Evolutionary Features in the Structure and Function of Bacterial Toxins.

Toxins can function both as a harmful and therapeutic molecule, depending on their concentrations. The diversity in their function allows us to ask some very pertinent questions related to their origin and roles: (a) What makes them such effective molecules? (b) Are there evolutionary features encoded within the structures of the toxins for their function? (c) Is structural hierarchy in the toxins important for maintaining their structure and function? (d) Do protein dynamics play a role in the function of toxins? and (e) Do the evolutionary connections to these unique features and functions provide the fundamental points in driving evolution? In light of the growing evidence in structural biology, it would be appropriate to suggest that protein dynamics and flexibility play a much bigger role in the function of the toxin than the structure itself. Discovery of IDPs (intrinsically disorder proteins), multifunctionality, and the concept of native aggregation are shaking the paradigm of the requirement of a fixed three-dimensional structure for the protein's function. Growing evidence supporting the above concepts allow us to redesign the structure-function aspects of the protein molecules. An evolutionary model is necessary and needs to be developed to study these important aspects. The criteria for a well-defined model would be: (a) diversity in structure and function, (b) unique functionality, and (c) must belong to a family to define the evolutionary relationships. All these characteristics are largely fulfilled by bacterial toxins. Bacterial toxins are diverse and widely distributed in all three forms of life (Bacteria, Archaea and Eukaryotes). Some of the unique characteristics include structural folding, sequence and functional combination of domains, targeting a cellular process to execute their function, and most importantly their flexibility and dynamics. In this work, we summarize certain unique aspects of bacterial toxins, including role of structure in defining toxin function, uniqueness in their enzymatic function, and interaction with their substrates and other proteins. Finally, we have discussed the evolutionary aspects of toxins in detail, which will help us rethink the current evolutionary theories. A careful study, and appropriate interpretations, will provide answers to several questions related to the structure-function relationship of proteins, in general. Additionally, this will also allow us to refine the current evolution theories.

Differential role of molten globule and protein folding in distinguishing unique features of botulinum neurotoxin.

Botulinum neurotoxins (BoNTs) are proteins of great interest not only because of their extreme toxicity but also paradoxically for their therapeutic applications. All the known serotypes (A-G) have varying degrees of longevity and potency inside the neuronal cell. Differential chemical modifications such as phosphorylation and ubiquitination have been suggested as possible mechanisms for their longevity, but the molecular basis of the longevity remains unclear. Since the endopeptidase domain (light chain; LC) of toxin apparently survives inside the neuronal cells for months, it is important to examine the structural features of this domain to understand its resistance to intracellular degradation. Published crystal structures (both botulinum neurotoxins and endopeptidase domain) have not provided adequate explanation for the intracellular longevity of the domain. Structural features obtained from spectroscopic analysis of LCA and LCB were similar, and a PRIME (PReImminent Molten Globule Enzyme) conformation appears to be responsible for their optimal enzymatic activity at 37°C. LCE, on the other hand, was although optimally active at 37°C, but its active conformation differed from the PRIME conformation of LCA and LCB. This study establishes and confirms our earlier finding that an optimally active conformation of these proteins in the form of PRIME exists for the most poisonous poison, botulinum neurotoxin. There are substantial variations in the structural and functional characteristics of these active molten globule related structures among the three BoNT endopeptidases examined. These differential conformations of LCs are important in understanding the fundamental structural features of proteins, and their possible connection to intracellular longevity could provide significant clues for devising new countermeasures and effective therapeutics.

Botulinum neurotoxin: unique folding of enzyme domain of the most-poisonous poison.

Botulinum neurotoxin (BoNT), the most toxic substance known to mankind, is the first example of the fully active molten globule state. To understand its folding mechanism, we performed urea denaturation experiments and theoretical modeling using BoNT serotype A (BoNT/A). We found that the extent of BoNT/A denaturation from the native state (N) shows a nonmonotonic dependence on urea concentration indicating a unique multistep denaturation process, N → I1 [Formula: see text] I2 [Formula: see text] U, with two intermediate states I1 and I2. BoNT/A loses almost all its secondary structure in 3.75 M urea (I1), yet it displays a native-like secondary structure in 5 M urea (I2). This agrees with the results of theoretical modeling, which helped to determine the molecular basis of unique behavior of BoNT/A in solution. Except for I2, all the states revert back to full enzymatic activity for SNAP-25 including the unfolded state U stable in 7 M urea. Our results stress the importance of structural flexibility in the toxin's mechanism of survival and action, an unmatched evolutionary trait from billion-year-old bacteria, which also correlates with the long-lasting enzymatic activity of BoNT inside neuronal cells. BoNT/A provides a rich model to explore protein folding in relation to functional activity.

Molecular basis of activation of endopeptidase activity of botulinum neurotoxin type E.

Botulinum neurotoxins (BoNTs) are a group of large proteins that are responsible for the clinical syndrome of botulism. The seven immunologically distinct serotypes of BoNTs (A-G), each produced by various strains of Clostridium botulinum, act on the neuromuscular junction by blocking the release of the neurotransmitter acetylcholine, thereby resulting in flaccid muscle paralysis. BoNTs are synthesized as single inactive polypeptide chains that are cleaved by endogenous or exogenous proteases to generate the active dichain form of the toxin. Nicking of the single chain BoNT/E to the dichain form is associated with 100-fold increase in toxicity. Here we investigated the activation mechanism of botulinum neurotoxin type E upon nicking and subsequent reduction of disulfide bond. It was observed that nicking of BoNT/E significantly enhances its endopeptidase activity and that at the physiological temperature of 37 degrees C the reduced form of nicked BoNT/E adopts a dynamically flexible conformation resulting from the exposure of hydrophobic segments and facilitating optimal cleavage of its substrate SNAP-25. Such reduction-induced increase in the flexibility of the polypeptide folding provides a rationale for the mechanism of BoNT/E endopeptidase against its intracellular substrate, SNAP-25, and complements current understanding of the mechanistics of interaction between the substrate and BoNT endopeptidase.

Comparative role of neurotoxin-associated proteins in the structural stability and endopeptidase activity of botulinum neurotoxin complex types A and E.

Seven serotypes of botulinum neurotoxins, the most toxic substances known to mankind, are each produced by different strains of Clostridium botulinum along with a group of neurotoxin-associated proteins (NAPs). NAPs play a critical role in the toxicoinfection process of botulism in addition to their role in protecting the neurotoxin from proteolytic digestion in the GI tract as well as from adverse environmental conditions. In this study we have investigated the effect of temperature on the structural and functional stability of BoNT/A complex (BoNT/AC) and BoNT/E complex (BoNT/EC). Although the NAPs in the two complexes are quite different, both groups of NAPs activate the endopeptidase activities of their BoNTs without any need to reduce the disulfide bonds between light and heavy chains of respective BoNTs. BoNT/AC attains optimum enzyme activity at the physiological temperature of 37 degrees C whereas BoNT/EC is maximally active at 45 degrees C, and this is accompanied by conformational alterations in its polypeptide folding at this temperature, leading to favorable binding with its intracellular substrate, SNAP-25, and subsequent cleavage of the latter. BoNT/A in its complex form is found to be structurally more stable against temperature whereas BoNT/E in its complex form is functionally better protected against temperature. Based on the analysis of isolated NAPs we have observed that the structural stability of the BoNT/AC is contributed by the NAPs. In addition to the unique structural conditions in which the enzyme remains active, functional stability of botulinum neurotoxins against temperature plays a critical role in the survival of the agent in cooked food and in food-borne botulism.

Role of two active site Glu residues in the molecular action of botulinum neurotoxin endopeptidase.

Botulinum neurotoxin type A (BoNT/A) light chain (LC) is a zinc endopeptidase that causes neuroparalysis by blocking neurotransmitter release at the neuromuscular junctions. The X-ray crystal structure of the toxin reveals that His223 and His227 of the Zn(2+) binding motif HEXXH directly coordinate the active site zinc. Two Glu residues (Glu224 and Glu262) are also part of the active site, with Glu224 coordinating the zinc via a water molecule whereas Glu262 coordinates the zinc directly as the fourth ligand. In the past we have investigated the topographical role of Glu224 by replacing it with Asp thus reducing the side chain length by 1.4 A that reduced the endopeptidase activity dramatically [L. Li, T. Binz, H. Niemann, and B.R. Singh, Probing the role of glutamate residue in the zinc-binding motif of type A botulinum neurotoxin light chain, Biochemistry 39 (2000) 2399-2405]. In this study we have moved the Glu 224 laterally by a residue (HXEXH) to assess its positional influence on the endopeptidase activity, which was completely lost. The functional implication of Glu262 was investigated by replacing this residue with aspartate and glutamine using site-directed mutagenesis. Substitution of Glu262 with Asp resulted in a 3-fold decrease in catalytic efficiency. This mutation did not induce any significant structural alterations in the active site and did not interfere with substrate binding. Substitution of Glu262 with Gln however, dramatically impaired the enzymatic activity and this is accompanied by global alterations in the active site conformation in terms of topography of aromatic amino acid residues, zinc binding, and substrate binding, resulting from the weakened interaction between the active site zinc and Gln. These results suggest a pivotal role of the negatively charged carboxyl group of Glu262 which may play a critical role in enhancing the stability of the active site with strong interaction with zinc. The zinc may thus play structural role in addition to its catalytic role.