Dataset Information

The NC2 domain of collagen IX provides chain selection and heterotrimerization.

ABSTRACT: The mechanism of chain selection and trimerization of fibril-associated collagens with interrupted triple helices (FACITs) differs from that of fibrillar collagens that have special C-propeptides. We recently showed that the second carboxyl-terminal non-collagenous domain (NC2) of homotrimeric collagen XIX forms a stable trimer and substantially stabilizes a collagen triple helix attached to either end. We then hypothesized a general trimerizing role for the NC2 domain in other FACITs. Here we analyzed the NC2 domain of human heterotrimeric collagen IX, the only member of FACITs with all three chains encoded by distinct genes. Upon oxidative folding of equimolar amounts of the alpha1, alpha2, and alpha3 chains of NC2, a stable heterotrimer with a disulfide bridge between alpha1 and alpha3 chains is formed. Our experiments show that this heterotrimerization domain can stabilize a short triple helix attached at the carboxyl-terminal end and allows for the proper oxidation of the cystine knot of type III collagen after the short triple helix.

SUBMITTER: Boudko SP

PROVIDER: S-EPMC2911272 | biostudies-literature | 2010 Jul

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

The NC2 domain of collagen IX provides chain selection and heterotrimerization.

Boudko Sergei P SP Zientek Keith D KD Vance Jesse J Hacker Jessica L JL Engel Jürgen J Bächinger Hans Peter HP

The Journal of biological chemistry 20100527 31

The mechanism of chain selection and trimerization of fibril-associated collagens with interrupted triple helices (FACITs) differs from that of fibrillar collagens that have special C-propeptides. We recently showed that the second carboxyl-terminal non-collagenous domain (NC2) of homotrimeric collagen XIX forms a stable trimer and substantially stabilizes a collagen triple helix attached to either end. We then hypothesized a general trimerizing role for the NC2 domain in other FACITs. Here we a ...[more]

PMID: 20507993

Similar Datasets

Project description:BACKGROUND: Collagen IX is an integral cartilage extracellular matrix component important in skeletal development and joint function. RESULTS: Proteomic analysis and validation studies revealed novel alterations in collagen IX null cartilage. CONCLUSION: Matrilin-4, collagen XII, thrombospondin-4, fibronectin, ?ig-h3, and epiphycan are components of the in vivo collagen IX interactome. SIGNIFICANCE: We applied a proteomics approach to advance our understanding of collagen IX ablation in cartilage. The cartilage extracellular matrix is essential for endochondral bone development and joint function. In addition to the major aggrecan/collagen II framework, the interacting complex of collagen IX, matrilin-3, and cartilage oligomeric matrix protein (COMP) is essential for cartilage matrix stability, as mutations in Col9a1, Col9a2, Col9a3, Comp, and Matn3 genes cause multiple epiphyseal dysplasia, in which patients develop early onset osteoarthritis. In mice, collagen IX ablation results in severely disturbed growth plate organization, hypocellular regions, and abnormal chondrocyte shape. This abnormal differentiation is likely to involve altered cell-matrix interactions but the mechanism is not known. To investigate the molecular basis of the collagen IX null phenotype we analyzed global differences in protein abundance between wild-type and knock-out femoral head cartilage by capillary HPLC tandem mass spectrometry. We identified 297 proteins in 3-day cartilage and 397 proteins in 21-day cartilage. Components that were differentially abundant between wild-type and collagen IX-deficient cartilage included 15 extracellular matrix proteins. Collagen IX ablation was associated with dramatically reduced COMP and matrilin-3, consistent with known interactions. Matrilin-1, matrilin-4, epiphycan, and thrombospondin-4 levels were reduced in collagen IX null cartilage, providing the first in vivo evidence for these proteins belonging to the collagen IX interactome. Thrombospondin-4 expression was reduced at the mRNA level, whereas matrilin-4 was verified as a novel collagen IX-binding protein. Furthermore, changes in TGF?-induced protein ?ig-h3 and fibronectin abundance were found in the collagen IX knock-out but not associated with COMP ablation, indicating specific involvement in the abnormal collagen IX null cartilage. In addition, the more widespread expression of collagen XII in the collagen IX-deficient cartilage suggests an attempted compensatory response to the absence of collagen IX. Our differential proteomic analysis of cartilage is a novel approach to identify candidate matrix protein interactions in vivo, underpinning further analysis of mutant cartilage lacking other matrix components or harboring disease-causing mutations.

Project description:The cytoplasmic membrane protein TonB couples the protonmotive force of the cytoplasmic membrane to active transport across the outer membrane of Escherichia coli. The uncleaved amino-terminal signal anchor transmembrane domain (TMD; residues 12 to 32) of TonB and the integral cytoplasmic membrane proteins ExbB and ExbD are essential to this process, with important interactions occurring among the several TMDs of all three proteins. Here, we show that, of all the residues in the TonB TMD, only His(20) is essential for TonB activity. When alanyl residues replaced all TMD residues except Ser(16) and His(20), the resultant "all-Ala Ser(16) His(20)" TMD TonB retained 90% of wild-type iron transport activity. Ser(16)Ala in the context of a wild-type TonB TMD was fully active. In contrast, His(20)Ala in the wild-type TMD was entirely inactive. In more mechanistically informative assays, the all-Ala Ser(16) His(20) TMD TonB unexpectedly failed to support formation of disulfide-linked dimers by TonB derivatives bearing Cys substitutions for the aromatic residues in the carboxy terminus. We hypothesize that, because ExbB/D apparently cannot efficiently down-regulate conformational changes at the TonB carboxy terminus through the all-Ala Ser(16) His(20) TMD, the TonB carboxy terminus might fold so rapidly that disulfide-linked dimers cannot be efficiently trapped. In formaldehyde cross-linking experiments, the all-Ala Ser(16) His(20) TMD also supported large numbers of apparently nonspecific contacts with unknown proteins. The all-Ala Ser(16) His(20) TMD TonB retained its dependence on ExbB/D. Together, these results suggest that a role for ExbB/D might be to control rapid and nonspecific folding that the unregulated TonB carboxy terminus otherwise undergoes. Such a model helps to reconcile the crystal/nuclear magnetic resonance structures of the TonB carboxy terminus with conformational changes and mutant phenotypes observed at the TonB carboxy terminus in vivo.

Dataset Information

The NC2 domain of collagen IX provides chain selection and heterotrimerization.

Publications

The NC2 domain of collagen IX provides chain selection and heterotrimerization.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets