Stabilizing Isopeptide Bonds Revealed in Gram-Positive Bacterial Pilus Structure

Kang, Hae Joo; Coulibaly, F.; Clow, Fiona; Proft, Thomas; Baker, Edward N.

doi:10.1126/science.1145806

Cited by 312 publications

(518 citation statements)

References 25 publications

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“…2a). A mutein of PS whose catalytic residue essential for the isopeptide bond formation 31 was changed to alanine, E258A, did not polymerize under either oxidative or reductive conditions (Fig. 2a).…”

Section: Resultsmentioning

confidence: 99%

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Hyperthin nanochains composed of self-polymerizing protein shackles

et al. 2013

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Protein fibrils are expected to have applications as functional nanomaterials because of their sophisticated structures; however, nanoscale ordering of the functional units of protein fibrils remains challenging. Here we design a series of self-polymerizing protein monomers, referred to as protein shackles, derived from modified recombinant subunits of pili from Streptococcus pyogenes. The monomers polymerize into nanochains through spontaneous irreversible covalent bond formation. We design the protein shackles so that their reactions can be controlled by altering redox conditions, which affect disulphide bond formation between engineered cysteine residues. The interaction between the monomers improves their polymerization reactivity and determines morphologies of the polymers. In addition, green fluorescent protein-tagged protein shackles can polymerize, indicating proteins can be stably attached to the nanochains with its functionality preserved. Furthermore we demonstrate that a molecular-recognizable nanochain binds to its partner with an enhanced binding ability in solution. These characteristics are expected to be applied for novel protein nanomaterials.

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Section: Resultsmentioning

confidence: 99%

“…However, the biosynthesis of pili is difficult to duplicate in vitro, so we designed novel protein monomers based on the amino-acid sequence of a Streptococcus pyogenes pilus subunit (Spy0128) consisting of two domains, an N domain and a C domain 31 (Fig. 1a).…”

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Hyperthin nanochains composed of self-polymerizing protein shackles

et al. 2013

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“…These structures have aroused great interest because of their direct roles in infection and pathogenesis and their importance as vaccine candidates (2,3). They also use covalent isopeptide (amide) bonds, both intermolecular and intramolecular, to give strength and stability, and thus present a new paradigm among protein polymers (4)(5)(6).…”

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confidence: 99%

The Corynebacterium diphtheriae shaft pilin SpaA is built of tandem Ig-like modules with stabilizing isopeptide and disulfide bonds

Kang

Paterson

Gaspar

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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Cell-surface pili are important virulence factors that enable bacterial pathogens to adhere to specific host tissues and modulate host immune response. Relatively little is known about the structure of Gram-positive bacterial pili, which are built by the sortase-catalyzed covalent crosslinking of individual pilin proteins. Here we report the 1.6-Å resolution crystal structure of the shaft pilin component SpaA from Corynebacterium diphtheriae , revealing both common and unique features. The SpaA pilin comprises 3 tandem Ig-like domains, with characteristic folds related to those typically found in non-pilus adhesins. Whereas both the middle and the C-terminal domains contain an intramolecular Lys–Asn isopeptide bond, previously detected in the shaft pilins of Streptococcus pyogenes and Bacillus cereus , the middle Ig-like domain also harbors a calcium ion, and the C-terminal domain contains a disulfide bond. By mass spectrometry, we show that the SpaA monomers are cross-linked in the assembled pili by a Lys–Thr isopeptide bond, as predicted by previous genetic studies. Together, our results reveal that despite profound dissimilarities in primary sequences, the shaft pilins of Gram-positive pathogens have strikingly similar tertiary structures, suggesting a modular backbone construction, including stabilizing intermolecular and intramolecular isopeptide bonds.

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“…and B. burgdorferi is unknown, but to a large extent this crosslinking resembles the cross-linking of phage head proteins of HK-97 and the recently reported cross-linking of pilus proteins in Streptococcus pyogenes (27,32,52). As observed for HK-97 phage head proteins (52), the high-molecular-weight complex was found to be stable when it was boiled and when it was exposed to a number of protein-denaturing agents.…”

Section: Vol 190 2008 B Burgdorferi Flagellar Synthesis 1919mentioning

confidence: 99%

Borrelia burgdorferi Uniquely Regulates Its Motility Genes and Has an Intricate Flagellar Hook-Basal Body Structure

et al. 2008

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Borrelia burgdorferi is a flat-wave, motile spirochete that causes Lyme disease. Motility is provided by periplasmic flagella (PFs) located between the cell cylinder and an outer membrane sheath. The structure of these PFs, which are composed of a basal body, a hook, and a filament, is similar to the structure of flagella of other bacteria. To determine if hook formation influences flagellin gene transcription in B. burgdorferi, we inactivated the hook structural gene flgE by targeted mutagenesis. In many bacteria, completion of the hook structure serves as a checkpoint for transcriptional control of flagellum synthesis and other chemotaxis and motility genes. Specifically, the hook allows secretion of the anti-sigma factor FlgM and concomitant late gene transcription promoted by 28 . However, the control of B. burgdorferi PF synthesis differs from the control of flagellum synthesis in other bacteria; the gene encoding 28 is not present in the genome of B. burgdorferi, nor are any 28 promoter recognition sequences associated with the motility genes. We found that B. burgdorferi flgE mutants lacked PFs, were rod shaped, and were nonmotile, which substantiates previous evidence that PFs are involved in both cell morphology and motility. Although most motility and chemotaxis gene products accumulated at wild-type levels in the absence of FlgE, mutant cells had markedly decreased levels of the flagellar filament proteins FlaA and FlaB. Further analyses showed that the reduction in the levels of flagellin proteins in the spirochetes lacking FlgE was mediated at the posttranscriptional level. Taken together, our results indicate that in B. burgdorferi, the completion of the hook does not serve as a checkpoint for transcriptional regulation of flagellum synthesis. In addition, we also present evidence that the hook protein in B. burgdorferi forms a high-molecular-weight complex and that formation of this complex occurs in the periplasmic space.Borrelia burgdorferi, the causative agent of Lyme disease, is a spirochete with a characteristic flat-wave morphology (24, 25; for a review, see reference 12). As a result of their unusual morphology, B. burgdorferi and other spirochete species have a highly specialized ability that allows them to traverse viscous gellike media (8). This unique swimming ability enables these bacteria to penetrate into specific host connective tissues and ecological niches (43). The importance of motility as a virulence factor has been implicated in several spirochete species, including Treponema denticola (42), Brachyspira hyodysenteriae (54), Borrelia garinii (60), and B. burgdorferi (10, 56).Motility in B. burgdorferi is provided by bundles of between 7 and 11 periplasmic flagella (PFs) that are subterminally attached near the cell ends. These PFs extend inward along the cell cylinder beneath an outer membrane sheath (6,24,28,46). B. burgdorferi PFs have a structure similar to that of flagella of other bacteria; a PF is composed of a basal body, a hook, and a filament containing a single major ...

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Stabilizing Isopeptide Bonds Revealed in Gram-Positive Bacterial Pilus Structure

Cited by 312 publications

References 25 publications

Hyperthin nanochains composed of self-polymerizing protein shackles

Hyperthin nanochains composed of self-polymerizing protein shackles

The Corynebacterium diphtheriae shaft pilin SpaA is built of tandem Ig-like modules with stabilizing isopeptide and disulfide bonds

Borrelia burgdorferi Uniquely Regulates Its Motility Genes and Has an Intricate Flagellar Hook-Basal Body Structure

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