Structural and functional analysis of APOBEC-related editing enzymes, and the structural basis of antibody specificity in blood type recognition

Activation Induced Deaminase (AID) uses base deamination for class-switch recombination and somatic hypermutation and is related to the mammalian RNA-editing enzyme APOBEC-1. CDD1 is a yeast ortholog of APOBEC-1 that exhibits cytidine deaminase and RNA-editing activity, as well as targets DNA sequences in a bacterial deaminase assay. The crystal structure of CDD1 was solved to 2.0-A resolution and it was used in comparative modeling of APOBEC-1 and AID. The models explain dimerization and the need for trans-acting loops that contribute to active site formation. Substrate selectivity appears to be regulated by a central active site "flap" whose size and flexibility accommodate large substrates in contrast to deaminases of pyrimidine metabolism that bind only small nucleosides or free bases. Most importantly, the results suggested both AID and APOBEC-1 are equally likely to bind single-stranded DNA or RNA, which has implications for the identification of natural AID targets.; In a second study, the molecular basis of antibody specificity was analyzed. The NNA7 Fab recognizes the N-type antigen located at the N-terminus of glycophorin A. This system is of interest because little is known about the molecular basis of antibody-glycopeptide recognition and there is a need to improve diagnostic tools that discern blood-type compatibilities for transfusion reactions. Therefore, native and mutant structures of the NNA7 Fab were solved and refined to 1.83 A and 1.97 A resolution, respectively. The mutant G91S mapped to the light chain variable region and was shown previously to reduce antigen binding. Comparison of the two structures indicated the mutation led to differences in crystal packing that could be linked to the antigen binding pocket. The antigen-binding site itself was occupied by a morpholino-ethane sulfonic (MES) acid molecule that suggested a possible mode of galactose binding. A detailed model for antibody-antigen recognition is described that features a consideration of both peptide and glycogen specificity determinants.