Department of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama, USA.
mBio. 2011 May 31;2(3):e00084-11. doi: 10.1128/mBio.00084-11. Print 2011.
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.
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.
炭疽芽孢杆菌（炭疽的病原体）孢子的最外层外壁层由一个基层和一个外部毛发状的绒毛组成。绒毛包括由胶原样糖蛋白 BclA 的三聚体组成的细丝。基本上所有的 BclA 三聚体都紧密地附着在孢子上，这个过程需要基层蛋白 BxpB（也称为 ExsFA）。BclA 和 BxpB 都被整合到稳定的高分子质量复合物中，这表明 BclA 直接附着在 BxpB 上。BclA 的 38 个残基氨基末端结构域通常在残基 19 和 20 之间被蛋白水解切割，这对于基层附着是必要的和充分的。在这项研究中，我们证明 BclA 的附着是通过 BclA 残基 A20 的游离氨基与 BxpB 的酸性残基侧链羧基之间形成异肽键来实现的。BxpB 的 13 个酸性残基中的 10 个可以参与异肽键的形成，并且至少三个 BclA 多肽链可以附着到单个 BxpB 分子上。我们还证明，在纯化的重组 BclA 和 BxpB 之间，体外也发生类似的交联，表明该反应是自发的。BclA 附着的机制，特别是蛋白水解切割产生反应性氨基基团和内部酸性残基侧链羧基的随意选择，似乎与其他通过异肽键形成蛋白质交联的已知机制不同。类似的机制似乎参与了其他孢子蛋白的交联，并且可能存在于无关的生物体中。
异肽键是自然界中发现的蛋白质修饰，其中酰胺键是在两个氨基酸的功能基团之间形成的，至少有一个功能基团是由氨基酸侧链提供的。异肽键在蛋白质内部和蛋白质之间产生交联，这对于蛋白质的正确结构和功能是必要的。在这项研究中，我们发现，Bacillus anthracis 孢子外部绒毛的主要结构蛋白 BclA 通过一种与先前描述的异肽键形成机制根本不同的机制附着在下面的外孢子基层蛋白 BxpB 上。这种机制的最不寻常的特征是蛋白水解切割产生反应性氨基基团和酸性侧链的随意选择。这种机制显然只依赖于蛋白质底物中的短肽序列，可能是体内的一种普遍机制，并适用于体外的蛋白质交联。
