DNA deamination and binding properties of wild type and HIGM2 mutants of activation induced cytidine deaminase

AID is required for SHM and CSR as an initiator protein. Hyper IgM syndrome 2, a primary immunological deficiency disorder caused by AICDA gene (encoding AID) mutations, is characterized by severe decrease in CSR, which is often accompanied with more or less down-regulated SHM.;Here we purified 23 missense AID HIGM2 mutants, which naturally occur in the human population and a few C terminus truncated mutants, one of which is a C-10 amino acid deletion and is known to only cause a defect in CSR but retains WT level or higher activity in SHM.;All the mutants containing one single point mutation showed no detectable deamination activity on ssDNA substrate except for S43P, L98R, and R174S. Comparison of specific activity indicates that they are about 3, 26, 211 fold less active than WT AID respectively. Although these 3 mutants have reduced deamination specific activity, they have similar mutation spectrum to WT AID. To the contrary DeltaC15 and DeltaC10 deletion mutants have about 2 fold increase in specific activity, whereas DeltaC18 and DeltaC17 have reduced specific activity. Sequence alignment indicates that helix 6 is a conserved structural feature of all APOBEC proteins. In agreement with this, a C terminus truncation of 19 or more amino acids disrupted the integrity of helix 6 of AID, resulting in the loss of deamination.;Additionally DNA binding activity of some mutants was tested through rotational anisotropy and their dissociation constants were measured. Compared with WT AID R174S displayed 5 fold less binding affinity and the binding affinity of the DeltaC10 is comparable to WT AID. Interestingly when we fit the data points from DNA binding by plotting the change in anisotropy as a function of enzyme concentration, we found that the ssDNA binding of WT AID fit better with cooperativity, which is abrogated in the case of C terminus deletion mutants, for which a rectangular hyperbolic curve is the best fit.;Through the comparison of AID to some artificial mutants of APOBEC3G-CD2 domain of which the structure has been solved, the molecular mechanism of AID mutants and the relationship of structure and functions will be discussed.