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An unusual mechanism of isopeptide bond formation attaches the collagenlike glycoprotein BclA to the exosporium of Bacillus anthracis.

Tan L, Li M, Turnbough CL - MBio (2011)

Bottom Line: Analogous mechanisms appear to be involved in the cross-linking of other spore proteins and could be found in unrelated organisms.Isopeptide bonds are protein modifications found throughout nature in which amide linkages are formed between functional groups of two amino acids, with at least one of the functional groups provided by an amino acid side chain.This mechanism, which apparently relies only on short peptide sequences in protein substrates, could be a general mechanism in vivo and adapted for protein cross-linking in vitro.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama, USA.

ABSTRACT

Unlabelled: The outermost exosporium layer of spores of Bacillus anthracis, the causative agent of anthrax, is comprised of a basal layer and an external hairlike nap. The nap includes filaments composed of trimers of the collagenlike glycoprotein BclA. Essentially all BclA trimers are tightly attached to the spore in a process requiring the basal layer protein BxpB (also called ExsFA). Both BclA and BxpB are incorporated into stable, high-molecular-mass complexes, suggesting that BclA is attached directly to BxpB. The 38-residue amino-terminal domain of BclA, which is normally proteolytically cleaved between residues 19 and 20, is necessary and sufficient for basal layer attachment. In this study, we demonstrate that BclA attachment occurs through the formation of isopeptide bonds between the free amino group of BclA residue A20 and a side chain carboxyl group of an acidic residue of BxpB. Ten of the 13 acidic residues of BxpB can participate in isopeptide bond formation, and at least three BclA polypeptide chains can be attached to a single molecule of BxpB. We also demonstrate that similar cross-linking occurs in vitro between purified recombinant BclA and BxpB, indicating that the reaction is spontaneous. The mechanism of BclA attachment, specifically, the formation of a reactive amino group by proteolytic cleavage and the promiscuous selection of side chain carboxyl groups of internal acidic residues, appears to be different from other known mechanisms for protein cross-linking through isopeptide bonds. Analogous mechanisms appear to be involved in the cross-linking of other spore proteins and could be found in unrelated organisms.

Importance: Isopeptide bonds are protein modifications found throughout nature in which amide linkages are formed between functional groups of two amino acids, with at least one of the functional groups provided by an amino acid side chain. Isopeptide bonds generate cross-links within and between proteins that are necessary for proper protein structure and function. In this study, we discovered that BclA, the dominant structural protein of the external nap of Bacillus anthracis spores, is attached to the underlying exosporium basal layer protein BxpB via isopeptide bonds formed through a mechanism fundamentally different from previously described mechanisms of isopeptide bond formation. The most unusual features of this mechanism are the generation of a reactive amino group by proteolytic cleavage and promiscuous selection of acidic side chains. This mechanism, which apparently relies only on short peptide sequences in protein substrates, could be a general mechanism in vivo and adapted for protein cross-linking in vitro.

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Model for the formation of isopeptide bonds that attach BclA to BxpB during exosporium assembly. (A) BclA NTD localization signals direct the binding of a BclA trimer to BxpB present in the basal layer of the exosporium. (B) Each NTD of a bound BclA trimer is proteolytically cleaved between residues S19 and A20, which produces a new and reactive amino terminus. The protein(s) required for cleavage remains to be identified. (C) The amino group of BclA residue A20 forms an isopeptide bond with an appropriately positioned side chain carboxyl group of an internal BxpB acidic residue. (D) Each strand of the BclA trimer can form an isopeptide bond with 1 of 10 acidic residues of BxpB, with each trimer presumably attaching to 3 neighboring acidic residues. There is no requirement, however, that all strands of the BclA trimer participate in isopeptide bond formation. The 13 acidic residues of BxpB are represented by red tick marks, and their positions within the protein are approximate.
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f5: Model for the formation of isopeptide bonds that attach BclA to BxpB during exosporium assembly. (A) BclA NTD localization signals direct the binding of a BclA trimer to BxpB present in the basal layer of the exosporium. (B) Each NTD of a bound BclA trimer is proteolytically cleaved between residues S19 and A20, which produces a new and reactive amino terminus. The protein(s) required for cleavage remains to be identified. (C) The amino group of BclA residue A20 forms an isopeptide bond with an appropriately positioned side chain carboxyl group of an internal BxpB acidic residue. (D) Each strand of the BclA trimer can form an isopeptide bond with 1 of 10 acidic residues of BxpB, with each trimer presumably attaching to 3 neighboring acidic residues. There is no requirement, however, that all strands of the BclA trimer participate in isopeptide bond formation. The 13 acidic residues of BxpB are represented by red tick marks, and their positions within the protein are approximate.

Mentions: The results presented in this study demonstrate that BclA attachment to the surface of B. anthracis spores occurs through the formation of isopeptide bonds to the exosporium basal layer protein BxpB. The mechanism of isopeptide bond formation in this case appears to be unlike any previously described mechanism. Our current results and those provided by recent related studies (12, 13) suggest the following model for BclA attachment to BxpB (Fig. 5). Following the synthesis of BclA and BxpB in the mother cell, BclA forms glycosylated trimers and monomeric BxpB is incorporated into the outer region of the developing basal layer of the exosporium. Directed by its NTD localization signal(s), each strand of a BclA trimer binds to BxpB or perhaps to an adapter protein associated with BxpB. Up to three BclA NTDs can associate with a single molecule of BxpB (Fig. 5A). Within this protein complex, the NTD of each strand of the BclA trimer is cleaved between residues S19 and A20 in a reaction catalyzed by a mother cell protease (Fig. 5B). After cleavage, the amino group of BclA residue A20 is appropriately positioned to permit the formation of an isopeptide bond to 1 of 10 acidic residues in BxpB. Formation of this bond then occurs spontaneously (Fig. 5C). The final reaction product includes up to three strands of the BclA trimer covalently linked to side chains of neighboring acidic amino acids of BxpB (Fig. 5D).


An unusual mechanism of isopeptide bond formation attaches the collagenlike glycoprotein BclA to the exosporium of Bacillus anthracis.

Tan L, Li M, Turnbough CL - MBio (2011)

Model for the formation of isopeptide bonds that attach BclA to BxpB during exosporium assembly. (A) BclA NTD localization signals direct the binding of a BclA trimer to BxpB present in the basal layer of the exosporium. (B) Each NTD of a bound BclA trimer is proteolytically cleaved between residues S19 and A20, which produces a new and reactive amino terminus. The protein(s) required for cleavage remains to be identified. (C) The amino group of BclA residue A20 forms an isopeptide bond with an appropriately positioned side chain carboxyl group of an internal BxpB acidic residue. (D) Each strand of the BclA trimer can form an isopeptide bond with 1 of 10 acidic residues of BxpB, with each trimer presumably attaching to 3 neighboring acidic residues. There is no requirement, however, that all strands of the BclA trimer participate in isopeptide bond formation. The 13 acidic residues of BxpB are represented by red tick marks, and their positions within the protein are approximate.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3104494&req=5

f5: Model for the formation of isopeptide bonds that attach BclA to BxpB during exosporium assembly. (A) BclA NTD localization signals direct the binding of a BclA trimer to BxpB present in the basal layer of the exosporium. (B) Each NTD of a bound BclA trimer is proteolytically cleaved between residues S19 and A20, which produces a new and reactive amino terminus. The protein(s) required for cleavage remains to be identified. (C) The amino group of BclA residue A20 forms an isopeptide bond with an appropriately positioned side chain carboxyl group of an internal BxpB acidic residue. (D) Each strand of the BclA trimer can form an isopeptide bond with 1 of 10 acidic residues of BxpB, with each trimer presumably attaching to 3 neighboring acidic residues. There is no requirement, however, that all strands of the BclA trimer participate in isopeptide bond formation. The 13 acidic residues of BxpB are represented by red tick marks, and their positions within the protein are approximate.
Mentions: The results presented in this study demonstrate that BclA attachment to the surface of B. anthracis spores occurs through the formation of isopeptide bonds to the exosporium basal layer protein BxpB. The mechanism of isopeptide bond formation in this case appears to be unlike any previously described mechanism. Our current results and those provided by recent related studies (12, 13) suggest the following model for BclA attachment to BxpB (Fig. 5). Following the synthesis of BclA and BxpB in the mother cell, BclA forms glycosylated trimers and monomeric BxpB is incorporated into the outer region of the developing basal layer of the exosporium. Directed by its NTD localization signal(s), each strand of a BclA trimer binds to BxpB or perhaps to an adapter protein associated with BxpB. Up to three BclA NTDs can associate with a single molecule of BxpB (Fig. 5A). Within this protein complex, the NTD of each strand of the BclA trimer is cleaved between residues S19 and A20 in a reaction catalyzed by a mother cell protease (Fig. 5B). After cleavage, the amino group of BclA residue A20 is appropriately positioned to permit the formation of an isopeptide bond to 1 of 10 acidic residues in BxpB. Formation of this bond then occurs spontaneously (Fig. 5C). The final reaction product includes up to three strands of the BclA trimer covalently linked to side chains of neighboring acidic amino acids of BxpB (Fig. 5D).

Bottom Line: Analogous mechanisms appear to be involved in the cross-linking of other spore proteins and could be found in unrelated organisms.Isopeptide bonds are protein modifications found throughout nature in which amide linkages are formed between functional groups of two amino acids, with at least one of the functional groups provided by an amino acid side chain.This mechanism, which apparently relies only on short peptide sequences in protein substrates, could be a general mechanism in vivo and adapted for protein cross-linking in vitro.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama, USA.

ABSTRACT

Unlabelled: The outermost exosporium layer of spores of Bacillus anthracis, the causative agent of anthrax, is comprised of a basal layer and an external hairlike nap. The nap includes filaments composed of trimers of the collagenlike glycoprotein BclA. Essentially all BclA trimers are tightly attached to the spore in a process requiring the basal layer protein BxpB (also called ExsFA). Both BclA and BxpB are incorporated into stable, high-molecular-mass complexes, suggesting that BclA is attached directly to BxpB. The 38-residue amino-terminal domain of BclA, which is normally proteolytically cleaved between residues 19 and 20, is necessary and sufficient for basal layer attachment. In this study, we demonstrate that BclA attachment occurs through the formation of isopeptide bonds between the free amino group of BclA residue A20 and a side chain carboxyl group of an acidic residue of BxpB. Ten of the 13 acidic residues of BxpB can participate in isopeptide bond formation, and at least three BclA polypeptide chains can be attached to a single molecule of BxpB. We also demonstrate that similar cross-linking occurs in vitro between purified recombinant BclA and BxpB, indicating that the reaction is spontaneous. The mechanism of BclA attachment, specifically, the formation of a reactive amino group by proteolytic cleavage and the promiscuous selection of side chain carboxyl groups of internal acidic residues, appears to be different from other known mechanisms for protein cross-linking through isopeptide bonds. Analogous mechanisms appear to be involved in the cross-linking of other spore proteins and could be found in unrelated organisms.

Importance: Isopeptide bonds are protein modifications found throughout nature in which amide linkages are formed between functional groups of two amino acids, with at least one of the functional groups provided by an amino acid side chain. Isopeptide bonds generate cross-links within and between proteins that are necessary for proper protein structure and function. In this study, we discovered that BclA, the dominant structural protein of the external nap of Bacillus anthracis spores, is attached to the underlying exosporium basal layer protein BxpB via isopeptide bonds formed through a mechanism fundamentally different from previously described mechanisms of isopeptide bond formation. The most unusual features of this mechanism are the generation of a reactive amino group by proteolytic cleavage and promiscuous selection of acidic side chains. This mechanism, which apparently relies only on short peptide sequences in protein substrates, could be a general mechanism in vivo and adapted for protein cross-linking in vitro.

Show MeSH
Related in: MedlinePlus