Figure 5. Features of structurally significant PfA-M1 limited proteolysis coupled with mass spectrometry (LiP-MS) peptides.

(A) Relative abundance of the significant LiP peptides commonly identified across two LiP-MS experiments following P. falciparum proteome lysate treatment with MIPS2673. The mean ± SEM of at least three independent lysate incubations per condition and experiment are shown. (B) Median distances between atoms of the nine significant LiP peptides or all detected PfA-M1 peptides and the PfA-M1 binding cleft residues. *p<0.05, Mann-Whitney test. (C) MIPS2673 binding site on PfA-M1 determined by X-ray crystallography (PDB: 8SLO), and approximation of the MIPS2673 binding site using the significant PfA-M1 LiP peptides and centre of mass calculation. The significant LiP peptides are mapped onto the PfA-M1 structure with MIPS2673 bound and are shown in pink. The drug ligand is shown in blue and the centre of mass of the significant LiP peptides is shown by a magenta star. The area in cyan represents the neighbourhood of the drug binding site determined as residues within 6.44 Å of bound MIPS2673. The neighbourhood of the LiP peptide centre of mass (residues within 6.44 Å of the centre of mass) is depicted in magenta.

Figure 5—figure supplement 1. Minimum distance of significant limited proteolysis coupled with mass spectrometry (LiP-MS) or all other PfA-M1 peptides from bound MIPS2673.

Among the 108 PfA-M1 peptides commonly identified across both LiP-MS experiments, nine peptides were significantly dysregulated in the presence of MIPS2673 at all concentrations tested. The atoms of these LiP peptides were used to measure the distribution of distances to bound MIPS2673 compared to all detected PfA-M1 peptides. *p<0.05, Mann-Whitney test.

Figure 5—figure supplement 2. Minimum distance measurements and mapping of significant limited proteolysis coupled with mass spectrometry (LiP-MS) peptides to structures of other putative MIPS2673-interacting proteins.

Distance measurement analyses were performed for other putative MIPS2673-interacting proteins identified with thermal stability proteomics and LiP-MS (experiment one) where a protein structure was available and significant LiP-MS peptides were identified. Only peptides detected in both LiP-MS experiments were considered for analysis. Significant LiP peptides were defined as any peptide that passed the relative abundance (absolute fold-change >1.5), statistical significance (q<0.01), and proteolytic peptide pattern (i.e. increased fully tryptic or decreased half tryptic) filters at any concentration in both LiP-MS experiments. For the metalloproteins PfA-M17, PfAPP, and PfADA (PDB: 7RIE, 5JR6, and 6II7, respectively) the active site metal ions were used as a reference point for minimum distance measurements. For PfMIF (PDB: 4P7S), the bound ligand 20K and TRXR1 (PDB: 2ZZC), the bound ligands FAD and NADP, were used for minimum distance measurements. For RAB39A, which is not a metalloprotein and where no ligand-bound structure is available, the GTP binding site residues (blue) were used (AlphaFold: AF-Q14964-F1). Significant LiP-MS peptides are in pink, metal ions are in light blue, and ligands are in dark blue or orange (NADP only). Peptide mapping for multimeric proteins was performed for one chain only (chain A for PfA-M17, PfAPP and TRXR1, and chain B for PfMIF). The putative PfApiAP2 transcription factor, PF3D7_1239200, was excluded as no protein structure is available and the AlphaFold predicted structure is of very low quality (pLDDT score 36.67). No peptides met the significance criteria for PfA-M18 and the conserved parasite proteins, PF3D7_1026000 and PF3D7_0604300. *p<0.05, Mann-Whitney test. For RAB39A and TRXR1, where only one significant LiP-MS peptide was identified per protein, statistical significance was not determined.