Catalytic antibody model and mutagenesis implicate arginine in transition-state stabilization.
To probe the mechanism of the catalytic antibody NPN43C9, we have constructed a three-dimensional model of the NPN43C9 variable region using our antibody structural database (ASD), which takes maximal advantage of immunoglobulin sequence and structural information. The ASD contains separately superimposed variable light and variable heavy chains, which reveal not only conserved backbone structure, but also structurally conserved side-chain conformations. The NPN43C9 model revealed that the guanidinium group of light chain Arg L96 was positioned at the bottom of the antigen-binding site and formed a salt bridge with the antigen's phosphonamidate group, which mimics the negatively charged, tetrahedral transition states in the hydrolysis reaction. Thus, the model predicts both binding and catalytic functions for Arg L96, which previously had not been implicated in either. First, Arg L96 should enhance antigen binding by electrostatically complementing the negative charge of the antigen, which is buried upon complex formation. Second, Arg L96 should promote catalysis by electrostatically stabilizing the negatively charged transition states formed during catalysis. These hypotheses were tested experimentally by design and characterization of the R-L96-Q mutant, in which Arg L96 was replaced with Gln by site-directed mutagenesis. As predicted, antigen binding in the R-L96-Q mutant was decreased relative to that in the parent NPN43C9 antibody, but binding of antigen fragments lacking the phosphonamidate group was retained. In addition, the R-L96-Q mutant had no detectable esterase activity. Thus, the computational model and experimental results together suggest a mechanism by which the catalytic antibody NPN43C9 stabilizes high-energy transition states during catalysis.
Roberts
V A
VA
Department of Molecular Biology, Scripps Research Institute, La Jolla, CA 92037.
Stewart
J
J
Benkovic
S J
SJ
Getzoff
E D
ED
D000595:Amino Acid Sequence; D017151:Antibodies, Catalytic; D000937:Antigen-Antibody Reactions; D001120:Arginine / Q000737:chemistry*; D001666:Binding Sites, Antibody; D002384:Catalysis; D007128:Immunoglobulin Fragments / Q000737:chemistry / Q000648:ultrastructure; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry / Q000648:ultrastructure; D007147:Immunoglobulin Light Chains / Q000737:chemistry / Q000648:ultrastructure; D007135:Immunoglobulin Variable Region / Q000737:chemistry* / Q000648:ultrastructure; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D013816:Thermodynamics
D017151:Antibodies, Catalytic; D007128:Immunoglobulin Fragments; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; C055966:immunoglobulin Fv; D001120:Arginine