Mutations in human AID differentially affect its ability to deaminate cytidine and 5-methylcytidine in ssDNA substrates in vitro

Activation-induced cytidine deaminase (AID) is known for its established role in antibody production. AID induces the diversification of antibodies by deaminating deoxycytidine (C) within immunoglobulin genes. The capacity of AID to deaminate 5-methyldeoxycytidine (5 mC) and/or 5-hydroxymethyldeoxycytidine (5 hmC), and consequently AID involvement in active DNA demethylation, is not fully resolved. For instance, structural determinants of AID activity on different substrates remain to be identified. To better understand the latter issue, we tested how mutations in human AID (hAID) influence its ability to deaminate C, 5 mC, and 5 hmC in vitro. We showed that each of the selected mutations differentially affects hAID’s ability to deaminate C and 5 mC. At the same time, we did not observe hAID activity on 5 hmC. Surprisingly, we found that the N51A hAID mutant, with no detectable activity on C, efficiently deaminated 5 mC, which may suggest different requirements for C and 5 mC deamination. Homology modeling and molecular dynamics simulations revealed that the pattern of enzyme-substrate recognition is one of the important factors determining enzyme activity on C and 5 mC. Consequently, we have proposed mechanisms that explain why wild type hAID more efficiently deaminates C than 5 mC in vitro and why 5 hmC is not deaminated.

N1 -number of residues in our model (only residues 5-19, 23-37, 40-181 that have their counterparts in X-ray structure were analyzed) N2 -number of residues in X-ray structure DIST -selected distance cutoff [Å] N -number of residues superimposed under distance cutoff (DIST) GDT_TS -Global Distance Test LGA_S -structure similarity score (0.00 -100.00) LGA_Q -quality score Supplementary Figure S5. Comparison of the generated hAID models with X-ray structure of AID variant -AIDv(Δ15) (PDB ID: 5JJ4). (a) Comparison of conformations sampled during MD simulation of the generated wt hAID model (colored by the secondary structure) with the recently published X-ray structure of AID variant (AIDv(Δ15)) 7 (shown in green). The gray spheres represent zinc ions. (b) Spatial occupancy maps for CA, CB, ND2, and OD1 atoms of N51 residue in the compared structures. The structure of wt hAID model (a representative frame) is shown in beige. The X-ray structure of AIDv(Δ15) is shown in green.
The great similarity of compared catalytic centers and Loops3 proves that the generated model is suitable to propose a function of the N51 residue. (c) A set of representative parameters calculated by LGA server 8 showing quality of the generated models in comparison with the X-ray structure of AIDv(Δ15). c (a), 5hmC (b) and 5mC (c). The deaminase activity was tested for hAID variants: wt, R50A, N51A, R190X. Full-length gel images from Figure 1b,e,g (see main text) are presented. In the case of deamination, a 40-nucleotide-long product was expected. Positive and negative control reactions are indicated by "+" or "-", respectively.

Supplementary Methods
To confirm that the recombinant hAID obtained in a bacterial system (see Methods, main text) represents the true activity of hAID, a detailed characterization of the protein was performed, which included (i) MALDI-TOF analysis; (ii) western blot analysis; (iii) a test of inhibition of hAID activity by 1,10-phenanthroline; (iv) a test of inhibition of hAID activity by tetrahydrouridine; and (v) a detailed characterization of deamination activity on Ccontaining and non-C-containing substrates after each step of deamination activity assay.

(iv) Test of inhibition of hAID activity by tetrahydrouridine (THU)
THU is an analog of the transition state of the enzymatic reaction catalyzed by deaminases of free cytidine. Therefore, THU inhibits bacterial cytidine deaminase but does not inhibit hAID 6 . The test of inhibition was performed for the protein extract after purification on a Glutathione Sepharose 4 FastFlow column. The reactions were carried out according to the standard hAID activity assay (see Methods, main text) and THU was added to the reaction mixtures at a concentration ranging from 2 μM to 10 mM (it has been shown that bacterial deaminase of free cytidine is inhibited by THU at concentrations ranging from 20 to 50 μM 16 ). As presented in Supplementary Figure S12, the observed deamination activity was not influenced by THU. Thus, one can conclude that the observed activity was not the activity of bacterial deaminase.
Supplementary Figure S12. Denaturing PAGE analysis of the products of the hAID activity assay for C deamination performed in the presence of THU at concentrations ranging from 2 μM to 10 mM. In the case of deamination, a 40-nucleotide-long product was expected. "+" -positive control reaction (the activity assay performed without the protein extract and on ssDNA substrate containing U instead of C in the position of the 40th nucleotide); "-"negative control reaction (the activity assay performed without the protein extract and on ssDNA substrate containing C in the position of the 40th nucleotide); "0" -the activity assay for C deamination performed in the presence of the protein extract but without THU; next lanes -the activity assay for C deamination performed in the presence of the protein extract and in the presence of THU at concentrations ranging from 2 μM to 10 mM.

(v) Characteristics of deamination activity on C-containing and non-Ccontaining substrates after each step of deamination activity assay
The hAID activity assay for C deamination (see Methods, main text) is a three-stage reaction wherein a 40-nucleotide product is generated from an 80-nucleotide ssDNA substrate. It was assumed that the presence of the 40-nucleotide product indicates the presence of deamination activity. To unequivocally confirm that the observed activity is the activity specific to cytidine, the activity of the protein extract on ssDNA not containing cytidine was tested (ssDNA containing T instead of C in the position of the 40th nucleotide was used). The analysis showed no effect of the tested protein extract on the ssDNA molecule not containing cytidine (Supplementary Figure S13d), which proved the reaction specificity. Moreover, products formed after the 1 st , 2 nd and 3 rd stages of the activity assay were analyzed to determine the stage at which the final product appeared. In the case of a nuclease