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One Vaccine Leads to Another Print

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.

One Vaccine Leads to Another

Diphtheria is a respiratory disease that is rare in the developed world, thanks to vaccines that emerged in the early part of the twentieth century. These vaccines have been extremely effective, so why study how they work? The answer to this question isn't just important to expand scientific knowledge; studies on one vaccine—the nontoxic form of the diphtheria toxin (DT), in particular—have led to vaccines against other diseases, like the flu and bacterial meningitis.

Often, nontoxic forms of various disease toxins are used to make vaccines, delivering antigens to raise an immune response. These are called toxoids and are not able to cause disease themselves.

The structure of NAD-bound DT (yellow) is superposed onto NCA-bound CRM197 (shown as red sticks and labeled). The last visible residues of the active-site loop of CRM197 and DT complexed with NAD are shown as magenta-colored spheres.

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.

Comparison of NAD binding pocket in DT (active-site loop is shown in orange). The mutation at residue 52 is shown in yellow for DT and in grey for CRM197. The magenta spheres represent the last visible residues of the disordered active-site loop in CRM197 (K37 and W50).

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

 

ALSNews Vol. 342