Computational 3-D modeling and site-directed mutation of an antibody that binds Neu2en5Ac, a transition state analogue of a sialic acid

An antibody against a transition state analog (TSA) may share some common f eatures with an enzyme that produces such a transition state. SIC172 antibo dy binds specifically to Neu2en5Ac, a TSA of Neu5Ac in the sialidase reacti on, but has no catalytic activity. To understand how the antibody recognize s Neu2en5Ac and to find out if it is possible to convert it to a catalytic antibody, we made and sequenced the SIC172 ScFv, and constructed a 3-D mode l of it. The VH-CDR3 contains a unique sequence with Cys at H95. The 3-D mo del showed that Cys-H95 is exposed inside the antigen-binding cavity. After affinity docking, 4 types emerged. In type I, the carboxyl group of Neu2en 5Ac is located near the Cys-H95 and neighboring positively charged residues . The change of Cys-H95 to Asp by site-directed mutation decreased the bind ing activity, while a change to Arg did not. These and other mutation exper iments, and further modeling of mutant Fv, support the 3-D model and dockin g type I. A comparison with sialidase indicates that SIC172 antibody appear s to have some groups of residues that are conserved at the active site of the enzyme. The possibility of Neu2en5Ac-binding antibody being converted t o a catalytic antibody is discussed. (C) 2001 Wiley-Liss, Inc.