Analysis of the Structure and Function of FOX-4 Cephamycinase.

Class C ß-lactamases poorly hydrolyze cephamycins (e.g., cefoxitin, cefotetan, and moxalactam). In the past 2 decades, a new family of plasmid-based AmpC ß-lactamases conferring resistance to cefoxitin, the FOX family, has grown to include nine unique members descended from the Aeromonas caviae chromosomal AmpC. To understand the basis for the unique cephamycinase activity in the FOX family, we determined the first X-ray crystal structures of FOX-4, apo enzyme and the acyl-enzyme with its namesake compound, cefoxitin, using the Y150F deacylation-deficient variant. Notably, recombinant expression of N-terminally tagged FOX-4 also yielded an inactive adenylylated enzyme form not previously observed in ß-lactamases. The posttranslational modification (PTM), which occurs on the active site Ser64, would not seem to provide a selective advantage, yet might present an opportunity for the design of novel antibacterial drugs. Substantial ligand-induced changes in the enzyme are seen in the acyl-enzyme complex, particularly the R2 loop and helix H10 (P289 to N297), with movement of F293 by 10.3 Å. Taken together, this study provides the first picture of this highly proficient class C cephamycinase, uncovers a novel PTM, and suggests a possible cephamycin resistance mechanism involving repositioning of the substrate due to the presence of S153P, N289P, and N346I substitutions in the ligand binding pocket.

A comparative analysis of the immunological evolution of antibody 28B4.

In an effort to gain greater insight into the evolution of the redox active, catalytic antibody 28B4, the germline genes used by the mouse to generate this antibody were cloned and expressed, and the X-ray crystal structures of the unliganded and hapten-bound germline Fab of antibody 28B4 were determined. Comparison with the previously determined structures of the unliganded and hapten-bound affinity-matured Fab [Hsieh-Wilson, L. C., Schultz, P. G., and Stevens, R. C. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 5363] shows that the germline antibody binds the p-nitrophenyl ring of hapten 3 in an orientation significantly different from that seen in the affinity-matured antibody, whereas the phosphonate moiety is bound in a similar mode by both antibodies. The affinity-matured antibody 28B4 has more electrostatic and hydrophobic interactions with hapten 3 than the germline antibody and binds the hapten in a lock-and-key fashion. In contrast, significant conformational changes occur in the loops of CDR H3 and CDR L1 upon hapten binding to the germline antibody, consistent with the notion of structural plasticity in the germline antibody-combining site [Wedemayer, G. J., Patten, P. A., Wang, L. H., Schultz, P. G., and Stevens, R. C. (1997) Science 276, 1665]. The structural differences are reflected in the differential binding affinities of the germline Fab (K(d) = 25 microM) and 28B4 Fab (K(d) = 37 nM) to hapten 3. Nine replacement mutations were found to accumulate in the affinity-matured antibody 28B4 compared to its germline precursor. The effects of each mutation on the binding affinity of the antibody to hapten 3 were characterized in detail in the contexts of both the germline and the affinity-matured antibodies. One of the mutations, Asp95(H)Trp, leads to a change in the orientation of the bound hapten, and its presence is a prerequisite for other somatic mutations to enhance the binding affinity of the germline antibody for hapten 3. Thus, the germline antibody of 28B4 acquired functionally important mutations in a stepwise manner, which fits into a multicycle mutation, affinity selection, and clonal expansion model for germline antibody evolution. Two other antibodies, 20-1 and NZA6, with very different antigen specificities were found to be highly homologous to the germline antibody of 28B4, consistent with the notion that certain germline variable-region gene combinations can give rise to polyspecific hapten binding sites [Romesberg, F. E., Spiller, B., Schultz, P. G., and Stevens, R. C. (1998) Science 279, 1929]. The ultimate specificity of the polyspecific germline antibody appears to be defined by CDR H3 variability and subsequent somatic mutation. Insights into the evolution of antibody-combining sites provided by this and other structural studies are discussed.

Conformational effects in biological catalysis: an antibody-catalyzed oxy-cope rearrangement.

Antibody AZ-28 was generated against the chairlike transition-state analogue (TSA) 1 and catalyzes the oxy-Cope rearrangement of substrate 2 to product 3. The germline precursor to AZ-28 catalyzes the reaction with a 35-fold higher rate (k(cat)/k(uncat) = 163 000), despite a 40-fold lower binding affinity for TSA.1 (K(D) = 670 nM). To determine the structural basis for the differences in the binding and catalytic properties of the germline and affinity-matured antibodies, the X-ray crystal structures of the unliganded and TSA.1 complex of antibody AZ-28 have been determined at 2.8 and 2.6 A resolution, respectively; the structures of the unliganded and TSA.1 complex of the germline precursor to AZ-28 were both determined at 2. 0 A resolution. In the affinity-matured antibody.hapten complex the TSA is fixed in a catalytically unfavorable conformation by a combination of van der Waals and hydrogen-bonding interactions. The 2- and 5-phenyl substituents of TSA.1 are almost perpendicular to the cyclohexyl ring, leading to decreased orbital overlap and decreased stabilization of the putative transition state. The active site of the germline antibody appears to have an increased degree of flexibility-CDRH3 moves 4.9 A outward from the active site upon binding of TSA.1. We suggest that this conformational flexibility in the germline antibody, which results in a lower binding affinity for TSA.1, allows dynamic changes in the dihedral angle of the 2-phenyl substituent along the reaction coordinate. These conformational changes in turn lead to enhanced orbital overlap and increased catalytic rate. These studies suggest that protein and substrate dynamics play a key role in this antibody-catalyzed reaction.

The interplay between binding energy and catalysis in the evolution of a catalytic antibody.

Antibody catalysis provides an opportunity to examine the evolution of binding energy and its relation to catalytic function in a system that has many parallels with natural enzymes. Here we report such a study involving an antibody AZ-28 that catalyses an oxy-Cope rearrangement, a pericyclic reaction that belongs to a well studied and widely used class of reactions in organic chemistry. Immunization with transition state analogue 1 results in a germline-encoded antibody that catalyses the rearrangement of hexadiene 2 to aldehyde 3 with a rate approaching that of a related pericyclic reaction catalysed by the enzyme chorismate mutase. Affinity maturation gives antibody AZ-28, which has six amino acid substitutions, one of which results in a decrease in catalytic rate. To understand the relationship between binding and catalytic rate in this system we characterized a series of active-site mutants and determined the three-dimensional crystal structure of the complex of AZ-28 with the transition state analogue. This analysis indicates that the activation energy depends on a complex balance of several stereoelectronic effects which are controlled by an extensive network of binding interactions in the active site. Thus in this instance the combinatorial diversity of the immune system provided both an efficient catalyst for a reaction where no enzyme is known, as well as an opportunity to explore the mechanisms and evolution of biological catalysis.