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Minicollagen cysteine-rich domains encode distinct modes of polymerization to form stable nematocyst capsules.

Tursch A, Mercadante D, Tennigkeit J, Gräter F, Özbek S - Sci Rep (2016)

Bottom Line: Our combined experimental and computational analyses reveal the cysteines in the C-CRD fold to exhibit a higher structural propensity for disulfide bonding and a faster kinetics of polymerization.During nematocyst maturation, the highly reactive C-CRD is instrumental in efficient cross-linking of minicollagens to form pressure resistant capsules.The higher ratio of C-CRD folding types evidenced in the medusozoan lineage might have fostered the evolution of novel, predatory nematocyst types in cnidarians with a free-swimming medusa stage.

View Article: PubMed Central - PubMed

Affiliation: University of Heidelberg, Centre for Organismal Studies, Department of Molecular Evolution and Genomics, Im Neuenheimer Feld 329, 69120 Heidelberg, Germany.

ABSTRACT
The stinging capsules of cnidarians, nematocysts, function as harpoon-like organelles with unusual biomechanical properties. The nanosecond discharge of the nematocyst requires a dense protein network of the capsule structure withstanding an internal pressure of up to 150 bar. Main components of the capsule are short collagens, so-called minicollagens, that form extended polymers by disulfide reshuffling of their cysteine-rich domains (CRDs). Although CRDs have identical cysteine patterns, they exhibit different structures and disulfide connectivity at minicollagen N and C-termini. We show that the structurally divergent CRDs have different cross-linking potentials in vitro and in vivo. While the C-CRD can participate in several simultaneous intermolecular disulfides and functions as a cystine knot after minicollagen synthesis, the N-CRD is monovalent. Our combined experimental and computational analyses reveal the cysteines in the C-CRD fold to exhibit a higher structural propensity for disulfide bonding and a faster kinetics of polymerization. During nematocyst maturation, the highly reactive C-CRD is instrumental in efficient cross-linking of minicollagens to form pressure resistant capsules. The higher ratio of C-CRD folding types evidenced in the medusozoan lineage might have fostered the evolution of novel, predatory nematocyst types in cnidarians with a free-swimming medusa stage.

No MeSH data available.


Related in: MedlinePlus

Divergent cross-linking properties of minicollagen CRDs.(A) An mCherry fusion protein carrying a single C-terminal CRD is still able to form extended polymers. (B–E) Polymer formation of mCherry-C-CRD is inhibited by the addition of a GFP fusion protein with a single C-terminal N-CRD in a dose-dependent manner as indicated by increased pore formation. (F) The GFP-N-CRD fusion protein is not able to form polymers by reoxidation indicating a restriction to single cysteine cross-links. Scale bars: 200 μm.
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f4: Divergent cross-linking properties of minicollagen CRDs.(A) An mCherry fusion protein carrying a single C-terminal CRD is still able to form extended polymers. (B–E) Polymer formation of mCherry-C-CRD is inhibited by the addition of a GFP fusion protein with a single C-terminal N-CRD in a dose-dependent manner as indicated by increased pore formation. (F) The GFP-N-CRD fusion protein is not able to form polymers by reoxidation indicating a restriction to single cysteine cross-links. Scale bars: 200 μm.

Mentions: To exclude that the apparent heterophilic assembly mode demonstrated in Fig. 3C is an artifact, we designed GFP and mCherry fusion proteins carrying only a single C-terminal N- or C-CRD domain. Our aim was to demonstrate polymer inhibition of the N-CRD-GFP-N-CRD fusion protein by addition of the mCherry-C-CRD fusion and vice versa. Surprisingly, when reoxidized as a homogenous solution, the GFP-N-CRD protein did not form any polymers (Fig. 4F) while the single C-CRD fused to mCherry was still able to induce polymerization in a similar manner as the mCherry construct flanked by two C-CRDs (Fig. 4A). When we mixed the two single CRD fusion proteins at different ratios before reoxidation, we observed dose-dependent mCherry-C-CRD polymer inhibition with increasing amounts of the GFP-N-CRD protein, which could be visualized as pores of increasing number and size in the polymer texture (Fig. 4B–E). These data not only confirm that N- and C-CRDs can interact with each other and form intermolecular disulfide links, but they clearly confirm a functional diversification of the two CRDs. While the N-CRD is restricted to single intermolecular cross-links, the C-CRD is able to engage in at least two intermolecular disulfide links simultaneously.


Minicollagen cysteine-rich domains encode distinct modes of polymerization to form stable nematocyst capsules.

Tursch A, Mercadante D, Tennigkeit J, Gräter F, Özbek S - Sci Rep (2016)

Divergent cross-linking properties of minicollagen CRDs.(A) An mCherry fusion protein carrying a single C-terminal CRD is still able to form extended polymers. (B–E) Polymer formation of mCherry-C-CRD is inhibited by the addition of a GFP fusion protein with a single C-terminal N-CRD in a dose-dependent manner as indicated by increased pore formation. (F) The GFP-N-CRD fusion protein is not able to form polymers by reoxidation indicating a restriction to single cysteine cross-links. Scale bars: 200 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4863159&req=5

f4: Divergent cross-linking properties of minicollagen CRDs.(A) An mCherry fusion protein carrying a single C-terminal CRD is still able to form extended polymers. (B–E) Polymer formation of mCherry-C-CRD is inhibited by the addition of a GFP fusion protein with a single C-terminal N-CRD in a dose-dependent manner as indicated by increased pore formation. (F) The GFP-N-CRD fusion protein is not able to form polymers by reoxidation indicating a restriction to single cysteine cross-links. Scale bars: 200 μm.
Mentions: To exclude that the apparent heterophilic assembly mode demonstrated in Fig. 3C is an artifact, we designed GFP and mCherry fusion proteins carrying only a single C-terminal N- or C-CRD domain. Our aim was to demonstrate polymer inhibition of the N-CRD-GFP-N-CRD fusion protein by addition of the mCherry-C-CRD fusion and vice versa. Surprisingly, when reoxidized as a homogenous solution, the GFP-N-CRD protein did not form any polymers (Fig. 4F) while the single C-CRD fused to mCherry was still able to induce polymerization in a similar manner as the mCherry construct flanked by two C-CRDs (Fig. 4A). When we mixed the two single CRD fusion proteins at different ratios before reoxidation, we observed dose-dependent mCherry-C-CRD polymer inhibition with increasing amounts of the GFP-N-CRD protein, which could be visualized as pores of increasing number and size in the polymer texture (Fig. 4B–E). These data not only confirm that N- and C-CRDs can interact with each other and form intermolecular disulfide links, but they clearly confirm a functional diversification of the two CRDs. While the N-CRD is restricted to single intermolecular cross-links, the C-CRD is able to engage in at least two intermolecular disulfide links simultaneously.

Bottom Line: Our combined experimental and computational analyses reveal the cysteines in the C-CRD fold to exhibit a higher structural propensity for disulfide bonding and a faster kinetics of polymerization.During nematocyst maturation, the highly reactive C-CRD is instrumental in efficient cross-linking of minicollagens to form pressure resistant capsules.The higher ratio of C-CRD folding types evidenced in the medusozoan lineage might have fostered the evolution of novel, predatory nematocyst types in cnidarians with a free-swimming medusa stage.

View Article: PubMed Central - PubMed

Affiliation: University of Heidelberg, Centre for Organismal Studies, Department of Molecular Evolution and Genomics, Im Neuenheimer Feld 329, 69120 Heidelberg, Germany.

ABSTRACT
The stinging capsules of cnidarians, nematocysts, function as harpoon-like organelles with unusual biomechanical properties. The nanosecond discharge of the nematocyst requires a dense protein network of the capsule structure withstanding an internal pressure of up to 150 bar. Main components of the capsule are short collagens, so-called minicollagens, that form extended polymers by disulfide reshuffling of their cysteine-rich domains (CRDs). Although CRDs have identical cysteine patterns, they exhibit different structures and disulfide connectivity at minicollagen N and C-termini. We show that the structurally divergent CRDs have different cross-linking potentials in vitro and in vivo. While the C-CRD can participate in several simultaneous intermolecular disulfides and functions as a cystine knot after minicollagen synthesis, the N-CRD is monovalent. Our combined experimental and computational analyses reveal the cysteines in the C-CRD fold to exhibit a higher structural propensity for disulfide bonding and a faster kinetics of polymerization. During nematocyst maturation, the highly reactive C-CRD is instrumental in efficient cross-linking of minicollagens to form pressure resistant capsules. The higher ratio of C-CRD folding types evidenced in the medusozoan lineage might have fostered the evolution of novel, predatory nematocyst types in cnidarians with a free-swimming medusa stage.

No MeSH data available.


Related in: MedlinePlus