 2.5 m urea). [31,32] However,e ngineering of highly activeS aSrtA variants with enhanced tolerance against thermaland chemical stress has not been reported yet.
In this study,afirst engineering campaign of the SaSrtA variant rM4 (P94S/D160N/D165A/K196T) [9] towards high thermal stabilityw as performed. SaSrtA rM4 showeds ignificantly higher activity (> 75-fold vs. WT at ambient temperature) [9,25] but remarkablyl ow thermal stability (T m = 48.6 vs. 59.4 8Co f WT,F igure S3). [31] In the protein engineering campaign, ah igh throughput screening (HTS) assay of SaSrtA was employed based on the reconstitution of the self-sufficient cytochrome P450 BM3 (CYP102A1) monooxygenase from Bacillusm egaterium (Figure 1a). The P450 BM3 monooxygenase consists of a heme and ar eductase domain, which are fused by af lexible linker regioni nasingle polypeptide chain. [33] We separated the heme and the reductase domain at af lexible linker andt agged the heme domain with the C-terminal LPETG motif and the reductased omain with the N-terminal triglycine (Table S1). Heme and reductase domains were individually expressed and purified ( Figure S1). By using SaSrtA, the heme and reductase domain are reconstituted into the active and full-length P450 BM3 monooxygenase, which forms ad imer preventing ab ack reaction( Figure 1a). [34,35] The amount of reconstituted P450 BM3 was detectedb yareported fluorogenica ssay using 7benzoxy-3-carboxycoumarin ethyl ester (BCCE) as the substrate ( Figure 1a). [36] Reconstitution of heme and reductase domain to full-length P450 BM3 using SaSrtA WT was previously reported. [37] However,t he utilization of the P450 BM3 reconstitution in screening of activity improved sortase Ah as not been reported yet.
The generation of full-length P450 BM3 by SaSrtA rM4 was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (Figure 1b/S2). In comparison to SaSrtA WT,r M4 catalyzed as ignificantly higher amount of reconstituted full-length P450 BM3 after sortagging (Figure 1b/S2). Samples were subsequently employedi nt he BCCE assay.I n sample 3, background activity in BCCE assay was observed ( Figure 1c). It is suggested that the copresence of heme and reductase domains also contributes the intermoleculare lectron transfer [33] and thusl ead to increased activity (vs. samples 1 and 2). SaSrtA rM4 showed a5 .8-fold improved activity (vs. WT) in the BCCE assay (Figure 1c). The latter highlights the applicability of the P450 BM3 reconstitution assay to screen for activity improved SaSrtA variants.
Head-to-tailb ackbonec yclization (HtTBC)h as been reported to improve tolerance of proteins (e.g. green fluorescentp rotein, [45] cytokines [46] )t owards thermali ncubation, protease digestion, or chaotropic agents. [43] HtTBC of SaSrtA WT was previously implementedf or enhanced stability against urea using an intein-mediated posttranslationalm odification. [32] To further improves tabilityp roperties, HtTBC of SaSrtA M6 was performed using as ortase Af rom Streptococcus pyogenes (SpSrtA). SpSrtA recognizes orthogonal sorting motifs (e.g. LPELA, Figure 3a)w hich differ from the preferred SaSrtA motif (LPETG). [21] The generation of cyclized SaSrtA M6 (hereafter CyM6)w as initially confirmed via SDS-PAGE. After as ix-hour reaction time, am ajority (! 80 %, data is not shown) of SaSrtA M6 was cyclized ( Figure 3b). Upon the cyclization, the C-terminal Strep II in M6 is cleaved and subsequently removed from the reaction mixture using column based chromatography ( Figure S7). The correct mass of CyM6 was determined through matrix-assisted laser desorption/ionization mass spectrum analysis (MALDI-MS, Figure 3c). Owing to the flexible and spatial proximity N-and C-termini of SaSrtA (Figure 2b), the fusion of terminis howed little effect on the overall structural conformation of the enzyme. CyM6 retained 99 %o fi ts specific activity after cyclization when compared to the linear SaSrtA M6 ( Figure S8). This result is in agreement with ap revious report, showingt hat SaSrtA WT retained full activity after cyclization. [32] Circular dichroism (CD) spectra revealed no detectable differences between CyM6 and SaSrtA M6, rM4 and WT at ambient temperature ( Figure 3d). The T m of SaSrtA variants were determined by CD spectroscopy under gradient temperatures (from 30 to 80 8C, FigureS9). M6 gains 6.0 8Ci nT m when compared to the parentr M4 (T m of M6 and rM4 is 54.6 and 48.6 8C, respectively) and interestingly,C yM6 has af urther1 .5 8Ci n-creasedT m compared to M6 (T m of CyM6 is 56.1 8C; Figure 3e). In summary,t he engineered CyM6 shows ar emarkably improvedt hermal stability( DT m =+7.5 8C) when compared to the starting parentrM4.
Thermal stabilityo fe nzymes often goes in hand with storage stabilitya sw ell as resistance against denaturants. [45] Therefore, storage stabilityo fC yM6 at room temperature was investigated. CyM6 and M6 retained more than 80 %o fi ts initial activity after fourteen days at room temperature (Figure 4a). In comparison, rM4 only retained 14 %a ctivity under the same conditions (Figure 4a). The significantly enhanced storage stability of M6 andC yM6 facilitates their utilization at ambient temperature over longerprocess time.
In summary,w ee stablisheda nd validated aP 450 BM3 reconstitution assay for its suitability in high-throughput screening systemsf or SaSrtA. Engineering of highly active SaSrtA rM4 in the b6/b7l oop identifiedt wo key beneficial substitutions (R159N and K162P) for enhancedt hermal stability.T he key substitutions werer ecombined in the variant M6 resulting in increased thermals tability( DT m =+6.0 8C, T m :m eltingt emper-ature) when compared to the parentr M4.H ead-to-tail backbone cyclization of M6 yieldedacyclized variant CyM6 which retained full specific activity (99 %v s. M6) and further gained thermals tability (DT m =+7.5 8Cv s. parent rM4). CyM6 retained 83 %( vs. 14 %o ft he parent rM4) activity after fourteen days of storagea tr oom temperature. Additionally,C yM6 showed improveda ctivity (up to 8.8-fold vs. rM4), resistance (up to 4.6fold vs. rM4) andf acilitated higher production (up to 2.6-fold vs. rM4) of sortagged conjugates under thermal and denaturants stress. In essence, we generated robust SaSrtA variants with improved stability, which facilitates ortagging reactions in presence of denaturants, organic solvento ver significantly enhanced thermal/storage stability.T he latter advances sortagging reactions for synthetically attractive applications, such as continuous protein labelling/functionalizationi nf low-based systemsa nd large scale macro-cyclization of pharmaceutical peptides.