A structural basis for the biochemical behavior of activation-induced deoxycytidine deaminase class-switch recombination-defective hyper-IgM-2 mutants.
Hyper-IgM syndrome type 2 stems from mutations in activation-induced deoxycytidine deaminase (AID) that abolish immunoglobulin class-switch recombination, causing an accumulation of IgM and absence of IgG, IgA, and IgE isotypes. Although hyper-IgM syndrome type 2 is rare, the 23 missense mutations identified in humans span almost the entire gene for AID resulting in a recessive phenotype. Using high resolution x-ray structures for Apo3G-CD2 as a surrogate for AID, we identify three classes of missense mutants as follows: catalysis (class I), substrate interaction (class II), and structural integrity (class III). Each mutant was expressed and purified from insect cells and compared biochemically to wild type (WT) AID. Four point mutants retained catalytic activity at 1/3rd to 1/200th the level of WT AID. These "active" point mutants mimic the behavior of WT AID for motif recognition specificity, deamination spectra, and high deamination processivity. We constructed a series of C-terminal deletion mutants (class IV) that retain catalytic activity and processivity for deletions ≤18 amino acids, with ΔC(10) and ΔC(15) having 2-3-fold higher specific activities than WT AID. Deleting 19 C-terminal amino acids inactivates AID. WT AID and active and inactive point mutants bind cooperatively to single-stranded DNA (Hill coefficients ∼1.7-3.2) with microscopic dissociation constant values (K(A)) ranging between 10 and 250 nm. Active C-terminal deletion mutants bind single-stranded DNA noncooperatively with K(A) values similar to wild type AID. A structural analysis is presented that shows how localized defects in different regions of AID can contribute to loss of catalytic function.
Department of Biological Sciences, University of Southern California, Los Angeles, California 90089-2910, USA.
Yunxiang
Y
Mu
Courtney
C
Prochnow
Phuong
P
Pham
Xiaojiang S
XS
Chen
Myron F
MF
Goodman
D000595:Amino Acid Sequence; D018801:CD2 Antigens / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002384:Catalysis; D003564:Cytidine Deaminase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004277:DNA, Single-Stranded; D006801:Humans; D053306:Hyper-IgM Immunodeficiency Syndrome / Q000201:enzymology / Q000235:genetics; D017578:Immunoglobulin Class Switching; D007075:Immunoglobulin M; D020125:Mutation, Missense; D017354:Point Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017384:Sequence Deletion; D013329:Structure-Activity Relationship; D000098830:AICDA (Activation-Induced Cytidine Deaminase)
D018801:CD2 Antigens; D004277:DNA, Single-Stranded; D007075:Immunoglobulin M; D000098830:AICDA (Activation-Induced Cytidine Deaminase); D003564:Cytidine Deaminase