Fig. 4: Selectivity of wt AM0627^A21-E506 for  peptides containing different O-glycan forms.

a Scheme of the different (glyco)peptides used in this study. Note that both the GalNAc and Gal moieties are indicated as a yellow square and circle, respectively, whereas galactosamine is indicated as a white/yellow square. The sugar moieties are displayed in different colored symbols according to the SNFG⁶⁰. b Time course of the cleavage assay using the 0.4 μM of wt AM0627^A21-E506 with the different (glyco)peptides (500 μM) incubated at 37 °C. See the raw spectra of MALDI-TOF analysis in Supplementary Fig. 4. The specific activity of AM0627 was calculated using the P1 glycopeptide at a time point 5 min where 38% P1 cleavage took place (rate of conversion 5.7 nmol substrate/360 nmol enzyme/5 min). Note that substrate inhibition might be present for the wt AM0627^A21-E506 at higher concentrations of P1, explaining that the slope is less-steeper for P1 in Fig. 4 than the slope for P1 in Fig. 3 (P1 is at 57 μM in Fig. 3). The concentrations of the (glyco)peptides were higher than that of P1 in Fig. 3 to figure out whether other (glyco)peptides might be substrates for the wt AM0627^A21-E506. All experiments were done in duplicate (n = 2 independent experiments). Source data are provided as a Source Data file. Note that each time point represents the average of two independent determinations. c Evolution of several distances (His324-N_ε···Zn²⁺, His329-N_ε···Zn²⁺ and N_R291···O_Q4 and N_R291···N_Q4) during the MD simulation of AM0627 in complex with P1, P2, and P9. The simulations were performed in triplicate (3 independent experiments). d–f Overall structure of AM0627 complexed to P9, P2, and P1 showing the dynamics of these (glyco)peptides during the MD simulations. The initial positions of the glycopeptides, the β-sheet formed by Met289-Asp292 and Zn²⁺ are shown in green, while the final positions (after one of the 500 ns replicas) are shown in cyan. The N- and C-termini of the glycopeptides are colored in blue and red, respectively. A closer view of e and f is shown in Supplementary Fig. 5, and more detailed interactions between AM0627 and these (glyco)peptides are shown in Supplementary Figs. 7 and 8.