Structural disorder throws new light on moonlighting

The study of botulinum neurotoxins (BoNT) is rapidly progressing in many aspects. Novel BoNTs are being discovered owing to next generation sequencing, but their biologic and pharmacological properties remain largely unknown. The molecular structure of the large protein complexes that the toxin forms with accessory proteins, which are included in some BoNT type A1 and B1 pharmacological preparations, have been determined. By far the largest effort has been dedicated to the testing and validation of BoNTs as therapeutic agents in an ever increasing number of applications, including pain therapy. BoNT type A1 has been also exploited in a variety of cosmetic treatments, alone or in combination with other agents, and this specific market has reached the size of the one dedicated to the treatment of medical syndromes. The pharmacological properties and mode of action of BoNTs have shed light on general principles of neuronal transport and protein-protein interactions and are stimulating basic science studies. Moreover, the wide array of BoNTs discovered and to be discovered and the production of recombinant BoNTs endowed with specific properties suggest novel uses in therapeutics with increasing disease/symptom specifity. These recent developments are reviewed here to provide an updated picture of the biologic mechanism of action of BoNTs, of their increasing use in pharmacology and in cosmetics, and of their toxicology.

https://pharmrev.aspetjournals.org/content/pharmrev/69/2/200.full.pdf

Botulinum Neurotoxins: Biology, Pharmacology, and Toxicology.

Recognition requires protein flexibility because it facilitates conformational rearrangements and induced-fit mechanisms upon target binding. Intrinsic disorder is an extreme on the continuous spectrum of possible protein dynamics and its role in recognition may seem counterintuitive. However, conformational disorder is widely found in many eukaryotic regulatory proteins involved in processes such as signal transduction and transcription. Disordered protein regions may in fact confer advantages over folded proteins in binding. Rapidly interconverting and diverse conformers may create mean electrostatic fields instead of presenting discrete charges. The resultant "polyelectrostatic" interactions allow for the utilization of post-translational modifications as a means to change the net charge and thereby modify the electrostatic interaction of a disordered region. Plasticity of disordered protein states enables steric advantages over folded proteins and allows for unique binding configurations. Disorder may also have evolutionary advantages, as it facilitates alternative splicing, domain shuffling and protein modularity. As proteins exist in a continuous spectrum of disorder, so do their complexes. Indeed, disordered regions in complexes may control the degree of motion between domains, mask binding sites, be targets of post-translational modifications, permit overlapping binding motifs, and enable transient binding of different binding partners, making them excellent candidates for signal integrators and explaining their prevalence in eukaryotic signaling pathways. "Dynamic" complexes arise if more than two transient protein interfaces are involved in complex formation of two binding partners in a dynamic equilibrium. "Disordered" complexes, in contrast, do not involve significant ordering of interacting protein segments but rely exclusively on transient contacts. The nature of these interactions is not well understood yet but advancements in the structural characterization of disordered states will help us gain insights into their function and their implications for health and disease.

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Protein dynamics and conformational disorder in molecular recognition

Botulinum neurotoxin (BoNT), the causative agent of botulism, is acknowledged to be the most poisonous protein known. BoNT proteases disable synaptic vesicle exocytosis by cleaving their cytosolic SNARE (soluble NSF attachment protein receptor) substrates. BoNT is a modular nanomachine: an N-terminal Zn(2+)-metalloprotease, which cleaves the SNAREs; a central helical protein-conducting channel, which chaperones the protease across endosomes; and a C-terminal receptor-binding module, consisting of two subdomains that determine target specificity by binding to a ganglioside and a protein receptor on the cell surface and triggering endocytosis. For BoNT, functional complexity emerges from its modular design and the tight interplay between its component modules--a partnership with consequences that surpass the simple sum of the individual component's action. BoNTs exploit this design at each step of the intoxication process, thereby achieving an exquisite toxicity. This review summarizes current knowledge on the structure of individual modules and presents mechanistic insights into how this protein machine evolved to this level of sophistication. Understanding the design principles underpinning the function of such a dynamic modular protein remains a challenging task.

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Botulinum Neurotoxin: A Marvel of Protein Design

In this review, we focus on significant developments in our understanding of the mechanisms that control the cutaneous vasculature in humans, with emphasis on the literature of the last half-century To provide a background for subsequent sections, we review methods of measurement and techniques of importance in elucidating control mechanisms for studying skin blood flow In addition, the anatomy of the skin relevant to its thermoregulatory function is outlined The mechanisms by which sympathetic nerves mediate cutaneous active vasodilation during whole body heating and cutaneous vasoconstriction during whole body cooling are reviewed, including discussions of mechanisms involving cotransmission, NO, and other effectors Current concepts for the mechanisms that effect local cutaneous vascular responses to local skin warming and cooling are examined, including the roles of temperature sensitive afferent neurons as well as NO and other mediators Factors that can modulate control mechanisms of the cutaneous vasculature, such as gender, aging, and clinical conditions, are discussed, as are nonthermoregulatory reflex modifiers of thermoregulatory cutaneous vascular responses

Cutaneous vasodilator and vasoconstrictor mechanisms in temperature regulation.

Clostridal neurotoxins (CNTs) are the causative agents of the neuroparalytic diseases botulism and tetanus1,2. CNTs impair neuronal exocytosis through specific proteolysis of essential proteins called SNAREs3. SNARE assembly into a low-energy ternary complex is believed to catalyse membrane fusion, precipitating neurotransmitter release; this process is attenuated in response to SNARE proteolysis4,5,6,7. Site-specific SNARE hydrolysis is catalysed by the CNT light chains, a unique group of zinc-dependent endopeptidases3. The means by which a CNT properly identifies and cleaves its target SNARE has been a subject of much speculation; it is thought to use one or more regions of enzyme–substrate interaction remote from the active site (exosites)8,9,10. Here we report the first structure of a CNT endopeptidase in complex with its target SNARE at a resolution of 2.1 A: botulinum neurotoxin serotype A (BoNT/A) protease bound to human SNAP-25. The structure, together with enzyme kinetic data, reveals an array of exosites that determine substrate specificity. Substrate orientation is similar to that of the general zinc-dependent metalloprotease thermolysin11. We observe significant structural changes near the toxin's catalytic pocket upon substrate binding, probably serving to render the protease competent for catalysis. The novel structures of the substrate-recognition exosites could be used for designing inhibitors specific to BoNT/A.

/pdf/substrate-recognition-strategy-for-botulinum-neurotoxin-2iz5y8z3rf.pdf

Substrate recognition strategy for botulinum neurotoxin serotype A

Author(s): Brunger, Axel T; Breidenbach, Mark A; Jin, Rongsheng; Fischer, Audrey; Santos, Jose S; Montal, Mauricio

/pdf/botulinum-neurotoxin-heavy-chain-belt-as-an-intramolecular-2ixr86j1sn.pdf

Botulinum neurotoxin heavy chain belt as an intramolecular chaperone for the light chain.

New insights into clostridial neurotoxin–SNARE interactions

Despite its extreme toxicity, botulinum neurotoxin is widely utilized in low doses as a treatment for several neurological disorders; higher doses cause the neuroparalytic syndrome botulism. The toxin blocks neurotransmitter release by preferentially attaching to pre-synaptic membrane receptors at neuromuscular junctions and subsequently delivering a Zn2+-dependent protease component to presynaptic neuronal cytosol. These highly specialized enzymes exclusively hydrolyze peptide bonds within SNARE (soluble N-ethylmaleiamide sensitive factor attachment protein receptor) proteins. In this review we discuss the structural basis for botulinum toxin’s exquisite specificity for its neuronal cell-surface receptors and intracellular SNARE targets.

Highly specific interactions between botulinum neurotoxins and synaptic vesicle proteins

TeNT is the causative agent of the neuroparalytic disease tetanus. A key component of TeNT is its light chain, a Zn2+ endopeptidase that targets SNAREs. Recent structural studies of closely related BoNT endopeptidases indicate that substrate-binding exosites remote from a conserved active site are the primary determinants of substrate specificity. Here we report the 2.3 A X-ray crystal structure of TeNT-LC, determined by combined molecular replacement and MAD phasing. As expected, the overall structure of TeNT-LC is similar to the other known CNT light chain structures, including a conserved thermolysin-like core inserted between structurally distinct amino- and carboxy-terminal regions. Differences between TeNT-LC and the other CNT light chains are mainly limited to surface features such as unique electrostatic potential profiles. An analysis of surface residue conservation reveals a pattern of relatively high variability matching the path of substrate binding around BoNT/A, possibly serving to accommoda...

Mark A. Breidenbach

Papers

Substrate recognition strategy for botulinum neurotoxin serotype A

Botulinum neurotoxin heavy chain belt as an intramolecular chaperone for the light chain.

New insights into clostridial neurotoxin–SNARE interactions

Highly specific interactions between botulinum neurotoxins and synaptic vesicle proteins

2.3 A crystal structure of tetanus neurotoxin light chain.