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Substrate binding mode and its implication on drug design for botulinum neurotoxin A.

Kumaran D, Rawat R, Ahmed SA, Swaminathan S - PLoS Pathog. (2008)

Bottom Line: The most potent among them, botulinum neurotoxin type A (BoNT/A), cleaves its substrate synaptosome-associated protein of 25 kDa (SNAP-25).P5'-Met202 binds in the hydrophobic pocket formed by the residues from the 250 and 200 loop.Knowledge of interactions between the enzyme and substrate peptide from these complex structures should form the basis for design of potent inhibitors for this neurotoxin.

View Article: PubMed Central - PubMed

Affiliation: Biology Department, Brookhaven National Laboratory, Upton, New York, USA.

ABSTRACT
The seven antigenically distinct serotypes of Clostridium botulinum neurotoxins, the causative agents of botulism, block the neurotransmitter release by specifically cleaving one of the three SNARE proteins and induce flaccid paralysis. The Centers for Disease Control and Prevention (CDC) has declared them as Category A biowarfare agents. The most potent among them, botulinum neurotoxin type A (BoNT/A), cleaves its substrate synaptosome-associated protein of 25 kDa (SNAP-25). An efficient drug for botulism can be developed only with the knowledge of interactions between the substrate and enzyme at the active site. Here, we report the crystal structures of the catalytic domain of BoNT/A with its uncleavable SNAP-25 peptide (197)QRATKM(202) and its variant (197)RRATKM(202) to 1.5 A and 1.6 A, respectively. This is the first time the structure of an uncleavable substrate bound to an active botulinum neurotoxin is reported and it has helped in unequivocally defining S1 to S5' sites. These substrate peptides make interactions with the enzyme predominantly by the residues from 160, 200, 250 and 370 loops. Most notably, the amino nitrogen and carbonyl oxygen of P1 residue (Gln197) chelate the zinc ion and replace the nucleophilic water. The P1'-Arg198, occupies the S1' site formed by Arg363, Thr220, Asp370, Thr215, Ile161, Phe163 and Phe194. The S2' subsite is formed by Arg363, Asn368 and Asp370, while S3' subsite is formed by Tyr251, Leu256, Val258, Tyr366, Phe369 and Asn388. P4'-Lys201 makes hydrogen bond with Gln162. P5'-Met202 binds in the hydrophobic pocket formed by the residues from the 250 and 200 loop. Knowledge of interactions between the enzyme and substrate peptide from these complex structures should form the basis for design of potent inhibitors for this neurotoxin.

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Binding of the substrate peptide in the active site of Balc424.(A) Figure shows the substrate peptide (QRATKM) binding in the active site of Balc424. Balc424 is shown in solid blue-colored surface. Substrate peptide is shown in sphere (CPK) model. Carbon, oxygen, nitrogen, sulfur and zinc atoms are shown in green, red, blue, yellow and magenta, respectively. (B). Superposed stick models of QRATKM and RRATKM are shown in green, and gray, respectively. Carbon atoms are shown in green (QRATKM) and gray (RRATKM). Balc424 is shown in semi-transparent surface (blue) representation with secondary elements at the active site pocket. Only a few substrate-binding residues are shown as markers. Pymol (DeLano, W.L. The PyMOL Molecular Graphics System (2002) on World Wide Web http://www.pymol.org) was used to prepare these figures.
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ppat-1000165-g002: Binding of the substrate peptide in the active site of Balc424.(A) Figure shows the substrate peptide (QRATKM) binding in the active site of Balc424. Balc424 is shown in solid blue-colored surface. Substrate peptide is shown in sphere (CPK) model. Carbon, oxygen, nitrogen, sulfur and zinc atoms are shown in green, red, blue, yellow and magenta, respectively. (B). Superposed stick models of QRATKM and RRATKM are shown in green, and gray, respectively. Carbon atoms are shown in green (QRATKM) and gray (RRATKM). Balc424 is shown in semi-transparent surface (blue) representation with secondary elements at the active site pocket. Only a few substrate-binding residues are shown as markers. Pymol (DeLano, W.L. The PyMOL Molecular Graphics System (2002) on World Wide Web http://www.pymol.org) was used to prepare these figures.

Mentions: The crystal structure has been determined to 1.5Å resolution. The model refined with R and R free of 18.4 and 20.1%, respectively. The final refined model contains 423 protease residues, 6 substrate residues, one sulfate and one zinc ions and 375 waters. More than 91% of residues are within the most allowed region of the Ramachandran plot. The electron density in the residual map (Fo-Fc) was well defined for the hexapeptide and QRATKM could be modeled unambiguously except for the side chains of K and M (Figure 1A). It appears that K could take two rotamer positions. This is the first time an uncleavable substrate bound structure of an active botulinum neurotoxin has been reported and it has helped in unequivocally defining S1 to S5′ sites. Most notably, the amino nitrogen and carbonyl oxygen of P1 residue (Gln197) chelate the zinc ion (Figures 2 and 3). The amino nitrogen has replaced the nucleophilic water as was shown earlier [16].


Substrate binding mode and its implication on drug design for botulinum neurotoxin A.

Kumaran D, Rawat R, Ahmed SA, Swaminathan S - PLoS Pathog. (2008)

Binding of the substrate peptide in the active site of Balc424.(A) Figure shows the substrate peptide (QRATKM) binding in the active site of Balc424. Balc424 is shown in solid blue-colored surface. Substrate peptide is shown in sphere (CPK) model. Carbon, oxygen, nitrogen, sulfur and zinc atoms are shown in green, red, blue, yellow and magenta, respectively. (B). Superposed stick models of QRATKM and RRATKM are shown in green, and gray, respectively. Carbon atoms are shown in green (QRATKM) and gray (RRATKM). Balc424 is shown in semi-transparent surface (blue) representation with secondary elements at the active site pocket. Only a few substrate-binding residues are shown as markers. Pymol (DeLano, W.L. The PyMOL Molecular Graphics System (2002) on World Wide Web http://www.pymol.org) was used to prepare these figures.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2533696&req=5

ppat-1000165-g002: Binding of the substrate peptide in the active site of Balc424.(A) Figure shows the substrate peptide (QRATKM) binding in the active site of Balc424. Balc424 is shown in solid blue-colored surface. Substrate peptide is shown in sphere (CPK) model. Carbon, oxygen, nitrogen, sulfur and zinc atoms are shown in green, red, blue, yellow and magenta, respectively. (B). Superposed stick models of QRATKM and RRATKM are shown in green, and gray, respectively. Carbon atoms are shown in green (QRATKM) and gray (RRATKM). Balc424 is shown in semi-transparent surface (blue) representation with secondary elements at the active site pocket. Only a few substrate-binding residues are shown as markers. Pymol (DeLano, W.L. The PyMOL Molecular Graphics System (2002) on World Wide Web http://www.pymol.org) was used to prepare these figures.
Mentions: The crystal structure has been determined to 1.5Å resolution. The model refined with R and R free of 18.4 and 20.1%, respectively. The final refined model contains 423 protease residues, 6 substrate residues, one sulfate and one zinc ions and 375 waters. More than 91% of residues are within the most allowed region of the Ramachandran plot. The electron density in the residual map (Fo-Fc) was well defined for the hexapeptide and QRATKM could be modeled unambiguously except for the side chains of K and M (Figure 1A). It appears that K could take two rotamer positions. This is the first time an uncleavable substrate bound structure of an active botulinum neurotoxin has been reported and it has helped in unequivocally defining S1 to S5′ sites. Most notably, the amino nitrogen and carbonyl oxygen of P1 residue (Gln197) chelate the zinc ion (Figures 2 and 3). The amino nitrogen has replaced the nucleophilic water as was shown earlier [16].

Bottom Line: The most potent among them, botulinum neurotoxin type A (BoNT/A), cleaves its substrate synaptosome-associated protein of 25 kDa (SNAP-25).P5'-Met202 binds in the hydrophobic pocket formed by the residues from the 250 and 200 loop.Knowledge of interactions between the enzyme and substrate peptide from these complex structures should form the basis for design of potent inhibitors for this neurotoxin.

View Article: PubMed Central - PubMed

Affiliation: Biology Department, Brookhaven National Laboratory, Upton, New York, USA.

ABSTRACT
The seven antigenically distinct serotypes of Clostridium botulinum neurotoxins, the causative agents of botulism, block the neurotransmitter release by specifically cleaving one of the three SNARE proteins and induce flaccid paralysis. The Centers for Disease Control and Prevention (CDC) has declared them as Category A biowarfare agents. The most potent among them, botulinum neurotoxin type A (BoNT/A), cleaves its substrate synaptosome-associated protein of 25 kDa (SNAP-25). An efficient drug for botulism can be developed only with the knowledge of interactions between the substrate and enzyme at the active site. Here, we report the crystal structures of the catalytic domain of BoNT/A with its uncleavable SNAP-25 peptide (197)QRATKM(202) and its variant (197)RRATKM(202) to 1.5 A and 1.6 A, respectively. This is the first time the structure of an uncleavable substrate bound to an active botulinum neurotoxin is reported and it has helped in unequivocally defining S1 to S5' sites. These substrate peptides make interactions with the enzyme predominantly by the residues from 160, 200, 250 and 370 loops. Most notably, the amino nitrogen and carbonyl oxygen of P1 residue (Gln197) chelate the zinc ion and replace the nucleophilic water. The P1'-Arg198, occupies the S1' site formed by Arg363, Thr220, Asp370, Thr215, Ile161, Phe163 and Phe194. The S2' subsite is formed by Arg363, Asn368 and Asp370, while S3' subsite is formed by Tyr251, Leu256, Val258, Tyr366, Phe369 and Asn388. P4'-Lys201 makes hydrogen bond with Gln162. P5'-Met202 binds in the hydrophobic pocket formed by the residues from the 250 and 200 loop. Knowledge of interactions between the enzyme and substrate peptide from these complex structures should form the basis for design of potent inhibitors for this neurotoxin.

Show MeSH
Related in: MedlinePlus