Structural evidence for substrate strain in antibody catalysis

Abstract

The crystal structure of the Michaelis complex between the Fab fragment of ferrochelatase antibody 7G12 and its substrate mesoporphyrin has been solved to 2.6-Å resolution. The antibodybound mesoporphyrin clearly adopts a nonplanar conformation and reveals that the antibody catalyzes the porphyrin metallation reaction by straining͞distorting the bound substrate toward the transition-state configuration. The crystal structures of the Fab fragment of the germ-line precursor antibody to 7G12 and its complex with the hapten N-methylmesoporphyrin have also been solved. A comparison of these structures with the corresponding structures of the affinity-matured antibody 7G12 reveals the molecular mechanism by which the immune system evolves binding energy to catalyze this reaction.M odern theories of biological catalysis date from Haldane's theory of strain: ''using Fischer's lock and key simile, the key does not fit the lock perfectly but exercises a certain strain on it'' (1). Although the notion that enzymes use binding energy to strain or distort substrates is a fundamental theory of enzyme catalysis (2), it has proven difficult to experimentally validate (3). One approach that has proven effective in testing theories of biological catalysis involves the use of the programmable nature of antibody binding energy to evolve selective catalysts. Antibody catalysis has allowed us to dissect the energetic contributions of transition state stabilization, general base and covalent catalysis, and proximity effects to catalysis (4-9, 29). More recently, we showed that an antibody (7G12) raised against a strained ground-state mimic acted as an efficient porphyrin metallation catalyst (10). We now report the x-ray crystal structure of the Michaelis complex formed between the Fab fragment of the ferrochelatase antibody 7G12 and its substrate mesoporphyrin IX (MP), in which the bound substrate is distorted toward the transition-state conformation for metal insertion. The structure of this complex provides unequivocal structural evidence for the strain theory proposed by Haldane more than 70 years ago. Moreover, the detailed structural and biophysical characterization of the germ-line and affinity-matured antibodies has provided a detailed mechanistic picture of the immunological evolution of this strain mechanism.The enzyme ferrochelatase catalyzes the insertion of Fe 2ϩ into protoporphyrin IX as the last step in heme biosynthesis pathway (11). It was proposed that the enzyme catalyzes the porphyrin metallation reaction by distorting the porphyrin substrate toward a transition state-like geometry in which the pyrrole nitrogen lone pairs are exposed for metal chelation (12). To test this notion, antibody 7G12 was generated against N-methylmesoporphyrin (NMP) in which the porphyrin macrocycle is distorted due to alkylation at one of the pyrrole nitrogens. The resulting antibody was found to catalyze the metallation of MP by Zn 2ϩ with a catalytic efficiency comparable to that of the natural enzyme (10). The same antibody also catal...

RETRACTED: Somatic hypermutation maintains antibody thermodynamic stability during affinity maturation

“…Genetic, biochemical, and structural studies have revealed the molecular mechanisms that result in antibody variable region diversity and its role in antigen recognition. More recently, detailed structural and biophysical studies have shown that germline antibodies have significant combining-site conformational variability compared with affinity-matured antibodies, and this structural plasticity also plays a critical role in determining the enormous binding potential of the germ-line repertoire (3)(4)(5)(6). Somatic hypermutation and subsequent B-cell clonal selection further optimize antibody-antigen affinity and selectivity.…”

Chelatases: distort to select?

“…Antibody 7G12, the result of five somatic mutations accumulated during affinity maturation of the germline precursor antibody, is an example of such a catalytic antibody [19,21,22]. Similar to ferrochelatase, antibody 7G12 has a porphyrin-binding cleft at the junction of two surfaces with different hydrophobicities [19,22].…”

“…The crystal structure of ferrochelatase complexed with N-methylmesoporphyrin (N-MeMP), a potent inhibitor of ferrochelatase (dissociation constant, K d ≈10 nM) that mimics a strained substrate [18][19][20], shows that porphyrin binds in a deep cleft between two surfaces: one hydrophobic and one hydrophilic (Figure 1b). When used as a hapten, non-planar N-MeMP induces the formation of antibodies that catalyze the insertion of divalent metal ions into mesoporphyrin [19,21,22].…”

“…In antibody 7G12, N-MeMP and mesoporphyrin are bound in a cleft formed by the complementarity-determining regions (CDRs) L2 and L3 on one side and CDR H3 on the other [19,22]. The side chain of Asp100 L (Asp100 of the light-chain variable region of the 7G12 antibody) has an important role in interactions with the porphyrin.…”

“…Similar to ferrochelatase, antibody 7G12 has a porphyrin-binding cleft at the junction of two surfaces with different hydrophobicities [19,22]. Whereas binding of N-MeMP to Bacillus subtilis ferrochelatase leads to a conformational transition in ferrochelatase with widening of the binding cleft in a characteristic 'induced-fit' reaction mechanism [20], however, antibody 7G12 binds porphyrin in a 'lock-and-key' fashion without undergoing a conformational transition [19,21]. These findings indicate that, in contrast to ferrochelatase, the binding site of antibody 7G12 is rigid and pre-organized to bind a distorted porphyrin.…”