|One Vaccine Leads to Another|
Diphtheria is a potentially lethal respiratory disease that is fairly well controlled by vaccines discovered early last century. These vaccines have been extremely effective; studies on one vaccine in particular, the nontoxic form of the diphtheria toxin (DT), have informed other vaccines. Recently, researchers at Novartis GNF solved several structures of a nontoxic DT using data obtained at ALS Beamline 5.0.3, resolving a long-standing scientific puzzle and leading the way to even better vaccines for a variety of bacterial diseases.
DT is extremely toxic — even a tiny amount can be lethal. It is produced by pathogenic bacterium and enters the cytoplasm where it uses the protein cofactor NAD (nicotinamide adenine dinucleotide) to inhibit protein synthesis, eventually leading to cell death. Mutations of the DT protein can render the toxin inactive, and these mutated forms, or cross reactive materials (CRMs), are important because they can be used as carriers for other vaccines, delivering antigens from other pathogenic organisms to provoke an immune response.
For example, CRM197 has been used to vaccinate millions of children and adults worldwide against influenza and several different forms of bacterial infection. CRM197 is different from the lethal form of DT by only one amino acid out of over 500: one glycine is mutated to a glutamate at position 52. In this study, the atomic resolution structures of CRM197 both with and without substrates were obtained to 2.0Å resolution, allowing analysis of fine details of the protein never seen before.
The structures of CRM197 and DT showed the same overall architecture, with a translocation domain, a receptor domain, and catalytic domain arranged in a similar configuration relative to each other (Figure 1). In addition, the topology of the NAD-binding pocket is nearly identical, with one major difference: in the DT structure an active site loop in the catalytic domain is positioned over the NAD binding pocket, whereas in CRM197 the loop is flexible (Figure 2). In particular, in the structure for CRM197 there is no electron density for residues 38-49, while there is clear density for residues 37 and 50. Researchers further solved the structure of CRM197 bound with nicotinamide (NCA), one of the catalysis products.
In comparison with DT bound to NAD, the positions of key residues in the binding pocket making contact with NCA or NAD were nearly identical, showing that the single point mutation at residue 52 does not affect the overall shape or binding configuration of the active site.
In CRM197 the mutation from glycine to glutamate is the key: glycine is a small amino acid that is often found in tight turns in proteins. Mutating this residue to a glutamate introduces steric clashes with neighboring residues, preventing the active-site loop from locking into place. Therefore, through high-resolution structural analysis, the researchers were able to delineate exactly how a single amino acid change can drastically affect the toxicity of a protein.
Research conducted by: E. Malito, (Genomics Institute of the Novartis Research Foundation; Novartis Vaccines and Diagnostics, Siena, Italy), B. Bursulaya, A. Brock, C. Chen, G. Spraggon (Genomics Institute of the Novartis Research Foundation), F. Berti, M.J. Bottomley, P. Costantino, M. Nissum, R. Rappouli, and P.L. Surdo, (Novartis Vaccines and Diagnostics, Siena, Italy), M. Biancucci, M. Picchianti, (Novartis Vaccines and Diagnostics, Siena, Italy; University of Siena, Italy), and E. Balducci, (University of Camerino, Italy).
Research funding: Operation of the ALS is supported by the U.S. Department of Energy, Office of Basic Energy Sciences.
Publication about this research: E. Malito, B. Bursulaya, C. Chen, P.L. Surdo, M. Picchianti, E. Balducci, M. Biancucci, A. Brock, F. Berti, M.J. Bottomley, M. Nissum, P. Costantino, R. Rappouli, and G. Spraggon, “Structural basis of toxicity of the diphtheria toxin mutant CRM197,” PNAS USA 109, 5229 (2012). 10.1073/pnas.1201964109
ALS Science Highlight #272