Characterization of the Artemisinin Binding Site for Translationally Controlled Tumor Protein (TCTP) by Bioorthogonal Click Chemistry

Abstract

Despite the fact that multiple artemisinin-alkylated proteins in Plasmodium falciparum have been identified in recent studies, the alkylation mechanism and accurate binding site of artemisinin-protein interaction have remained elusive. Here, we report the chemical-probe-based enrichment of the artemisinin-binding peptide and characterization of the artemisinin-binding site of P. falciparum translationally controlled tumor protein (TCTP). A peptide fragment within the N-terminal region of TCTP was enriched and found to be alkylated by an artemisinin-derived probe. MS2 fragments showed that artemisinin could alkylate multiple amino acids from Phe12 to Tyr22 of TCTP, which was supported by labeling experiments upon site-directed mutagenesis and computational modeling studies. Taken together, the "capture-and-release" strategy affords consolidated advantages previously unavailable in artemisinin-protein binding site studies, and our results deepened the understanding of the mechanism of protein alkylation via heme-activated artemisinin.

Artemisinin as an anticancer drug: Recent advances in target profiling and mechanisms of action

“…Furthermore, MS‐based proteomics enables the screening and identification of protein modification sites, including that of artemisinin‐induced alkylation . Xiao's group successfully made use of activity‐based artemisinin probes to identify the binding sites of artemisinin in the well‐known malarial target TCTP (translationally controlled tumor protein), establishing a methodology for identifying modification sites of key proteins of interest, which can provide relevant structural and functional information . Strikingly, this study highlighted a highly nonspecific pattern of protein alkylation, where the artemisinin probe modified multiple adjacent residues on the analyzed fragment including phenylalanine, asparagine, threonine, and aspartic acid, in addition to the expected cysteine residues.…”

Target Binding and Sequence Prediction With LSTMs

“…After the model was trained, the network was given three novel sequences separate from the original dataset, shown in figure four. TCTP was used as a positive control sequence since literature validates its binding ability to Artemisinin [1]. A broken TCTP sequence with arbitrarily inserted amino acids to render it nonfunctional was used for the negative control since the known literature binding site was manually changed into an unrelated sequence.…”

“…Next, we attempted to extend the application of these models to a more challenging and potentially useful task: proteins that bind to antimalarial drug Artemisinin. Although some binding motifs such as translationally controlled tumour protein (TCTP) and ferritin are known to bind effectively, there are not extensive collections of Artemisinin binding proteins and properties [1,3,9]. Each motif resides in a protein's primary structure, has position-specific residues, and similar architectural sequence components, yet may have residues that vary at unpredicted locations in the sequence.…”

“…The AUC of these datasets was 0.80 and 0.87 respectively, further indicating the models' effectiveness. Furthermore, using these trained models, it was possible to derive a protocol for sequence detection of homeodomain and binding motif, which are well-documented in literature, and a known Artemisinin binding site, respectively [1][2][3]. Along with these contributions, we developed a python API to directly connect to Uniprot data sourcing, train deep neural networks on this primary sequence data using TensorFlow, and uniquely visualize the results of this analysis.…”

Evaluation and Optimization of Chemically-Cleavable Linkers for Quantitative Mapping of Small Molecule-Protein Interactomes

“…The biotin portion of the AZO linker is cleaved off upon reduction of the azobenzene group using sodium dithionite resulting in the formation of a residual aminophenol moiety on the cleaved peptide. To date, the applications of the DADPS linker included: identification of glycosylation sites 23 , profiling the deubiquitinase family of proteins 24 , and mapping the sites of small-molecule protein interactions [25][26][27] . The biotin portion of the DADPS linker is cleaved using formic acid leaving a hydroxyl moiety on the peptide adduct.…”