The molecular features of non-peptidic nucleophilic substrates and acceptor proteins determine the efficiency of sortagging

Abstract

Sortase A-mediated ligation (SML) or "sortagging" has become a popular technology to selectively introduce structurally diverse protein modifications. Despite the great progress in the optimization of the reaction conditions and design of miscellaneous C- or N-terminal protein modification strategies, the reported yields of conjugates are highly variable. In this study, we have systematically investigated C-terminal protein sortagging efficiency using a combination of several rationally selected and modified acceptor proteins and a panel of incoming surrogate non-peptidic amine nucleophile substrates varying in the structural features of their amino linker parts and cargo molecules. Our data suggest that the sortagging efficiency is modulated by the combination of molecular features of the incoming nucleophilic substrate, including the ionization properties of the reactive amino group, structural recognition of the nucleophilic amino linker by the enzyme, as well as the molecular nature of the attached payload moiety. Previous reports have confirmed that the steric accessibility of the C-terminal SrtA recognition site in the acceptor protein is also the critical determinant of sortase reaction efficiency. We suggest a computational procedure for simplifying a priori predictions of sortagging outcomes through the structural assessment of the acceptor protein and introduction of a peptide linker, if deemed necessary.

Fig. 1. PyMOL visualizations of the three-dimensional structures of EGF, TNFα, CAHII and VHH 7D12 modelled with the recombinant C-terminal appendages. (A) Side view of the I-TASSER model of the anti-epidermal growth factor receptor nanobody (VHH_7D12-SRT-6H). (B) Side view of the I-TASSER model of human epidermal growth factor (EGF-SRT). (C) Side and front views of the I-TASSER model of human tumor necrosis factor-α (TNFα-SRT). (D) Side and front views of the I-TASSER model of human carbonic anhydrase II (CAHII-SRT-6H). Positions of the SrtA recognition sites are highlighted in red, and 6xHis tags are shown in yellow. In all cases, the initial recombinant structures with the shortest C-terminal additions (VHH_7D12-SRT-6H, EGF-SRT, TNFα-SRT and CAHII-SRT-6H in Table 1, correspondingly) are shown.

Fig. 2. PyMOL visualization of docking of the C-terminus of the CAHII-GS-SRT-6H construct in the active site of the SrtA enzyme. (A) Side view of the ClusPro Protein–Protein docking model between SrtA and the I-TASSER model of human carbonic anhydrase II (Sortase-hCAHII-2GS-SRT-6H). (B) Front view of the ClusPro Protein–Protein docking model between Sortase and the C-terminus of the I-TASSER model of hCAHII-2GS-SRT-6H. The C-terminus includes the 2xG4S linker (green), the Sortase recognition site LPETG (red), and a His-tag (yellow). Sulphur atom of the thioester intermediate forming Cys184 in the SrtA active site is coloured yellow.

Fig. 3. Structures of the amino linker equipped derivatives of biotin (compounds 1–15, arranged in the order of decreasing SML efficiency based on composite product yields with 4 protein acceptors) and DOTA (16, 17) used in this work.

Fig. 4. Comparative visualization of conjugate product yields’ distribution for all the tested combinations of the 6 acceptor proteins (CAHII highlighted in red, TNFα in blue, and EGF in green) and 14 incoming biotin-containing substrates (1–14), excluding the non-reactive biocytin (15) control.

Fig. 5. (A) Composite reaction yields (%, left scale) for 4 protein acceptors, excluding the non-reactive proteins, and 14 biotin derivatives (1–14), excluding the non-reactive biocytin (15). The stacked bars (EGF – grey, TNFα-GS-SRT – black, CAHII-GS-SRT-6H – light grey, CAHII-PAS-SRT-6H – brick pattern) are sorted left to right in the order of decreasing composite yield. The line connecting mean pK_a values (right scale) for each substrate is overlayed over the bar graph. Brackets connect the compounds for pairwise comparisons. (B) Same graphical representation as in (A) for comparing two DOTA derivatives (16 and 17) with their corresponding biotin counterparts (8 and 11). Yields correspond to SML reactions done at 0.5 mM (panel A) or 1 mM (panel B) nucleophile concentrations.