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J. Biol. Chem., Vol. 283, Issue 27, 18883-18891, July 4, 2008

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Structure- and Substrate-based Inhibitor Design for Clostridium botulinum Neurotoxin Serotype A^*

Desigan Kumaran, Richa Rawat, Matthew L. Ludivico, S. Ashraf Ahmed, and Subramanyam Swaminathan¹

From the Biology Department, Brookhaven National Laboratory, Upton, New York 11973 and the Department of Molecular Biology, Integrated Toxicology Division, United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Maryland 21702

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 1.6–1.8Å. 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 Tyr³⁶⁶ and NH₂ of Arg³⁶³ 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 Phe¹⁹⁴, Thr²¹⁵, Thr²²⁰, Asp³⁷⁰, and Arg³⁶³. The K_i of the best inhibitory tetrapeptide is 157 nM.

Received for publication, February 15, 2008 , and in revised form, April 8, 2008.

The atomic coordinates and structure factors (codes 3BWI, 3C88, 3C89, 3C8A, and 3C8B) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This project was supported by Joint Science and Technology Office (Chemical and Biological Defense) Project 3.10012_06_RD_B (to S. A. Ahmed). A subcontract to Brookhaven National Laboratory was through Military Interdepartmental Purchase Request 8CO890039 under United States Department of Energy Prime Contract DEAC02-98CH10886 (to S. Swaminathan). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed. Tel.: 631-344-3187; Fax: 631-344-3407; E-mail: swami{at}bnl.gov.

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				R. Agarwal and S. Swaminathan SNAP-25 Substrate Peptide (Residues 180-183) Binds to but Bypasses Cleavage by Catalytically Active Clostridium botulinum Neurotoxin E J. Biol. Chem., September 19, 2008; 283(38): 25944 - 25951. [Abstract] [Full Text] [PDF]