Limits...
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

Disulfide-linked oligomers formed by N-CRD and C-CRD fusion proteins with the tetrameric RHCC.(A) Schematic representation of minicollagen-1 domain structure. The N-CRD is shown as green and the C-CRD as a red oval, the central collagen domain as a black bar. Lower panel: N-CRD and C-CRD amino acid sequences. The conserved cysteines are highlighted in red. Amino acids favoring the C-CRD fold are shown in bold. (B) N-CRD (green) and C-CRD (red) structures, secondary structure is shown in ribbons, disulfide bonds and the P10 and V4 residues responsible for the C-CRD fold are shown as sticks and colored by atom types (carbon, sulfur and nitrogen atoms are shown in grey, yellow and blue respectively). (C) Schematic representation of the C-CRD and N-CRD RHCC fusion proteins. (D) Tetrameric coiled-coil formation by the RHCC protein can be visualized in SDS-PAGE using low SDS concentration (right lane, apparent MW of about 35 kDa). Monomeric RHCC (left lane) has a calculated MW of 7,6 kDa and an apparent MW of about 9 kDa. (E) Disulfide-linked oligomers of RHCC-CRD fusion proteins in non-reducing SDS-PAGE (left lanes). IAA treatment blocks cysteine cross-linking (right lanes). Note that some residual dimers are present indicating incomplete modification with IAA.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4863159&req=5

f1: Disulfide-linked oligomers formed by N-CRD and C-CRD fusion proteins with the tetrameric RHCC.(A) Schematic representation of minicollagen-1 domain structure. The N-CRD is shown as green and the C-CRD as a red oval, the central collagen domain as a black bar. Lower panel: N-CRD and C-CRD amino acid sequences. The conserved cysteines are highlighted in red. Amino acids favoring the C-CRD fold are shown in bold. (B) N-CRD (green) and C-CRD (red) structures, secondary structure is shown in ribbons, disulfide bonds and the P10 and V4 residues responsible for the C-CRD fold are shown as sticks and colored by atom types (carbon, sulfur and nitrogen atoms are shown in grey, yellow and blue respectively). (C) Schematic representation of the C-CRD and N-CRD RHCC fusion proteins. (D) Tetrameric coiled-coil formation by the RHCC protein can be visualized in SDS-PAGE using low SDS concentration (right lane, apparent MW of about 35 kDa). Monomeric RHCC (left lane) has a calculated MW of 7,6 kDa and an apparent MW of about 9 kDa. (E) Disulfide-linked oligomers of RHCC-CRD fusion proteins in non-reducing SDS-PAGE (left lanes). IAA treatment blocks cysteine cross-linking (right lanes). Note that some residual dimers are present indicating incomplete modification with IAA.

Mentions: As reported previously, the morphological complexity of nematocyst types, which is higher in medusozoans than in anthozoans, is related to their molecular complexity9. The protein composition of Hydra nematocysts has been analyzed by mass spectrometry and revealed a significant number of extracellular matrix-like proteins10. Minicollagens constitute a prominent fraction of the capsule wall and tubule proteome and have been subject of detailed structure-function analyses91112. Minicollagens comprise a short central collagen sequence (12–16 Gly-X-Y repeats) flanked by variable polyproline stretches and N- and C-terminal CRDs (Fig. 1A). The CRDs have a conserved pattern of 6 cysteines (CX3CX3CX3CX3CC), which have been proposed to function in the formation of intermolecular disulfide bridges between minicollagen monomers during nematocyst maturation131415.


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)

Disulfide-linked oligomers formed by N-CRD and C-CRD fusion proteins with the tetrameric RHCC.(A) Schematic representation of minicollagen-1 domain structure. The N-CRD is shown as green and the C-CRD as a red oval, the central collagen domain as a black bar. Lower panel: N-CRD and C-CRD amino acid sequences. The conserved cysteines are highlighted in red. Amino acids favoring the C-CRD fold are shown in bold. (B) N-CRD (green) and C-CRD (red) structures, secondary structure is shown in ribbons, disulfide bonds and the P10 and V4 residues responsible for the C-CRD fold are shown as sticks and colored by atom types (carbon, sulfur and nitrogen atoms are shown in grey, yellow and blue respectively). (C) Schematic representation of the C-CRD and N-CRD RHCC fusion proteins. (D) Tetrameric coiled-coil formation by the RHCC protein can be visualized in SDS-PAGE using low SDS concentration (right lane, apparent MW of about 35 kDa). Monomeric RHCC (left lane) has a calculated MW of 7,6 kDa and an apparent MW of about 9 kDa. (E) Disulfide-linked oligomers of RHCC-CRD fusion proteins in non-reducing SDS-PAGE (left lanes). IAA treatment blocks cysteine cross-linking (right lanes). Note that some residual dimers are present indicating incomplete modification with IAA.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: Disulfide-linked oligomers formed by N-CRD and C-CRD fusion proteins with the tetrameric RHCC.(A) Schematic representation of minicollagen-1 domain structure. The N-CRD is shown as green and the C-CRD as a red oval, the central collagen domain as a black bar. Lower panel: N-CRD and C-CRD amino acid sequences. The conserved cysteines are highlighted in red. Amino acids favoring the C-CRD fold are shown in bold. (B) N-CRD (green) and C-CRD (red) structures, secondary structure is shown in ribbons, disulfide bonds and the P10 and V4 residues responsible for the C-CRD fold are shown as sticks and colored by atom types (carbon, sulfur and nitrogen atoms are shown in grey, yellow and blue respectively). (C) Schematic representation of the C-CRD and N-CRD RHCC fusion proteins. (D) Tetrameric coiled-coil formation by the RHCC protein can be visualized in SDS-PAGE using low SDS concentration (right lane, apparent MW of about 35 kDa). Monomeric RHCC (left lane) has a calculated MW of 7,6 kDa and an apparent MW of about 9 kDa. (E) Disulfide-linked oligomers of RHCC-CRD fusion proteins in non-reducing SDS-PAGE (left lanes). IAA treatment blocks cysteine cross-linking (right lanes). Note that some residual dimers are present indicating incomplete modification with IAA.
Mentions: As reported previously, the morphological complexity of nematocyst types, which is higher in medusozoans than in anthozoans, is related to their molecular complexity9. The protein composition of Hydra nematocysts has been analyzed by mass spectrometry and revealed a significant number of extracellular matrix-like proteins10. Minicollagens constitute a prominent fraction of the capsule wall and tubule proteome and have been subject of detailed structure-function analyses91112. Minicollagens comprise a short central collagen sequence (12–16 Gly-X-Y repeats) flanked by variable polyproline stretches and N- and C-terminal CRDs (Fig. 1A). The CRDs have a conserved pattern of 6 cysteines (CX3CX3CX3CX3CC), which have been proposed to function in the formation of intermolecular disulfide bridges between minicollagen monomers during nematocyst maturation131415.

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