SCÅTTER SCÅTTER SCÅTTER SCÅTTER SCÅTTER Data processing PRIMUS/ SCÅTTER


Supplementary


Supplementary Note 1


Structural analysis of EndoS and EndoS2 in complex with the Fc region of Rituximab in solution by small angle X-ray scattering

To study the overall structure of EndoS and EndoS2 in complex with the Fc region of IgG1 in solution, we first performed in-line size-exclusion chromatography small-angle X-ray scattering (SEC-SAXS) (Supplementary Figs. 1 and6; Supplementary Table 2). We purified the catalytically inactive EndoS E235A and EndoS2 E186L variants and the Fc region of Rituximab, a chimeric IgG1 used to treat B cell lymphoma, by papain digestion. We mixed the EndoS E235A or EndoS2 E186L with the Fc region substrate in a 1:5 ratio and we isolated the enzyme-substrate complexes (EndoS E235A -Fc and EndoS2 E186L -Fc) from the free enzymes and substrate by SEC chromatography and conducted SAXS analyses of the individual proteins and the complexes (Supplementary Fig. 6). Comparison of the R g shows an increase when EndoS E235A -Fc (R g = 53.6 Å) and EndoS2 E186L -Fc (R g = 52.2 Å) complexes are formed compared to the unliganded EndoS E235A (R g = 42.9 Å) and EndoS2 E186L (R g = 38.7 Å) (Supplementary Table 2). In addition, examination of the P(r)-distribution shows that the maximum dimension, Dmax, also increases when the enzyme-substrate is formed, from 148 Å to 181 Å in the case of EndoS and from 125 Å to 176 Å in the case of EndoS2. The resulting P(r) function profile of both enzyme-substrate complexes exhibit a unimodal distribution of real-spaces distances that is characteristic of a globular protein, while the P(r) function profiles of the unbound EndoS and EndoS2 are bimodal consistent with the overall "V" shape of the crystal structures of both enzymes (Supplementary Fig. 6). Thus, the change from a bimodal to a monomodal distribution in the enzyme-substrate complexes strongly suggests that the Fc might bind to the cleft of the EndoS or EndoS2, promoting a more compact, globular state. The experimental determination of the EndoS E235A -Fc complex MW 3 (Supplementary Table 2) shows a 36.7% and a 26.5 % discrepancy to the calculated MW of the enzyme-substrate complex with 1:1 and 2:1 stoichiometries, respectively. In the case of EndoS2 E186L -Fc complex, the experimental determination of the MW from the SAXS data shows a 40 % and 1.1 % discrepancy to the calculated MW of the enzyme-substrate complex with 1:1 and 2:1 stoichiometries (Supplementary Table 2). These results, together with the fact that the R g values are decreasing across the SEC peak of the enzyme-substrate complexes (Supplementary Fig. 6) indicate that a mixture of complexes with different stoichiometries might be present in that SEC peak. 
Therefore, we used OLIGOMER 4 to calculate the volume fractions of the complexes with different stoichiometry contributing to the scattering profile. In the case of the mixture EndoS E235A -Fc, the results showed that the enzyme-substrate 2:1 and 1:1 complexes contributed 67% and 29% volume fractions, respectively, to the scattering. Similar ratio of volume fractions was found for the EndoS2 E186L -Fc mixture (Supplementary Table 2). 

Supplementary Note 2


Extended NMR results

To shine more light on the interaction of endo-β-N-acetylglucosaminidases (ENGases) with their natural ligands, we have employed NMR-based chemical shift perturbation (CSP) experiments 5 . CSPs are usually extracted from uniformly 2 H, 15 N-labeled proteins applying 1 H, 15 N Transverse Relaxation Optimized Spectroscopy (TROSY) Heteronuclear Single Quantum Coherence (HSQC) spectroscopy. However, at a molecular weight of 97 kDa 1 H, 15 N HSQC-TROSY spectra of EndoS2 suffered from incomplete deuterium-hydrogen back exchange of backbone H N . The difficulty becomes evident when comparing 1 H, 15 N HSQC-TROSY spectra of EndoS2 with those acquired from EndoBT-3987 glycosidase 6,7 , the latter having a molecular weight of "only" 47 kDa (Supplementary Fig. 16a,b). We have previously shown that deuterium-hydrogen back exchange is crucial to extract reliable information from 1 H, 15 N HSQC-TROSY spectra 8 . Yet, all attempts to develop an efficient unfolding-refolding protocol for complete deuterium-hydrogen back exchange of backbone H N in ENGases failed. 
Therefore, we turned to specific 13 C side chain methyl group labeling strategies 1,9,10 , which enabled the acquisition of 1 H, 13 C Heteronuclear Multiple Quantum Correlation (HMQC) spectra, also known as methyl-TROSY 11 . Such approach grants the extraction of precise CSPs upon, e.g., ligand binding and provides experimental access to binding thermodynamics and kinetics 1,5 . Knowledge of the corresponding on-and off-rate constants would provide a link to the conformational changes associated with substrate binding as observed by cryoEM and H/D exchange, 2 assisting the mechanistic understanding of ENGases. 
NMR lineshape analysis has been used in numerous instances for the evaluation of chemical shift titration experiments, yielding quantitative descriptions of chemical exchange processes such as conformational equilibria or binding of ligands to receptor proteins 12 . A novel approach for analyzing CSP titration data from HSQC-and HMQC-type 2D NMR spectra engages direct quantum mechanical simulation of a given pulse sequence, as it is implemented in the TITAN (TITration ANalysis) software package 13 . Here, we have used TITAN to analyze the kinetics of binding of EndoS2 to its substrate CT, Rituximab-Fc, the reaction product CT n-1 and Ca 2+ ions. The interaction of the related EndoBT-3987 with HM was also studied and used as comparison. 
The isotope labeling scheme selected for this study was [U-15 N, 2 H] MIL proS V proS [ 13 C, 1 H 3 ]-methyl labelling (or simply MILV). Isotopically labeled ENGases were the catalytically inactive EndoS2 E186L and EndoBT D312A/E314L , and their corresponding wildtype EndoS2 and EndoBT species 2,6 . This labeling scheme rendered methyl-TROSY spectra with little to no signal overlapping and excellent signal-to-noise (Supplementary Fig. 16c,d). In addition, the selected methyl probes are uniformly distributed around the putative glycan binding pockets in both enzymes, granting the extraction of CSPs upon ligand titration (Fig. 6a; Supplementary Fig. 17a). 1 H, 15 N-TRACT experiments 14 were used to estimate rotational correlation times ! " , affording values of 45.0 ns and 21.8 ns for EndoS2 E186L and EndoBT D312A/E314 , respectively, in good agreement with their corresponding molecular weights (Supplementary Fig. 22). Taken together with the good signal dispersion observed in NMR spectra, ! " values evidence the presence of folded protein in solution enabling protein-ligand interaction studies. 
At the present time no resonance assignment is available for ENGases. Therefore, we avoided any attempt to use CSPs for epitope mapping. Instead, we focused our efforts on the quantification of the overall thermodynamic and kinetic binding parameters for the interaction with selected ligands. For such goal it is enough to identify a subset NMR signals reporting for the binding event of interest. Here, methyl signals associated with methyl groups in the vicinity of the glycan binding site were identified according to the following two-step exclusion criteria: a) Signals must show CSPs between the wild-type species and the corresponding enzymatically inactive mutants (i.e. EndoS2 vs EndoS2 E186L and EndoBT-3987 vs EndoBT-3987 D312A/E314L ). 
Such mutations were designed to render inactive enzymes while retaining the protein folding and protein dynamics associated with ligand recognition, as previously disclosed by crystallographic and H/D exchange MS studies 2,6 . Therefore, observed CSPs can be linked to changes localized in the vicinity of the carbohydrate binding pockets, even in the event of longrange effects 15,16 . b) Signals must exhibit CSPs after saturation with CT, CT n-1 and Rituximab-Fc (for EndoS2 E186L ), or after saturation with HM (for EndoBT-3987 D312A/E314L ). A visual description of ligands used in this study can be found in Fig. 6C, Supplementary Figs. domain (Fig. 6a). Signals exhibiting CSPs greater than 2 standard deviations upon titration with calcium chloride were selected as reporters for calcium binding. 

Extraction of kinetic and thermodynamic parameters from methyl-TROSY spectra

Titration of catalytically dead ENGases with carbohydrates CT, CT n-1 or Rituximab-Fc produced CSPs in the slow-exchange regime in the NMR time-scale, leading to varying peak intensities during titration (Supplementary Figs. 252627282930). On the other hand, titration of EndoS2 with CaCl 2 produced CSPs in the fast-exchange regime, which translates into shifts in peak positions (Supplementary Fig. 31). Methyl-TROSY based titrations in slow exchange were subjected to lineshape analysis with TITAN software package. For each titration point, the corresponding methyl-TROSY spectrum was imported into MATLAB and then analyzed with the TITAN algorithm, applying a two-state ligand binding model. Quantitative lineshape analysis yielded dissociation constants K D and on-and off-rate constants k on and k off . A representative example of lineshape fitting can be found in Fig. 6D. 
In the main text, we have described the kinetics of binding of EndoS2 to its substrate CT, Rituximab-Fc and the reaction product CT n-1 . Here, we focus on the effect of calcium in the interaction of EndoS2 with CT and Rituximab-Fc. Calcium binding produced similar CSPs in EndoS2 and EndoS2 E186L species with virtually identical dissociation constants K D , suggesting no influence of the mutation E186L on calcium recognition (Table 1, entries 6,7, Supplementary Fig. 32). It is worth noting however, that some signals showed significant CSPs during the titration of EndoS2 E186L with CT, CT n-1 , Rituximab-Fc and CaCl 2 . Such promiscuity in CSPs suggests the presence of alternative calcium-binding sites or a cross-talking mechanism between the GH and BS domains, as it has already been proposed from functional studies with chimeric ENGases. 2 Here, we did not attempt any further analysis on such signals. 
Instead, they will be the subject of a future study, whenever a spectrum assignment is available for EndoS2. Titrations of CT and Rituximab-Fc were repeated in the presence of saturating concentrations of CaCl 2 , producing almost identical results as in its absence. Similarly, no significant differences were observed for the titration of CaCl 2 in the presence of saturating concentrations of CT. No interaction between EndoS2 D186L and CaCl 2 was observed when the enzyme was pre-saturated with Rituximab-Fc. This observation is in good agreement with the cryoEM structure reported in this study, where no calcium ion is bound to EndoS2 in the complex EndoS2-Fc (Supplementary Table 8). It appears that calcium is not directly involved in the recognition of IgGs by EndoS2, although its exact role remains yet to be identified. Our study was complemented with the extraction of the catalytic rate k cat and the Michaelis-Menten constant K M from the catalytic cleavage of complex-type carbohydrates from Rituximab-Fc by EndoS2. Kinetic parameters were approximated using the Michaelis-Menten equation ( 5) (steady-state kinetics), hypothesizing a nearly constant concentration of enzyme-substrate complex during the reaction course. This assumption was based on the two orders of magnitude K D difference observed for the binding of EndoS2 to Fc as compared to the reaction product CT n-1 (K D = 3.1 µM vs K D = 150.7 µM, respectively). Concentration of enzymatically released carbohydrates was measured from 1 H spectra. Background signals were readily suppressed by a perfect CPMG T 2 -filter, which is a relaxation filter that avoids Jmodulation 17 (Supplementary Fig. 18). Cleaved carbohydrate was quantified from the integral of the composite signal at 2.08-2.10 ppm, which reports for N-acetylglucosamine and Nacetylneuraminic acid moieties (Supplementary Fig. 19a). Reaction time-courses were measured to close-to-reaction completion for five Rituximab-Fc initial concentrations (Supplementary Fig. 19b). The reaction rate was calculated from the fitting of initial velocity rates (# $ ) to the equation of Michaelis-Menten (5) (Fig. 6e). Under these conditions, a catalytic rate k cat of 5.9 s -1 and a Michaelis-Menten constant K M of 26.7 µM were observed. It is important to remark that K M resembles K D only when k off >> k cat , according to Eq. 6. In this study this condition is not met (k off = 4.0 s -1 ≈ k cat = 5.9 s -1 ), indicating that the system is not in rapid equilibrium. Nevertheless, care has to be taken when combining results obtained from different EndoS2 species (wt and E186L), because non-identical protein-carbohydrate interactions can result in slightly different kinetic parameters. In conclusion, this study shows for the first time an overview of enzyme kinetics followed by EndoS2. We hope this information will be key for the future development of ENGases with improved activity and selectivity. 
domains of EndoS (residues 606-987). e Structural superposition of the X-ray crystal structures of EndoS D233A/E235L -CT (PDB code 6EN3) and the Fc region of IgG1 (PDB code 1H3X) and the cryoEM structure of EndoS E235A -Fc complex (PDB code 8A64). 
Fc. g P(r) functions distributions of EndoS2 E186E , IgG1(Fc) and EndoS2 E186A -Fc complex. h Normalized Kratky plot EndoS2 E186A , IgG1(Fc) and EndoS2 E186A -Fc complex. i Ab initio modelling with MONSA of the envelope of EndoS E235A -FC and superimposition of EndoS (yellow) and Fc region of IgGa (blue) X-ray crystal structures. j Ab initio modelling with MONSA of the envelope of EndoS2 E186A -FC and superimposition of EndoS2 (orange) and Fc region of IgGa (blue) X-ray crystal structures. trajectories. For aromatic residues and Pro, the center of the aromatic ring and the center of the ring are considered, respectively, for these calculations. For L314, S298 and E354 residues, the Cd, the OH group, and the oxygens of the carboxylate are considered, respectively, for the calculations. Cut-off distance: 5.5 Å -p-p stating and CH-p -, and 3. 

Supplementary


17a and 20 )

 20 . Fulfilling these criteria grants the selection of CSPs associated to ligand recognition, irrespective of whether they originate from the presence of a ligand in the vicinity, or from long-range effects. Only such methyl signals were selected for lineshape analysis with TITAN. Detailed examples from EndoS2 E186L and EndoBT D312A/E314L can be scrutinized in Fig. 6B and in Supplementary Figs. 17c, respectively. Following this methodology, we could safely identify 10 methyl signals reporting for the binding of EndoS2 E186L to CT, CT n-1 and Rituximab-Fc, and another 10 signals reporting for the interaction of EndoBT D312A/E314L with HM (signals 1-10 in Supplementary Fig. 23 and signals 11-20 in Supplementary Fig. 24, respectively). EndoS2 also possesses a calcium-binding pocket located in the b-sandwich (BS) 


Figure 12 |

 12 Average distances derived from 1µs MD simulations between different residues of EndoS and Fc for the simulated complexes. Box-and-whisker plots, showing the minimum, maximum, median, 1st quartile, and 3rd quartile bars, as well as the interquartile range, based on n = 250 evenly distributed values obtained from the MD 


Supplementary Figure 13 |

 13 5 Å -hydrogen bond-. Errors are given as SD. The numbers in red and in parentheses are the experimental values found in the cryo-EM structure. The dashed lines represent the distance values found in the cryo-EM structure of EndoS-Fc. Average distances derived from 1µs MD simulations for two salt-bridge interactions between EndoS and Fc for the simulated complexes. Box-andwhisker plots, showing the minimum, maximum, median, 1st quartile, and 3rd quartile bars, as well as the interquartile range, based on n = 250 evenly distributed values obtained from the MD trajectories. The side chain nitrogen of Lys and the carbon atom of the guanidino group of Arg, together with the oxygens of the carboxylate of Glu residues are considered for these calculations (cut-off distance 4.5 Å). Errors are given as SD. The numbers in red and in parentheses are the experimental values found in the cryoEM structure. The dashed lines represent the distance values found in the cryoEM structure of EndoS-Fc. 


2 )

 2 Supplementary Figure 15 | r.m.s.d. value (±SD) of the peptide backbone of the different domains derived from 1 µs MD simulations for the simulated complexes is shown. Boxand-whisker plots, showing the minimum, maximum, median, 1st quartile, and 3rd quartile bars, as well as the interquartile range, based on n = 250 evenly distributed values obtained. 




  




  




  




  




  




  




  




  




  




  




  




  




  




  




  




  




  




  




  




  




  




  




  




  




  




  




  




  




  




  




  




  




  




  




  




  




  




  




  




  


Table 3 . Sequence of primers used to generate EndoS, EndoS2 and Fc mutants.

 3 
	EndoS	Primers
	H236A_F	GGATGTTGAAgcaGATAGTATTCCAAAAGTTGAC
	H236A_R	ACATCTAAGCCATCAAGG
	W314A_F	GAAAGGTGGTgcaGAGCCTGTTTC
	W314A_R	TCTCCTTGTGAACCATAG
	E315A_F	AGGTGGTTGGgcaCCTGTTTCTAATC
	E315A_R	TTCTCTCCTTGTGAACCATAG
	N319A_F	GCCTGTTTCTgcaCGACCTGAAAAAAC
	N319A_R	TCCCAACCACCTTTCTCTC
	K323A_F	TCGACCTGAAgcaACAATGGAAGAAC
	K323A_R	TTAGAAACAGGCTCCCAAC
	E354A_F	AAATGCTCAAgcaGGGAATCTTTG
	E354A_R	TCCTCATAGAAAGAAAAACC
	W803A_F	CTCTTTAGGAgcaGATTCTAAGCAAAGTATTATATTTAAATTG
	W803A_R	ATATTATCAGTCTCACTGC
	Y909A_F	AGGAGATAGAgcaAGTTCGCCAGTAGTC
	Y909A_R	TTAGTATCAAAGACAACTCG
	Strings for EndoS alanine scan	Fragment
	ΔLoop411-428 1	GAAGTGCCATTCCGCCTGACCTGCGCGCGGAGCTCCTTACATCAACCTTC
		TCCTCGTTCAAGTGTACGGCTCGCAGGGCGAAAAGGGCGGGTGGGAACC
		GGTGTCAAACCGCCCGGAGAAGACCATGGAAGAGCGTTGGCAGGGCTAC
		TCGAAATACATCCGCCCGGAGCAGTATATGATCGGCTTCAGCTTTTACGA
		AGAGAACGCGCAGGAAGGCAACCTGTGGTACGACATCAACTCACGTAAA
		GATGAAGATAAGGCCAACGGCATCAATACCGATATTACCGGCACTCGCG
		CGGAGCGCTACGCCCGCTGGCAGCCGAAAACTGGCGGCGTGAAAGGTGG
		AATCTTTAGCTATGCCATCGATCGCGACGGAGTTGCGATTTTCCATAGCG
		ACTACTCAGTGTCGAAAGCCCTGAAAACTGTGATGCTGAAGGACAAATC
		ATACGACCTCATCGACGAAAAGGACTTTCCTGACAAAGCCCTTCGCGAGG
		CCGTTATGGCTCAAGTGGGGACGCGCAAAGGCGACCTGGAGCGCTTTAA
		CGGTACGCTGCGCCTGGACAACCCGGCTATCCAGAGCCTTGAGGGACTGA
		ACAAGTTCAAGAAGCTGGCGCAGCTGGATCTGATCGGTCTTAGCCGTATC
		ACCAAACTGGATCGCTCAGTCCTGCCTGCCAACATGAAACCGGGGAAGG
		ACACGCTCGAGACTGTGCTGGAGACATACAAGAAAGACAATAAAGAGGA
		GCCGGCGACCATTCCGCCGGTGAGTCTGAAAGTCTCGGGGTTGACAGGAC
		TCAAGGAGCTGGACCTTTCTGGCTTCGATCGCGAGACACTGGCAGGGCTC
		GACGCGGCGACATTAACTTCATTGGAGAAGGTGGACATCAGCGGGAATA
		AGCTGGACCTTGCGCCGGGTACGGAGAACCGCCAGATCTTCGACACGAT
		GCTTAGCACCATTTCCAACCACGTCGGCTCGAACGAGCAGACGGTCAAGT
		TCGATAAACAGAAGCCTACGGGTCACTATCCCGACACATACGGAAAGAC
		CTCATTACGTTTGCCGGTTGCCAACGAGAAGGTAGACCTGCAGTCACAAT
		TGCTGTTCGGCACCGTAACCAACCAGGGTACTCTGATTAACAGTGAGGCC
		GATTACAAAGCATATCAGAACCACAAGATCGCCGGTCGCAGTTTCGTGGA
		CTCGAATTACCACTATAACAATTTCAAGGTCAGCTACGAAAATTACACAG
		TGAAGGTTACAGACAGCACTCTGGGGACTACCACGGATAAGACGCTAGC
		AGCGCGCAGGCTAGGTGGAGGCTCAGTG
	K70A/Q73A/E74	GAAGTGCCATTCCGCCTGACCTGCGCGCCATATGGAAGAAAAGACAGTG
	A/Q76A/K77A 2	CAAGTTCAAAAGGGGCTCCCGTCCATTGACTCATTACACTACCTCTCCGA
		AAACAGTAAGAAGGAGTTCAAGGAAGAGTTGAGCGCCGCCGGCGCCGCT

Table 3

 3 (continued). 


	Supplementary	
		AACTGCTGTTCGGTACGGTTACGAACCAGGGTACGCTTATTAACTCGGAA
		GCGGATTACAAAGCCTATCAGAACCACAAGATCGCCGGTCGCAGTTTCGT
		GGACAGCAATTACCACTATAACAATTTCAAGGTCTCGTACGAAAATTACA
		CGGTAAAGGTCACGGACAGCACGTTAGGGACGACCACGGATAAGACGCT
		AGCAGCGCGCAGGCTAGGTGGAGGCTCAGTG
	Y416A/Q419A/K4	GAAGTGCCATTCCGCCTGACCTGCGCGCGGAGCTCCTTACATCAACCTGC
	20A/E421A/F422A	TGTTAGTCCAAGTGTACGGCAGCCAGGGCGAAAAGGGCGGCTGGGAACC
	/K423A 1	GGTATCCAACCGTCCAGAGAAGACCATGGAAGAGCGTTGGCAGGGCTAC
		AGTAAATACATCCGCCCGGAGCAGTATATGATCGGGTTCTCGTTTTACGA
		AGAGAACGCGCAGGAAGGCAACTTGTGGTACGACATCAACAGCCGTAAA
		GATGAAGATAAGGCGAACGGCATCAATACCGATATTACGGGTACTCGCG
		CAGAGCGCTACGCGCGCTGGCAGCCGAAAACCGGCGGCGTTAAAGGCGG
		AATCTTTAGTTATGCCATCGATCGCGACGGCGTCGCCCACCAGCCTAAGA
		AGGCCGCAAAGGCTGCCGCCGCCGCCGATGCTACCGACAATATCTTTCAT
		TCCGACTACTCCGTTTCTAAAGCGCTCAAAACGGTGATGCTGAAGGACAA
		AAGCTACGACTTGATCGACGAAAAGGACTTTCCGGACAAAGCCTTACGTG
		AGGCGGTAATGGCCCAAGTCGGCACTCGTAAGGGCGACCTCGAGCGCTTT
		AACGGTACTCTGCGCCTGGACAACCCTGCCATCCAGTCATTGGAAGGCTT
		AAACAAGTTCAAGAAGTTGGCCCAGTTGGATCTTATCGGGTTGTCCCGTA
		TCACCAAACTGGATCGCTCGGTACTGCCAGCAAACATGAAACCCGGTAA
		GGACACTCTGGAGACGGTGCTGGAGACTTACAAGAAAGACAATAAAGAG
		GAGCCGGCCACCATTCCGCCGGTTAGTCTTAAAGTGTCAGGACTGACCGG
		ACTCAAGGAGCTGGACCTGAGCGGGTTCGATCGCGAGACGCTTGCCGGC
		CTGGACGCAGCGACCCTGACATCGCTCGAAAAGGTGGACATCAGCGGTA
		ATAAGCTCGATCTGGCGCCGGGGACCGAGAACCGCCAGATCTTCGACAC
		GATGTTATCGACCATTTCTAACCACGTGGGCTCCAACGAGCAGACCGTAA
		AGTTCGATAAACAGAAGCCGACCGGCCACTATCCTGACACTTACGGTAAG
		ACGAGCCTTCGTCTCCCGGTAGCCAACGAGAAGGTGGACCTGCAGTCTCA
		ATTACTGTTCGGCACGGTAACGAACCAGGGCACGTTAATTAACAGCGAG
		GCGGATTACAAAGCGTATCAGAACCACAAGATCGCAGGCCGCTCATTCGT
		GGACTCCAATTACCACTATAACAATTTCAAGGTATCCTACGAAAATTACA
		CGGTAAAGGTCACAGACAGTACCTTAGGCACAACCACGGATAAGACGCT
		AGCAGCGCGCAGGCTAGGTGGAGGCTCAGTG
	Y541A/K543A/D5	GAAGTGCCATTCCGCCTGACCTGCGCGCGGAGCTCCTTACATCAACCTGC
	44A/N545A/K546	TTCTTGTTCAAGTGTACGGCAGCCAGGGCGAAAAGGGCGGGTGGGAACC
	A 1	AGTAAGTAACCGCCCGGAGAAGACGATGGAAGAGCGTTGGCAGGGCTAC
		TCCAAATACATCCGCCCGGAGCAGTATATGATCGGCTTCTCCTTTTACGA
		AGAGAACGCCCAAGAGGGTAACCTCTGGTACGACATCAACAGCCGTAAA
		GATGAAGATAAGGCCAACGGGATCAATACGGATATTACCGGCACCCGCG
		CAGAGCGTTACGCGCGCTGGCAGCCGAAAACTGGCGGTGTCAAAGGCGG
		AATCTTTAGCTATGCGATCGATCGCGACGGCGTGGCGCACCAGCCAAAGA
		AGTACGCCAAGCAAAAGGAATTCAAAGATGCCACCGACAATATCTTTCAT
		AGCGACTACTCCGTGAGTAAAGCTCTGAAAACCGTGATGCTGAAGGACA
		AAAGTTACGACCTTATCGACGAAAAGGACTTTCCGGACAAAGCCTTACGC
		GAGGCCGTTATGGCCCAAGTAGGTACGCGTAAGGGCGACCTGGAGCGCT
		TTAACGGGACCCTGCGCCTTGACAACCCGGCCATCCAGTCGCTTGAGGGG
		CTGAACAAGTTCAAGAAGCTGGCACAGCTTGATCTGATCGGTCTTAGTCG
		TATCACCAAACTGGATCGCTCCGTGCTGCCGGCAAACATGAAACCGGGTA
		AGGACACTCTGGAGACTGTGTTAGAGACGGCCAAGGCCGCCGCAGCGGA
		AGAGCCCGCCACGATTCCGCCGGTGAGCCTGAAAGTGAGTGGCTTAACA
		GGCTTGAAGGAGCTGGACTTAAGCGGCTTCGATCGCGAGACGCTGGCGG
		GGTTGGACGCGGCCACCCTGACTAGTCTTGAGAAGGTCGACATCAGCGGT
		AATAAGCTGGACCTGGCGCCGGGCACGGAGAACCGTCAGATCTTCGACA
		CGATGCTGAGTACCATTTCGAACCACGTGGGCTCTAACGAGCAGACCGTC
		AAGTTCGATAAACAGAAGCCGACCGGACACTATCCGGACACTTACGGTA
		AGACGTCACTCCGTCTGCCTGTCGCGAACGAGAAGGTAGACCTGCAGTCA
		CAACTGTTATTCGGAACGGTTACCAACCAGGGCACACTGATTAACTCTGA
		GGCCGATTACAAAGCGTATCAGAACCACAAGATCGCAGGGCGCTCATTC
		GTGGACTCTAATTACCACTATAACAATTTCAAGGTCTCGTACGAAAATTA

Table 3

 3 (continued). Supplementary 



Table 3

 3 (continued). 


	Supplementary	
		CACGGTGAAGGTGACCGACAGCACCCTGGGCACTACCACCGATAAGACG
		CTAGCAGCGCGCAGGCTAGGTGGAGGCTCAGTG
	ΔLoop 314-323 1	GAAGTGCCATTCCGCCTGACCTGCGCGCGGAGCTCCTTACATCAACCTGC
		TGCTCGTTCAAGTGTACGGCTCTCAGGGCGAAAAGGGCGGCACCATGGA
		AGAGCGTTGGCAGGGATACTCCAAATACATCCGCCCGGAGCAGTATATG
		ATCGGCTTCTCATTTTACGAAGAGAACGCCCAGGAAGGTAACCTGTGGTA
		CGACATCAACAGCCGTAAAGATGAAGATAAGGCGAACGGCATCAATACC
		GATATTACAGGGACTCGCGCGGAGCGTTACGCGCGTTGGCAGCCTAAAA
		CTGGTGGAGTCAAAGGCGGAATCTTTAGCTATGCAATCGATCGCGACGGC
		GTCGCGCACCAGCCGAAGAAGTACGCGAAGCAAAAGGAATTCAAAGATG
		CCACCGACAATATCTTTCATTCGGACTACTCAGTGAGCAAAGCGCTGAAA
		ACGGTGATGCTGAAGGACAAATCATACGACTTGATCGACGAAAAGGACT
		TTCCGGACAAAGCGTTACGCGAGGCCGTAATGGCACAAGTAGGTACACG
		CAAAGGGGACTTAGAGCGCTTTAACGGTACCCTGCGCTTAGACAACCCGG
		CCATCCAGAGCTTGGAAGGCCTCAACAAGTTCAAGAAGCTCGCCCAGCTC
		GATCTGATCGGACTGTCGCGTATCACCAAACTGGATCGCTCGGTGCTGCC
		TGCAAACATGAAACCGGGTAAGGACACTCTTGAGACCGTGCTGGAGACA
		TACAAGAAAGACAATAAGGAAGAGCCCGCAACCATTCCGCCGGTTAGCC
		TCAAAGTGAGTGGACTGACGGGGTTGAAGGAGCTGGACCTGAGTGGATT
		CGATCGCGAGACGCTGGCCGGCCTGGACGCGGCGACTCTTACTTCACTGG
		AGAAGGTGGACATCTCGGGGAATAAGCTGGACCTCGCCCCGGGTACCGA
		GAACCGCCAGATCTTCGACACCATGTTGAGCACCATTTCTAACCACGTGG
		GTTCGAACGAGCAGACCGTAAAGTTCGATAAACAGAAGCCGACCGGACA
		CTATCCGGACACGTACGGTAAGACCAGCCTTCGTCTGCCGGTAGCCAACG
		AGAAGGTCGACCTGCAGTCTCAACTGCTTTTCGGCACCGTCACGAACCAG
		GGCACGCTCATTAACTCTGAGGCCGATTACAAAGCGTATCAGAACCACAA
		GATCGCCGGTCGCTCGTTCGTAGACTCCAATTACCACTATAACAATTTCA
		AGGTCAGCTACGAAAATTACACGGTAAAGGTCACGGACAGCACTCTTGG
		GACTACCACGGATAAGACGCTAGCAGCGCGCAGGCTAGGTGGAGGCTCA
		GTG
	ΔLoop 828-836 3	GAAGTGCCATTCCGCCTGACCTGCGCGCACGCTAGCAACCGACAAAGAG
		GAAACTTACAAAGTCGATTTCTTCAGTCCTGCGGACAAAACCAAGGCGGT
		GCACACCGCAAAGGTAATCGTAGGCGATGAGAAGACAATGATGGTGAAC
		CTCGCGGAAGGGGCCACCGTGATTGGCGGCAGCGCCGATCCAGTCAACG
		CCCGCAAAGTGTTCGACGGCCAGCTTGGGTCCGAAACCGACAACATTAGC
		CTTGGCTGGGACAGCAAACAGTCCATCATTTTCAAGCTGAAAGAGGACG
		GTCTGATCAAACACTGGCGCTTCTTTAACGACAAGCCGATCCAAGAGGCG
		TCACTGCAGATCTTCAACATTAAGGACTACAACCTCGACAACCTGCTTGA
		GAACCCGAACAAGTTCGACGACGAGAAGTACTGGATCACCGTGGACACA
		TATTCCGCGCAAGGGGAACGCGCGACCGCCTTTAGCAACACCTTGAACAA
		CATCACCTCAAAGTACTGGCGCGTGGTGTTCGACACCAAGGGTGACCGTT
		ACTCGTCACCGGTTGTGCCCGAGCTGCAGATCCTTGGGTACCCCCTGCCG
		AATGCGGATACAATTATGAAGACCGTTACCACAGCCAAGGAACTGTCGC
		AGCAGAAGGACAAATTCTCGCAGAAAATGTTAGACGAACTGAAGATCAA
		GGAAATGGCGCTGGAGACCAGCCTGAATAGCAAAATCTTCGACGTTACG
		GCGATCAACGCCAACGCGGGTGTGCTGAAGGACTGCATCGAAAAGCGTC
		AACTCCTGAAGAAGGGATCCGGCGCGCGCAGGCTAGGTGGAGGCTCAGT
		G
	Fc region	Primers
	R292A_F	GACCAAGCCTgctGAGGAACAGTACAAC
	R292A_R	TTGGCGTTGTGCACTTCC
	E293A_F	CAAGCCTAGAgctGAACAGTACAACAGC
	E293A_R	GTCTTGGCGTTGTGCACT
	E294A_F	GCCTAGAGAGgctCAGTACAACAGC
	E294A_R	TTGGTCTTGGCGTTGTGC
	Q295A_F	TAGAGAGGAAgctTACAACAGCACCTACAGAGTGGTG
	Q295A_R	GGCTTGGTCTTGGCGTTG

Table 3

 3 (continued). 


	Supplementary	
	Y296A_F	AGAGGAACAGgctAACAGCACCTACAGAG
	Y296A_R	CTAGGCTTGGTCTTGGCG
	S298A_F	ACAGTACAACgctACCTACAGAGTGGTG
	S298A_R	TCCTCTCTAGGCTTGGTC
	S298A/Y296A/E29 4A/R292A_F	ggctaacgctACCTACAGAGTGGTGTCC
	S298A/Y296A/E29 4A/R292A_R	tgagcctcagcAGGCTTGGTCTTGGCGTT
	Y296F_F	AGAGGAACAGttcAACAGCACCTAC
	Y296F_R	CTAGGCTTGGTCTTGGCG
	Y300F_F	CAACAGCACCttcAGAGTGGTGT
	Y300F_R	TACTGTTCCTCTCTAGGC
	Y296_Y300F_F	caccttcAGAGTGGTGTCCGTGCTG
	Y296_Y300F_R	ctgttgaaCTGTTCCTCTCTAGGCTTGG
	I253A_F	CACCCTGATGgctAGCAGAACCC
	I253A_R	TCCTTAGGCTTTGGAGGA
	H310A_F	GACCGTGCTGgctCAGGATTGGCTG
	H310A_R	AGCACGGACACCACTCTG
	H310A/L314A_F	ttgggctAACGGCAAAGAGTACAAGTGC
	H310A/L314A_R	tcctgagcCAGCACGGTCAGCACGGA
	Q311A/N315A_F	gctggctGGCAAAGAGTACAAGTGCAAGG
	Q311A/N315A_R	caatcagcGTGCAGCACGGTCAGCAC
	E430A/H435A_F	cacaacgctTACACCCAGAAAAGCCTGTC
	E430A/H435A _R	cagggcagcGTGCATCACGCTGCAGCT
	H268A/E269A/D27	tcccgctGTGAAGTTCAATTGGTACGTG
	0A/E272A_F	
	H268A/E269A/D27	gcagcagcGGACACATCCACCACCAC
	0A/E272A_R	
	P329A_F	CAAGGCCCTGgccGCTCCTATCG
	P329A_R	TTGGACACCTTGCACTTGTAC
	A330G_F	GGCCCTGCCTggcCCTATCGAGA
	A330G_R	TTGTTGGACACCTTGCAC
	A330W_F	GGCCCTGCCTtggCCTATCGAGA
	A330W_R	TTGTTGGACACCTTGCAC
	P329A/A330G_F	CAAGGCCCTGgccggcCCTATCGAGAAAAC
	P329A/A330G_R	TTGGACACCTTGCACTTG
	P329A/A330W_F	CAAGGCCCTGgcctggCCTATCGAGAAAAC
	P329A/A330W_R	TTGGACACCTTGCACTTG
	Loop325-	
	331(NKALPAP--	cggagggggtATCGAGAAAACCATCAGC
	>GGGSGGG)_F	
	Loop325-	
	331(NKALPAP--	gaaccgccaccGGACACCTTGCACTTGTAC
	>GGGSGGG)_R	
	Loop325-	
	331(NKALPAP--	ccctgctgggATCGAGAAAACCATCAGC
	>GGGGPAG)_F	

Table 3 (

 3 continued). Restriction site for cloning: SacI and NheI Restriction site for cloning: NdeI and HindIII Restriction site for cloning: NheI and BamH Supplementary 


	Loop325-	
	331(NKALPAP--	ccaccgccaccGGACACCTTGCACTTGTAC
	>GGGGPAG)_R	

Table 3 (

 3 continued). 


	Supplementary

Table 5 . List of NMR experiments and experimental details used for the assignment of CT, CTn-1 and HM N-glycans. Experiments were acquired at 310 K.

 5 
	Experiment	Pulse program from	NS TD	SW	Carrier	Observations	
		Bruker library			(ppm)	F2/F1			
						(ppm)			
	1 H	zgesgp	64	32768	11.65	4.7	Water	suppression	with
							Squa100.1000 shaped pulse, 8
							ms	length	to	minimise
							suppression	of	anomeric
							signals 2 .	
	[ 1 H, 13 C]-	hsqcedetgpsisp2.2	32	2048x256 12.5x146 4.7/86	J CH = 145 Hz	
	Edited-HSQC								
	[ 1 H, 1 H]-	noesygpphpp	16	2048x256 10x10	4.7/4.7	700 ms mixing time
	NOESY								
	[ 1 H, 1 H]-	mlevgpph19	16	2048x256 10x10	4.7/4.7	13 ms or 200 ms mixing times.
	TOCSY						Distance between nulls = 5.2
							ppm		
	[ 1 H, 13 C]-	hmbcgplpndqf	128 2048x240 12x92	4.7/66	Long range J CH = 6 Hz
	HMBC								
	[ 1 H, 13 C]-	h2bcetgpl3	328 2048x302 12x92	4.7/66	J CH min -max = 129 -190 Hz
	HMQC-								
	COSY								
	Recovery delay was set to 1.5 -2.3 s in all experiments.					

Table 6 . Glycan 1 H/ 13 C chemical shifts of CT, CT n-1 and HM N-glycans (referenced to TSP). Experiments were acquired at 310 K.

 6 
	Residue with residue number C1/	C2/	C3/	C4/	C5/	C6/	Acetyl-CH 3 Ca/	Cb/
		H1	H2	H3	H4	H5	H6a;H6b		Ha	Hba;Hbb
	CT									
	Asn	-	-	-	-	-	-	-	53.8/	38.0/
									3.98	2.94;2.86
	GlcNAc 1	80.9/	56.4/	75.6/	81.6/	79.0/	62.8/	24.9/	-	-
		5.08	3.87	3.78	3.66	3.60	3.84;3.66	2.02		
	GlcNAc 2	104.0/	57.8/	74.7/	82.2/	77.3/	62.8/	25.0/	-	-
		4.66	3.79	3.80	3.74	3.63	3.89;3.76	2.09		
	Man 3	103.1/	72.9/	83.3/	68.5/	77.3/	68.7/	-	-	-
		4.78	4.25	3.79	3.79	3.62	3.97;3.81			
	Man 4	102.3/	79.3/	72.2/	70.1/	75.8/	64.4/	-	-	-
		5.14	4.20	3.91	3.51	3.63	3.92;3.64			
	Man 4'	99.8/	79.2/	72.2/	70.1/	75.8/	64.4/	-	-	-
		4.94	4.12	3.91	3.51	3.63	3.92;3.64			
	GlcNAc 5/5'	102.3/	57.6/	74.9/	81.5/	77.5/	62.8/	25.1/	-	-
		4.60	3.75	3.75	3.74	3.59	3.99;3.86	2.06		
	Gal 6/6'	105.7/	73.7/	75.4/	71.3/	76.3/	63.7/	-	-	-
		4.48	3.56	3.68	3.95	3.76	3.77;3.77			
	CT n-1									
	a-GlcNAc 2	93.2/	56.4/	72.5/	82.7/	77.1/	62.1/	24.6/	-	-
		5.21	3.90	3.93	3.91	3.62	3.73;3.73	2.06		
	b-GlcNAc 2	97.7/	58.9/	72.5/	82.7/	77.1/	62.8/	24.8/	-	-
		4.72	3.71	3.93	3.91	3.62	3.79;3.77	2.06		
	Man 3	103.2/	73.0/	83.2/	68.2/	77.1/	68.6/	-	-	-
		4.77	4.24	3.77	3.79	3.62	3.96;3.79			
	Man 4	102.2/	79.3/	72.2/	70.1/	76.3/	64.4/	-	-	-
		5.12	4.19	3.90	3.50	3.75	3.91;3.62			
	Man 4'	99.8/	79.2/	72.2/	70.1/	75.6/	64.4/	-	-	-
		4.92	4.11	3.90	3.50	3.71	3.91;3.62			
	GlcNAc 5/5'	102.2/	57.6/	74.9/	81.4/	77.4/	62.8/	25.1/	-	-
		4.58	3.74	3.72	3.72	3.57	3.98;3.84	2.05		
	Gal 6/6'	105.7/	73.8/	75.3/	71.3/	78.1/	63.7/	-	-	-
		4.47	3.54	3.66	3.93	3.72	3.75;3.75			
	HM									
	Asn	-	-	-	-	-	-	-	53.8/	38.0/
									3.99	2.93;2.88
	GlcNAc 1	80.8/	56.3/	75.0/	81.5/	79.1/	62.8/	24.9/	-	-
		5.09	3.87	3.79	3.66	3.61	3.85;3.66	2.03		
	GlcNAc 2	104.0/	57.8/	75.0/	81.9/	77.2/	62.8/	25.0/	-	-
		4.63	3.79	3.79	3.74	3.61	3.89;3.74	2.08		
	Man 3	103.0/	73.1/	84.0/	68.3/	77.2/	68.11/	-	-	-
		4.78	4.23	3.74	3.91	3.61	4.03;3.75			
	Man 4	103.5/	81.3/	72.8/	69.7/	76.1/	63.8/	-	-	-
		5.34	4.12	4.01	3.77	3.82	3.90;3.77			
	Man 4'	102.4/	81.6/	81.5/	67.8/	74.2/	68.7/	-	-	-
		4.88	3.90	3.90	3.88	3.85	3.95;3.80			
	Man 5	103.3/	81.3/	72.8/	69.7/	76.1/	63.8/	-	-	-
		5.31	4.12	4.01	3.71	3.82	3.90;3.77			
	Man 5'	103.3/	81.3/	72.9/	69.7/	76.1/	63.8/	-	-	-
		5.41	4.12	3.96	3.71	3.82	3.90;3.77			
	Man 5''	100.9/	81.3/	72.9/	69.7/	75.5/	63.8/	-	-	-
		5.14	4.03	3.96	3.71	3.71	3.90;3.77			
	Man 6/6'/6''	105.0/	72.8/	73.2/	69.6/	76.0/	63.8/	-	-	-
		5.07	4.09	3.86	3.67	3.78	3.90;3.77			

Table 7 . Example of shell script for NMR data processing with NMRPipe

 7 
	#!/bin/csh		
	foreach spec (1 3 5 7 9 11 12 13 14 15)
	cd spec/$spec		
	bruk2pipe -in ./ser \	
	-bad 0.0 -ext -aswap -AMX -decim 8330.66666666667 -dspfvs 20
	-grpdly 67.9856719970703 \	
	-xN	512 -yN	256 \
	-xT	256 -yT	128 \
	-xMODE	DQD -yMODE	States-TPPI \
	-xSW	2400.768 -ySW	3320.053 \
	-xOBS	600.230 -yOBS	150.931 \
	-xCAR	0.8578 -yCAR	19.7253 \
	-xLAB	1H -yLAB	13C \
	-ndim	2 -aq2D	TPPI \
	-out ./test.fid -verb -ov	
	nmrPipe -in test.fid \	
	| nmrPipe -fn EM -lb 1.0 -c 1.0		\
	| nmrPipe -fn ZF -auto		\
	| nmrPipe -fn FT -auto		\
	| nmrPipe -fn PS -p0 62 -p1 0 -di -verb	\
	| nmrPipe -fn BASE -nw 5 -nl 0% 2% 98% 100%	\
	| nmrPipe -fn CS -rs 0Hz -sw		\
	| nmrPipe -fn EXT -x1 -0.3ppm -xn 2.4ppm -sw	\
	| nmrPipe -fn TP		\
	| nmrPipe -fn EM -lb 1.0 -c 1.0		\
	| nmrPipe -fn ZF -auto		\
	| nmrPipe -fn FT -auto		\
	| nmrPipe -fn PS -p0 0 -p1 0 -di -verb	\
	| nmrPipe -fn BASE -nw 2 -nl 0% 2% 98% 100%	\
	| nmrPipe -fn CS -rs 0Hz -sw		\
	-ov -out ../test-$spec.ft2	
	rm test.fid		
	cd ..		
	cd ..		
	end		

Table 8 . Dissociation constants K D , on-and off-rate constants k on and k off for CT, Rituximab-Fc, HM and Ca 2+ binding to EndoS2 E186L and EndoBT D312A/E314L in the presence of constant additives from fitting a two-state binding model to methyl- TROSY titration data using TITAN algorithm.

 8 
	N° Enzyme	Ligand	Additives 1	K D (µM)	k off (s -1 )	k on (M -1 s -1 )
	1	EndoS2 E186L	CT	CaCl 2	111.2 ± 9.7	11.7 ± 1.9	1.1x10 5 ± 2.0x10 4
	2		Fc	CaCl 2	3.8 ± 1.0	4.5 ± 1.8	1.2x10 6 ± 5.7x10 5
	3		CaCl 2	CT	4100 ± 300	-	-
	4		CaCl 2	Fc	-	-	-
	5	EndoBT D312A/E314L HM	CaCl 2	0.4 ± 0.3	2.4 ± 1.1	5.5x10 6 ± 4.8x10 5
	6	EndoBT D312A/E314L CT	-	>755 (if present)	-	-



  At this point, it is instructive to compare dissociation constants, on-and off-rate constants from ENGases with diverse specificities.EndoBT-3987 is a globular ENGase secreted by the gram-negative symbiont Bacteroides thetaiotaomicron, which displays high specificity for mammalian HM-type N-glycans.Titrations of EndoBT D312A/E314L with HM in the absence and presence of CaCl 2 afforded dissociation constant, on-and off-rate constants in the same order of magnitude as those previously disclosed for EndoS2 E186L binding Rituximab-Fc.As expected, no affinity was observed for CT glycan, even at ~750 µM glycan concentrations.(Table1,entry6, lineshape fittings for the titration of HM can be scrutinized in Supplementary Figs.33 and 34). 



Samples containing additives were prepared at saturating concentrations and diluted a maximum of 10% during the course of the titrations. Final additive concentrations were: 

mM CaCl 2 , 830 µM CT, 56 µM Fc or 72 µM Fc aglycan in samples containing EndoS2 E186L , and 291mM CaCl 2 in samples containing EndoBT D312A/E314L . 

SUPPLEMENTARY INFORMATION


Mechanism of antibody-specific deglycosylation and immune evasion by Streptococcal IgG-specific endoglycosidases


Trastoy et al.


Supplementary Table 1. CryoEM data collection, refinement and validation statistics

EndoS E235A -Fc Supplementary Table 2. SAXS data collection and refinement parameters. Supplementary Table 4. Precursors and quantities used for the preparation of [U-15 N, 2 H] MIL proS V proS [ 13 C, 1 H 3 ]-methyl labeled endoglycosidases. A systematic review on precursors required for optimal methyl group isotope labelling in E. coli can be found somewhere else 1 . The frames used for data analysis of the complex are highlighted with a red square. b SEC-SAXS profile of EndoS2 E186L , IgG1(Fc) and EndoS2 E186L -Fc complex. The frames used for data analysis of the complex are highlighted with a red square. c SAXS scattering curve of EndoS E235A , IgG1(Fc) and EndoS E235A -Fc. d P(r) functions distributions of EndoS E235A , IgG1(Fc) and EndoS E235A -Fc complex. e Normalized Kratky plot EndoS E235A , IgG1(Fc) and EndoS E235A -Fc complex. f SAXS scattering curve of EndoS2 E186E , IgG1(Fc) and EndoS2 E186E -Supplementary Figure 7 Fc (PDB code 8A64) colored in grey. In order to have insights on the possible location of the side chain residues that could be involved in the enzyme-substrate interactions, we locally compared/superimposed the high-resolution EndoS (PDB code 6EN3; yellow) and Fc region (PDB code 2DTS; orange) crystal structures. Therefore, we visualize potential side chain interactions between residues that are within interaction distance based on flexible fitting of the superimposed crystal structures. b X-ray crystal structure of B-domain from Protein A in complex with IgG1 Fc IgG1(PDB code 5UAY). c X-ray crystal structure of the C2 fragment of Protein G in complex with IgG1 Fc (PDB code 1FCC) d X-ray crystal structure of the ternary complex between FcRn, serum albumin and IgG1 Fc-YTE (PDB code 4N0U). 

Labeled amino acid


Supplementary References
SCÅTTER SCÅTTER SCÅTTER SCÅTTER SCÅTTER Data processing PRIMUS/ SCÅTTER