Recombinant Collagen Studies Link the Severe Conformational Changes Induced by Osteogenesis Imperfecta Mutations to the Disruption of a Set of Interchain Salt Bridges

Abstract

The clinical severity of Osteogenesis Imperfecta (OI), also known as the brittle bone disease, relates to the extent of conformational changes in the collagen triple helix induced by Gly substitution mutations. The lingering question is why Gly substitutions at different locations of collagen cause different disruptions of the triple helix. Here, we describe markedly different conformational changes of the triple helix induced by two Gly substitution mutations placed only 12 residues apart. The effects of the Gly substitutions were characterized using a recombinant collagen fragment modeling the 63-residue segment of the ␣1 chain of type I collagen containing no Hyp (residues 877-939) obtained from Escherichia coli. Two Gly 3 Ser substitutions at Gly-901 and Gly-913 associated with, respectively, mild and severe OI variants were introduced by site-directed mutagenesis. Biophysical characterization and limited protease digestion experiments revealed that while the substitution at Gly-901 causes relatively minor destabilization of the triple helix, the substitution at Gly-913 induces large scale unfolding of an unstable region C-terminal to the mutation site. This extensive unfolding is caused by the intrinsic low stability of the C-terminal region of the helix and the mutation induced disruption of a set of salt bridges, which functions to lock this unstable region into the triple helical conformation. The extensive conformational changes associated with the loss of the salt bridges highlight the long range impact of the local interactions of triple helix and suggest a new mechanism by which OI mutations cause severe conformational damages in collagen.Considerable effort has been made to elucidate the mechanisms by which Gly substitution mutations of the collagen triple helix cause Osteogenesis Imperfecta (OI), 2 also known as brittle bone disease. The collagen triple helix consists of three polypeptide chains each in extended polyproline II conformation and with the characteristic (Gly-X-Y) n repeating amino acid sequence (1-3). The Gly at every third position is necessitated by the close packing of the helix; while the X and Y residues (where X and Y can be any amino acids) contribute directlytothestabilityofthetriplehelixandconferthesequencedependent properties of collagen (4). Missense mutations that replace the obligatory Gly by another amino acid residue in type I collagen, the major component of bones, are the most common cause of OI (5, 6). The triple helix domain of type I collagen is a heterotrimer composed of two ␣1 chains and one ␣2 chain each with more than 1000 amino acids in an uninterrupted (Gly-X-Y) n sequence (7). Nearly 800 Gly replacing mutations from both ␣1 and ␣2 chains have been linked to OI, yet, depending on the location and the identity of the Gly substitution, the clinical severity of OI varies from mild increase of bone fragility to the most severe type characterized by death at the prenatal stage (the Type II OI) (6). It remains unclear what molecular properties are related to the se...

NMR Conformational and Dynamic Consequences of a Gly to Ser Substitution in an Osteogenesis Imperfecta Collagen Model Peptide

“…However, as cited above, a Gly to Ser mutation can lead to a mild, a severe, or a lethal OI case, with no obvious molecular explanation. Other factors suggested to contribute to clinical phenotype include the rigidity of its immediate sequence environment; its location with respect to the C terminus; its proximity to salt bridges; and its presence at an interaction site, such as the binding site for proteoglycans on collagen fibrils (7,9). A recent study of the stability of OI collagens supported the importance of the domain location of the mutation (10), whereas a network analysis of the mutations suggested the importance of a destabilizing tripeptide sequence C-terminal to the mutation site (11).…”

Genetically Modified Collagen‐like Triple Helix Peptide as Biomimetic Template THIS CHAPTER HAS BEEN RETRACTED

“…46 All crystal structures show hydrogen bonds between the NH of Gly in one chain and the C¼O of the residue at the X position of the neighboring chain. 16,50,51 When the Y position is occupied by an amino acid rather than an imino acid, it is hydrated by water molecules and directed into the solvent, which reduces the helix stability. 19,24 In addition, peptides with the sequence where the X position is occupied by a residue other than Pro show second interchain hydrogen bonds …”

“…By using recombinant technology, one can design and amplify a collagen-like triple helix that is monodisperse, easily mineralized with metal ions, and can thus be applied as a rigid biomolecular template for metal/semiconductor nanowire fabrications. 4,16 …”

Inherited Connective Tissue Disorders of Collagens: Lessons from Targeted Mutagenesis

“…To circumvent the difficulty of producing a large repertoire of full-length mutated collagens I in order to undertake a genotype/phenotype analysis, a recombinant trimeric minicollagen I was recently expressed in an Escherichia coli system. Recombinant mini-collagens can be obtained by fusing the sequence encoding a fragment of the pro1(I) chain triplehelix to the sequence encoding the C-terminal domain (called "foldon") of the bacteriophage T4 fibritin, which is capable of trimerization (Xu et al, 2008). Two mutations (G901S and G913S), corresponding to mild and severe types of OI, respectively, were introduced into the recombinant mini-collagen I. Biophysical measurements and protease cleavage analysis revealed that the G913S mutant chain resulted in the formation of an unstable collagen I triple helix by disrupting salt bridges important for maintaining the chains in a triple-helix conformation (Yang et al, 1997;Xu et al, 2008).…”

“…Recombinant mini-collagens can be obtained by fusing the sequence encoding a fragment of the pro1(I) chain triplehelix to the sequence encoding the C-terminal domain (called "foldon") of the bacteriophage T4 fibritin, which is capable of trimerization (Xu et al, 2008). Two mutations (G901S and G913S), corresponding to mild and severe types of OI, respectively, were introduced into the recombinant mini-collagen I. Biophysical measurements and protease cleavage analysis revealed that the G913S mutant chain resulted in the formation of an unstable collagen I triple helix by disrupting salt bridges important for maintaining the chains in a triple-helix conformation (Yang et al, 1997;Xu et al, 2008). A very recent study utilized a recombinant bacterial collagen to develop a mutagenesis scheme in which a glycine residue within the triple-helix sequence is substituted with arginine or serine.…”