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Potassium Channel Binding by Kunitz Modules and Targeted Phospholipase Action Peter D Kwong1, Neil Q Mcdonaldi T, Paul B Sigler3 and Wayne a Hendricksonl,2*

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Potassium Channel Binding by Kunitz Modules and Targeted Phospholipase Action Peter D Kwong1, Neil Q Mcdonaldi T, Paul B Sigler3 and Wayne a Hendricksonl,2* View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Structure of 2-bungarotoxin: potassium channel binding by Kunitz modules and targeted phospholipase action Peter D Kwong1, Neil Q McDonaldi t, Paul B Sigler3 and Wayne A Hendricksonl,2* 1Department of Biochemistry and Molecular Biophysics and 2Howard Hughes Medical Institute, Columbia University, New York, NY 10032, USA and 3 Department of Molecular Biophysics and Biochemistry and Howard Hughes Medical Institute, Yale University, New Haven, CT 06511, USA Background: 3-bungarotoxin is a heterodimeric neuro- substrate-binding surface and reduced hydrophobicity. toxin consisting of a phospholipase subunit linked by a Conclusions: Molecular recognition by this Kunitz disulfide bond to a K+ channel binding subunit which module appears to diverge considerably from more is a member of the Kunitz protease inhibitor superfam- conventional superfamily members. The ion channel ily. Toxicity, characterized by blockage of neural trans- binding region identified here may mimic the regula- mission, is achieved by the lipolytic action of the tory interaction of endogenous neuropeptides. Adapta- phospholipase targeted to the presynaptic membrane by tions of the phospholipase subunit make it uniquely the Kunitz module. suited to targeting and explain the remarkable ability of Results: The crystal structure at 2.45 A resolution sug- the toxin to avoid binding to non-target membranes. gests that the ion channel binding region of the Kunitz Insight into the mechanism of -bungarotoxin gained subunit is at the opposite end of the module from the here may lead to the development of therapeutic strat- loop typically involved in protease binding. Analysis of egies against not only pathological cells, but also the phospholipase subunit reveals a partially occluded enveloped viruses. Structure 15 October 1995, 3:1109-1119 Key words: Kunitz module, molecular recognition, neurotoxin, phospholipase, potassium channel, targeted toxin Introduction pancreatic secretions, inflammatory exudates and certain The venoms of many poisonous animals contain a diverse toxins ([8], reviewed in [9]). It is the lipolytic action of cocktail of toxins, often neurotoxins, conferring potency 3-bungarotoxin, directly responsible for the depolarizing against a spectrum of targets and prey (reviewed in [1,2]). permeabilization of the synaptosomal plasma membrane, For example, venom from the snake Bungarus multicinctus that characterizes its toxicity [10]. Sequences of the contains both o-bungarotoxin, which blocks acetyl- smaller subunit identify them as members of the Kunitz choline receptors on the postsynaptic membrane of (Kunin or pancreatic trypsin type) protease inhibitor neuromuscular junctions, and P-bungarotoxin, a com- superfamily ([6], reviewed in [11,12]), although 13-bun- pletely unrelated protein, which inhibits neurotransmitter garotoxin has no protease inhibitor capacity [6]. Instead, release from presynaptic membranes [3,4]. Diversity in the 13-bungarotoxin Kunitz subunit serves to guide the targets of action is supplemented by further heterogene- toxin to its site of action on the presynaptic membrane ity in each toxic component. A K-homolog of ca-bun- by virtue of a high-affinity interaction (nanomolar Kd) garotoxin has specificity for neuronal over neuromuscular with a specific subclass of voltage-sensitive potassium receptors [5], and there are many isoforms of 1-bungaro- channels [13-15]. Although the phospholipase/Kunitz toxins. These 13-bungarotoxin isoforms are heterodimeric combination is unique to 13-bungarotoxin, other toxins proteins, and diversity arises from combinatorial associa- are known to be based separately on either phospholipase tions of variants of each subunit. Six different -bun- or Kunitz-type proteins. garotoxin isoforms (designated 1-I-6) have been identified by sequencing, and their differential activities A number of venom phospholipases are toxic (reviewed have been partially characterized [6,7]. in [16]) and, although they appear to share similar biochemical properties [17], they vary mechanistically. Each -bungarotoxin dimer has a phospholipase A2 sub- For example, the neurotoxin crotoxin is composed of a unit covalently coupled through a disulfide link to a standard phospholipase and an inhibitory chaperone, smaller subunit related to Kunitz-type protease inhibitors which blocks the phospholipase active site until binding [6]. Both components are members of well-studied pro- to a presynaptic receptor triggers the dissociation of the tein families, with more than a dozen atomic-level struc- chaperone [18]. In contrast, both the monomeric tures known for representatives of each. Sequences of the phospholipase from Oxyuranus scutellatus and notexin enzymatic subunit closely resemble those of other extra- from Notechis scutatus scutatus are unchaperoned; the cellular A2-specific phospholipases which are found in former has a specific skeletal muscle receptor [19], *Corresponding author. tPresent address: ICRF Unit for Structural Biology, Birkbeck College, Malet Street, London, WC1 E 7HX, UK. © Current Biology Ltd ISSN 0969-2126 1109 1110 Structure 1995, Vol 3 No 10 whereas notexin targets the neuromuscular junction at concentrations of only 1-2 nM, it specifically suppresses [20]. Among all known phospholipase toxins, only the in vitro growth of neuronal cells while maintaining a [3-bungarotoxin has a mechanism of action in which tar- high population of viable photoreceptor cells [32]. geting and phospholipase activity are separated into non- homologous subunits. We report here the crystal structure of the 32 -isoform of P3-bungarotoxin. Despite expected similarity in overall Other Kunitz toxins of known structure include oa-den- structure between the individual -bungarotoxin drotoxin and its relatives, toxin I and toxin K [21-23]. subunits and their respective homologs, each has distinc- The mode of action of these toxins is both similar and tive features. Comparative analyses, made in light of different from that of [-bungarotoxin. They also bind these features and the mode of intersubunit association tightly to certain voltage-gated potassium channels on defined by the structure, reveal surprisingly novel modes presynaptic membranes, but their mode of action is of interaction between this toxin and ion channels and channel blockage, which in turn facilitates neurotrans- lipid membranes. Insight into these interactions may be mitter release and leads to convulsions. Although their useful in other contexts: we discuss the relevance of effects are opposite, and ultimately toxic, binding by toxin binding to ion-channel regulation and the [3-bungarotoxin and ot-dendrotoxin is similar. Thus, therapeutic potential of a redirected phospholipase P-bungarotoxin binding can be inhibited by dendrotoxin against enveloped viruses such as HIV. [24] and the Kunitz subunit from 13-bungarotoxin (pre- pared by reduction and carboxymethylation) binds with high affinity and blocks both non-inactivating and inacti- Results vating voltage-gated channels [25]. Indeed, in the early Overall structure stage of -bungarotoxin action, miniature end-plate The structure of 132-bungarotoxin was solved at 2.45 A potentials are actually increased and only later, presum- resolution by a combination of molecular replacement ably associated with phospholipase action, are they abol- and multiple isomorphous replacement (MIR) phasing ished [26]. Because of its primary role in guiding the techniques and refined to an R value of 19.3%. The toxin, we have termed the Kunitz subunit the 'targeting structure of 132-bungarotoxin is shown schematically in subunit' and we refer to the mechanism by which Figure la. Although each subunit is essentially globular, [3-bungarotoxin acts as 'targeted phospholipase' action. their joining produces a relatively extended molecule of main-chain dimensions -60 A x 40 A x 20 A. There is interest in therapeutic applications of toxins, and Kunitz toxins have been the focus of drug design efforts As expected from sequence analysis [7], the structure of ([27], reviewed in [28]). By analogy with immunotoxins the phospholipase subunit (120 residues and 6 disulfide (reviewed in [29,30]), the bipartite structure of -bun- bonds) closely resembles other members of a large family garotoxin may make it useful in the design of appropri- of homologous phospholipases A2 [9]. Most of the back- ately directed toxins. 3-bungarotoxin has potential bone, including the three helices that constitute its core advantages in this respect. Unlike other highly toxic phos- as well as the calcium-binding loop, superimpose well pholipases, it is extremely specific in its effects and has nei- with the non-toxic class I phospholipase from Naja naja ther direct nor indirect hemolytic activity [31]. Moreover, atra [8] (root mean square deviation [rmsd] of 0.826 A for Fig. 1. Backbone structure of P2-bungarotoxin. (a) Schematic displaying secondary structural elements. The smaller Kunitz subunit is shown at the top right connected through a disulfide bond to the larger phopholipase subunit. (Drawn with SETOR 175].) (b) Stereo plot of the Cc backbone of 2-bungarotoxin shown superimposed with toxic (notexin and a-dendrotoxin; dashed line), and non-toxic (Naja naja atra phospholipase and BPTI; thin line) homologs of each subunit. Superpositions were made using all Cas that remained within 2.5 A of each other after least-squares alignment. Outliers are
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