Biochemistry 2006, 45, 8903-8911
8903
Light Chain of Botulinum Neurotoxin Serotype A: Structural Resolution of a Catalytic Intermediate†,‡ Zhuji Fu,| Sheng Chen,§ Michael R. Baldwin,§ Grant E. Boldt,¶ Adam Crawford,| Kim D. Janda,¶ Joseph T. Barbieri,§ and Jung-Ja P. Kim*,| Department of Biochemistry and Department of Microbiology and Molecular Genetics, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, Wisconsin 53226, and Departments of Chemistry and Immunology, The Skaggs Institute for Chemical Biology, and Worm Institute for Research and Medicine, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037 ReceiVed April 22, 2006; ReVised Manuscript ReceiVed May 21, 2006
ABSTRACT: Botulinum neurotoxin serotype A (BoNT/A, 1296 residues) is a zinc metalloprotease that cleaves SNAP25 to inhibit the fusion of neurotransmitter-carrying vesicles to the plasma membrane of peripheral neurons. BoNT/A is a disulfide-linked di-chain protein composed of an N-terminal, thermolysin-like metalloprotease light chain domain (LC/A, 448 residues) and a C-terminal heavy chain domain (848 residues) that can be divided into two subdomains, a translocation subdomain and a receptor binding subdomain. LC/A cleaves SNAP25 between residues Gln197-Arg198 and, unlike thermolysin, recognizes an extended region of SNAP25 for cleavage. The structure of a recombinant LC/A (1-425) treated with EDTA (No-Zn LC/A) was determined. The overall structure of No-Zn LC/A is similar to that reported for the holotoxin, except that it lacks the Zn ion, indicating that the role of Zn is catalytic not structural. In addition, structures of a noncatalytic mutant LC/A (Arg362Ala/Tyr365Phe) complexed with and without an inhibitor, ArgHX, were determined. The overall structure and the active site conformation for the mutant are the same as wild type. When the inhibitor binds to the active site, the carbonyl and N-hydroxyl groups form a bidentate ligand to the Zn ion and the arginine moiety binds to Asp369, suggesting that the inhibitor-bound structure mimics a catalytic intermediate with the Arg moiety binding at the P1′ site. Consistent with this model, mutation of Asp369 to Ala decreases the catalytic activity of LC/A by ∼600fold, and the residual activity is not inhibited by ArgHX. These results provide new information on the reaction mechanism and insight into the development of strategies for small molecule inhibitors of BoNTs.
The botulinum neurotoxins (BoNT)1 are zinc proteases that cleave SNARE proteins. There are seven serotypes of the botulinum toxins (A-G) which are designated by the neutralizing capacity of antitoxin sera (1). The BoNT serotypes A-G share ∼30-60% primary amino acid identity (2). The neurotoxins are synthesized as ∼150 kDa single † This work was supported by a grant from the Great Lakes Regional Center of Excellence (GLRCE) from NIH-NIAID U54 AI057153 (Z.F., S.C., M.R.B., J.T.B., and J.-J.P.K.) and grants NIH-NIAID AI066507 (G.E.B., K.D.J., and J.T.B.) and NIH-NIAID BT010-04 (K.D.J.). Use of the Advanced Photon Source was supported by the United States Department of Energy, Office of Science, Office of Basic Energy Sciences, under Contract W31-109-Eng-38, and use of the BioCARS was supported by National Center for Research Resources Grant RR07707 from the National Institutes of Health. ‡ The atomic coordinates and structure factors (PDB codes: 2G7K, 2G7P, and 2G7Q) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ. * Corresponding author. Phone, 414-456-8479; fax, 414-456-6510; e-mail,
[email protected]. | Department of Biochemistry, Medical College of Wisconsin. § Department of Microbiology and Molecular Genetics, Medical College of Wisconsin. ¶ The Scripps Research Institute. 1 Abbreviations: BoNT/A, bolulinum neurotoxin serotype A; LC/ A, BoNT/A light chain; SNAP25, synaptosomal-associated 25 kDa protein; LC/A RYM, BoNT light chain Arg362Ala/Tyr365Phe mutant; ArgHX, arginine hydroxamate.
chain proteins that are cleaved to produce disulfide linked di-chain proteins. The N-terminal ∼440 amino acids (termed the light chain, LC) include the zinc protease domain, while the C-terminal ∼800 amino acids (termed the heavy chain, HC) are organized into two domains, an internal translocation domain (HCT) and the C-terminal receptor binding domain (HCR). Flaccid paralysis associated with botulism is due to the cleavage of SNARE proteins in peripheral neurons. A twostep serotype-specific association of BoNT with neurons has been proposed where the initial association of the HCR domain with polysialogangliosides on the cell surface precedes high-affinity binding to vesicle-associated or surfacebound proteins (3). In the case of BoNT/B, the toxin associates with gangliosides, which facilitate binding to synaptotagmin I and II (4), while BoNT/G associates with synaptotagmin I and II independent of gangliosides (5). BoNT/C appears to utilize glycoproteins as receptor(s) (6). Following receptor-mediated endocytosis, the HCT of BoNTs assists in the translocation of LC across the endocytic membrane by a pH-dependent mechanism (7, 8). Each internalized LC then cleaves a specific SNARE protein, uncoupling neurotransmitter vesicle fusion capabilities. Spastic paralysis associated with tetanus toxin is due to the anterior transport of TeNT through the peripheral neuron
10.1021/bi060786z CCC: $33.50 © 2006 American Chemical Society Published on Web 06/27/2006
8904 Biochemistry, Vol. 45, No. 29, 2006 axon where the toxin crosses the inner neuron cell junction and enters spinal inhibitory neurons where the VAMP protein is cleaved to block the release of inhibitory neurotransmitters (9). BoNTs and TeNT cleave specific SNARE proteins (3). BoNT/A, BoNT/E, and BoNT/C cleave the membrane-bound synaptosomal-associated 25 kDa protein (SNAP25). BoNT/C also cleaves syntaxin, another membrane-bound SNARE protein. BoNT/B, BoNT/D, BoNT/F, BoNT/G, and TeNT cleave the vesicle-associated membrane protein (VAMP). Each neurotoxin cleaves a unique site on the SNARE protein with one exception; BoNT/B and TeNT cleave at the same amino acid on VAMP. Sequence recognition of the neurotoxins appears to be more complex than other zinc proteases with substrate recognition domains within and outside the active site. Both SNARE binding motifs (10) and an R-exosite and a β-exosite have been implicated in substrate recognition of SNARE proteins outside the active site (11). However, our understanding of the steps involved in substrate recognition is limited. The crystal structures of BoNT/A (12), and BoNT/B (13), along with LC/A (14), LC/B (15), LC/E (16), LC/F (17), LC/G (18), and TeNT, (19) have been determined. Overall, the LC structures are similar and provide a basis for comparative functional studies. Recently, the structure of a mutated LC/A complexed with a truncated form of SNAP25 was solved (11). The complex structure provides insight into substrate recognition distal to the active site, but had limited information on interactions between SNAP25 and the active site presumably due to structural perturbations caused by the mutations used to inactivate catalytic activity. Neurotoxin cleavage of SNARE proteins follows the basic molecular organization of the thermolysin family of zinc proteases. Zinc binding is coordinated by the His-Glu-X-XHis motif, a downstream glutamic acid, and a water molecule (2, 15). While the mechanism for peptide bond cleavage by the neurotoxins remains to be resolved, cleavage of the scissile peptide bond appears to follow a general basecatalyzed mechanism. Peptide bond cleavage is initiated by a water molecule polarized by the Glu within the Zinc binding motif (H-E-X-X-H) and Zn2+, which cause a nucleophilic attack on the carbonyl carbon of the scissile bond to form an oxyanion. Peptide bond cleavage is likely achieved by a proton transfer from the attacking water mediated by the carboxyl group of a downstream Glu to form a protonated amine. In addition, a conserved Arg and Tyr (Arg362 and Tyr365 in LC/A) are proposed to stabilize the oxyanion in the transition state (20). Mutation of either Arg or Tyr alone lowers the velocity of cleavage with only limited effects on substrate affinity. Unexpectedly, a double mutation of Arg362 and Tyr365 to LC/A eliminated cleavage of SNAP25 to undetectable levels. The synergistic affect of mutations to Arg and Tyr on peptide bond cleavage prompted an evaluation of how the double mutation affected the structure of the active site of LC/A. In addition, recent studies observed an inhibition of LC/A catalysis by ArgHX (Boldt,G. E. et al., J. Comb. Chem., in press). To study the substrate recognition at the active site (P1 and P1′), we have determined the structure of ArgHX bound to the double mutant (Arg362Ala/ Tyr365Phe), yielding insight into the catalytic mechanism of this bacterial neurotoxin.
Fu et al. MATERIALS AND METHODS Expression and Purification of BoNT LC/A. Protein expression and purification were carried out as previously described (21). Briefly, Escherichia coli BL-21 RIL (DE3) cells containing the LC/A expression plasmid, pET15bLC/A (1-425), were cultured overnight on L-agar with 100 µg/ mL ampicillin and 50 µg/mL chloramphenicol. Cells were inoculated into LB medium containing antibiotics and cultured at 30 °C for 2 h at 250 rpm when cells were induced with 0.75 mM IPTG, and then cultured at 250 rpm overnight at 16 °C. Cells were harvested and broken with a French Press in 40 mL of cold buffer A (20 mM Tris-HCl (pH 7.9), 1 mM DTT, 10 mM imidazole, and 0.5 M NaCl) containing EDTA-free protease inhibitor cocktail (Sigma) and 2.5 µg/ mL DNAse I and 2.5 µg/mL RNAse I. The lysate was clarified by centrifugation at 20 000g for 20 min at 4 °C and passed through a 0.45 µm filter. The filtered lysate was applied to Ni-NTA resin (5 mL bed volume per 2 L of bacterial culture in buffer A). The column was washed with 25 mL of buffer A and eluted with 20 mM Tris-HCl, pH 7.9, containing 250 mM imidazole and 0.5 M NaCl. Peak fractions were pooled and subjected to gel-filtration on a Sephacryl S200 HR column (150 mL of resin equilibrated in 10 mM Tris-HCl (pH 7.9) and 20 mM NaCl). Peak fractions were pooled and subjected to anion exchange chromatography (10 mL of resin of DEAE-Sephacel with a 10 mM Tris-HCl (pH 7.9) with a 20-500 mM NaCl gradient). Peak fractions were pooled and dialyzed overnight into 10 mM Tris-HCl (7.6) and 20 mM NaCl with 40% glycerol (v/v). The RYM mutant was engineered using Quick Change techniques, and purified using the same procedure as the wild-type protein. Purified proteins were stored at -20 °C until ready to use. Protease ActiVity of LC/A and CleaVage of SNAP25. Endopeptidase activity of the recombinant LC proteins was assayed in 40-µL of reaction mixture containing GSTSNAP25(146-205) in 20 mM K+-HEPES, pH 7.4, and 150 mM potassium glutamate at 37 °C for 2 h. Reactions were stopped by adding SDS-PAGE sample buffer and boiling. Samples were resolved by SDS-PAGE, and the extent of cleavage was determined by visual inspection or by densitometry. The Inhibition of LC/A and LC/A (Asp369Ala) CleaVage of SNAP25 by Arginine Hydroxamate (ArgHX). LC/A (3 nM) and LC/A(Asp369Ala) (3 µM) were incubated with the indicated concentration of ArgHX for 30 min on ice, and then 1 µg of GST-SNAP25(146-205) was added and the incubation continued for 10 min at 37 °C (total volume, 10 µL). Reactions were stopped by the addition of SDS-PAGE sample buffer and subjected to SDS-PAGE. Gels were stained, and the amount of SNAP25 cleavage was determined by densitometry. Steady-State Kinetic Parameters for the CleaVage of SNAP25 by LC/A and LC/A(Asp369Ala). Km and kcat determinations were made for LC/A and LC/A (Asp369Ala) with SNAP25(residues 146-206). The amount of LCs was adjusted to cleave
