Structural basis for lack of toxicity of the diphtheria toxin mutant CRM197

CRM197 is an enzymatically inactive and nontoxic form of diphtheria toxin that contains a single amino acid substitution (G52E). Being naturally nontoxic, CRM197 is an ideal carrier protein for conjugate vaccines against encapsulated bacteria and is currently used to vaccinate children globally against Haemophilus influenzae, pneumococcus, and meningococcus. To understand the molecular basis for lack of toxicity in CRM197, we determined the crystal structures of the full-length nucleotide-free CRM197 and of CRM197 in complex with the NAD hydrolysis product nicotinamide (NCA), both at 2.0-Å resolution. The structures show for the first time that the overall fold of CRM197 and DT are nearly identical and that the striking functional difference between the two proteins can be explained by a flexible active-site loop that covers the NAD binding pocket. We present the molecular basis for the increased flexibility of the active-site loop in CRM197 as unveiled by molecular dynamics simulations. These structural insights, combined with surface plasmon resonance, NAD hydrolysis, and differential scanning fluorimetry data, contribute to a comprehensive characterization of the vaccine carrier protein, CRM197.Diphtheria is a contagious respiratory disease that was a major cause of death among children around the world until mass vaccination was introduced in the 1920s. Although diphtheria has now been virtually eliminated in the industrialized world, rare outbreaks still occur worldwide (1, 2). Corynebacterium diphtheriae was shown to be the causative agent of diphtheria by Loeffler in 1885 (3), and Roux and Yersin showed that an extracellular toxin, diphtheria toxin (DT) secreted by C. diphtheriae is responsible for toxicity (4). The formaldehyde-treated detoxified form of DT, diphtheria toxoid, has been successfully used for mass vaccination and is still widely used as a component of combination vaccines (5, 6).A major contribution to the understanding of the mode of action of DT was the discovery of mutated forms in the early 1970s (7). Several phages encoding mutants of DT, named cross-reactive materials (CRMs), were isolated following nitrosoguanidine-based mutagenesis of the phage containing the gene encoding DT. Being naturally nontoxic, CRMs were immediately recognized as having great potential for vaccine development. The most important CRM identified was CRM197, an enzymatically inactive and nontoxic form of DT that contains a single amino acid substitution from Glycine to Glutamate in position 52 (8). Subsequently CRM197 was found to be an ideal carrier for conjugate vaccines against encapsulated bacteria. Here, the carrier protein is covalently linked to poorly immunogenic and T-cell-independent capsular polysaccharides, thus creating T-cell-dependent conjugate antigens that are highly immunogenic in infants (9–11).Vaccines containing CRM197 as a carrier protein have been successfully used to immunize hundreds of millions of children. Such vaccines currently include Menveo®, a recently approved tetravalent conjugate vaccine against serogroups A-C-W135-Y of Neisseria meningitidis, Menjugate® and Meningitec® (against serotype C of N. meningitidis), Vaxem-Hib® and HibTITER® (against Haemophilus influenzae type B, Hib), and the multivalent pneumococcal conjugate Prevnar™ (12).The widespread use of diphtheria toxoid and CRM197 has prompted many investigations of DT and related proteins. Diphtheria toxin is an ADP-ribosylating enzyme that is secreted as a proenzyme of 535 residues and is processed by trypsin-like proteases with release of two fragments (A and B). Fragment A uses NAD as a substrate, catalyzing the cleavage of the N-glycosidic bond between the nicotinamide ring and the N-ribose and mediating the covalent transfer of the ADP-ribose (ADPRT activity) to the modified Histidine 715 (diphthamide) of the elongation factor EF-2. This posttranslational diphthamide modification inactivates EF-2, halting protein synthesis and resulting in cell death. Extensive structural studies elucidated the molecular architecture of DT (13–19). The A fragment of DT (also named C domain) carries the catalytic active site and is the only fragment of the toxin required for the final step of intoxication, while the B fragment carries the R and T domains, which mediate binding to receptors on the host cell surface and promote the pH-dependent transfer of fragment A to the cytoplasm, respectively. An Arginine-rich disulfide-linked loop connects fragment A to fragment B (or domain C to domains TR), and this interchain disulfide bond is the only covalent link between the two fragments after proteolytic cleavage of the chain at position 186.While much progress has been made on the molecular characterization of DT over the last two decades, an understanding of the molecular basis for the lack of toxicity of CRM197 has so far been elusive. Here we present the crystal structures of full-length nucleotide-free (NF)-CRM197, and of CRM197 in complex with the NAD hydrolysis product nicotinamide (NCA). In addition, the differences between CRM197 and DT were elucidated using surface plasmon resonance (SPR), a NAD-glycohydrolase (NADase) activity assay, molecular dynamics (MD) simulations, and differential scanning fluorimetry (DSF).