The heavy chain complementarity-determining region 3 (HCDR3) of the anti-oligo(dT) recombinant antibody fragment, DNA-1, contributes significantly to antigen binding (Komissarov, A. A., Calcutt, M. J., Marchbank, M. T., Peletskaya, E. N., and Deutscher, S. L. (1996) J. Biol. Chem. 271, 12241-12246). In the present study, the role of separate HCDR3 residues of DNA-1 in interaction with oligo(dT) was elucidated. Based on a molecular model of the combining site, residues at the base (Arg 98 and Asp 108 ) and in the middle (Tyr
101-ArgPro-Tyr-Tyr 105 ) of HCDR3 were predicted to support the loop conformation and directly contact the ligand, respectively. Twenty-five site-specific mutants were produced as hexahistidine-tagged proteins, purified, and examined for binding to (dT) 15 Antibodies comprise a valuable class of protein produced in mammals and other species in response to antigen. The importance of antibodies is highlighted by their vital roles in immune protection. Mammalian immune systems are capable of generating millions of different antibody specificities in response to foreign antigens. For unknown reasons, a breakdown in immune tolerance can occur resulting in activation of self-reactive B and T cells. This abnormal cascade of events leads to autoimmune disease. A hallmark of the autoimmune disorders systemic lupus erythematosus (SLE) 1 and mixed connective tissue disease is the presence of serum antibodies that recognize nucleic acids (1-3). The presence of high levels of circulating antibodies that bind double-stranded (ds) DNA is diagnostic for SLE, and certain anti-DNA immune complexes contribute to disease pathology (4, 5). It is uncertain what are the distinguishing features of pathogenic versus non-pathogenic antibodies, however. Studies of the binding properties of anti-DNA antibodies have been facilitated by methods to select for DNA-binding antibody fragments (Fab) from bacteriophage display libraries and the ability to produce large quantities of the Fab in Escherichia coli. We previously isolated an anti-single-stranded (ss) DNA-binding Fab, DNA-1, from a bacteriophage display library derived from the immunoglobulin repertoire of an autoimmune MRL/MpJ-lpr/lpr (MRL/lpr) mouse (6). The MRL/lpr murine library was enriched in anti-DNA Fab since these mice spontaneously produce anti-ssDNA and anti-dsDNA antibodies and develop an SLE-like syndrome. DNA-1 was shown to preferentially bind to oligo(dT) of 15 nucleotides or greater in length, with an equilibrium dissociation constant (K d ) of 150 -200 nM (7, 8). The regions responsible for DNA binding are known to reside in the three heavy (H) and light (L) chain complementarity-determining regions (CDRs) of the Fab. It has been shown that the H chain contributes more to the interaction with DNA than the L chain (9). Comparison of HCDR transplantation mutants between DNA-1 and a Fab that bound poorly to DNA demonstrated that HCDR3 of DNA-1 was critical for oligo(dT) binding (7,8). The importance of HCDR3 in binding to other DNA molecules includin...