Structure- and substrate-based inhibitor design for Clostridium botulinum neurotoxin serotype A.

The seven antigenically distinct serotypes of Clostridium botulinum neurotoxins cleave specific soluble N-ethylmaleimide-sensitive factor attachment protein receptor complex proteins and block the release of neurotransmitters that cause flaccid paralysis and are considered potential bioweapons. Botulinum neurotoxin type A is the most potent among the clostridial neurotoxins, and to date there is no post-exposure therapeutic intervention available. To develop inhibitors leading to drug design, it is imperative that critical interactions between the enzyme and the substrate near the active site are known. Although enzyme-substrate interactions at exosites away from the active site are mapped in detail for botulinum neurotoxin type A, information about the active site interactions is lacking. Here, we present the crystal structures of botulinum neurotoxin type A catalytic domain in complex with four inhibitory substrate analog tetrapeptides, viz. RRGC, RRGL, RRGI, and RRGM at resolutions of 1.6-1.8 A. These structures show for the first time the interactions between the substrate and enzyme at the active site and delineate residues important for substrate stabilization and catalytic activity. We show that OH of Tyr(366) and NH(2) of Arg(363) are hydrogen-bonded to carbonyl oxygens of P1 and P1' of the substrate analog and position it for catalytic activity. Most importantly, the nucleophilic water is replaced by the amino group of the N-terminal residue of the tetrapeptide. Furthermore, the S1' site is formed by Phe(194), Thr(215), Thr(220), Asp(370), and Arg(363). The K(i) of the best inhibitory tetrapeptide is 157 nm.

Identification of residues surrounding the active site of type A botulinum neurotoxin important for substrate recognition and catalytic activity.

Type A botulinum neurotoxin is one of the most lethal of the seven serotypes and is increasingly used as a therapeutic agent in neuromuscular dysfunctions. Its toxic function is related to zinc-endopeptidase activity of the N-terminal light chain (LC) on synaptosome-associated protein-25 kDa (SNAP-25) of the SNARE complex. To understand the determinants of substrate specificity and assist the development of strategies for effective inhibitors, we used site-directed mutagenesis to investigate the effects of 13 polar residues of the LC on substrate binding and catalysis. Selection of the residues for mutation was based on a computational analysis of the three-dimensional structure of the LC modeled with a 17-residue substrate fragment of SNAP-25. Steady-state kinetic parameters for proteolysis of the substrate fragment were determined for a set of 16 single mutants. Of the mutated residues non-conserved among the serotypes, replacement of Arg-230 and Asp-369 by polar or apolar residues resulted in drastic lowering of the catalytic rate constant (k(ca)), but had less effect on substrate affinity (K(m)). Substitution of Arg-230 with Lys decreased the catalytic efficiency (k(cat)/K(m)) by 50-fold, whereas replacement by Leu yielded an inactive protein. Removal of the electrostatic charge at Asp-369 by mutation to Asn resulted in 140-fold decrease in k(cat)/K(m). Replacement of other variable residues surrounding the catalytic cleft (Glu-54, Glu-63, Asn-66, Asp-130, Asn-161, Glu-163, Glu-170, Glu-256), had only marginal effect on decreasing the catalytic efficiency, but unexpectedly the substitution of Lys-165 with Leu resulted in fourfold increase in k(cat)/K(m). For comparison purposes, two conserved residues Arg-362 and Tyr-365 were investigated with substitutions of Leu and Phe, respectively, and their catalytic efficiency decreased 140- and 10-fold, respectively, whereas substitution of the tyrosine ring with Asn abolished activity. The altered catalytic efficiencies of the mutants were not due to any significant changes in secondary or tertiary structures, or in zinc content and thermal stability. We suggest that, despite the large minimal substrate size for catalysis, only a few non-conserved residues surrounding the active site are important to render the LC competent for catalysis or provide conformational selection of the substrate.

Factors affecting autocatalysis of botulinum A neurotoxin light chain.

The light chain of botulinum neurotoxin serotype A undergoes autocatalytic fragmentation into two major peptides during purification and storage (Ahmed S. A. et al. 2001, J. Protein Chem. 20:221-231) by both intermolecular and intramolecular mechanisms (Ahmed S. A. et al. 2003, Biochemistry 42:12539 12549). In this study, we investigated the effects of buffers and salts on this autocatalytic reaction in the presence and absence of zinc chloride. In the presence of zinc chloride, the fragmentation reaction was enhanced in each of acetate, MES, HEPES and phosphate buffers with maximum occurring in acetate when compared to those in the absence of zinc chloride. Adding sodium chloride in phosphate buffer in the presence of zinc chloride increased the extent of proteolysis. Irrespective of the presence of zinc chloride, adding sodium chloride or potassium chloride in phosphate buffer elicited an additional proteolytic reaction. Higher concentrations of sodium phosphate buffer enhanced the autocatalytic reaction in the absence of zinc chloride. In contrast, in the presence of zinc chloride, higher concentrations of sodium phosphate decreased the autocatalytic reaction. Optimum pH of autocatalysis was not affected significantly by the absence or presence of zinc chloride. Like zinc chloride, other chlorides of divalent metals, such as magnesium, cobalt, iron and calcium also enhanced the autocatalytic reaction. Polyols such as ethylene glycol protected the light chain from fragmentation. Exposure of light chain to UV radiation led to enhanced fragmentation. In order to avoid fragmentation, the protein should be stored frozen in a low concentration buffer of neutral or higher pH devoid of any metal. Our results provide a choice of buffers and salts for isolation, purification and storage of intact botulinum neurotoxin serotype A light chain.