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

Close packing of three chains in a standard collagen triple helix requires Gly as every third residue. Missense mutations replacing one Gly by a larger residue in the tripeptide repeating sequence in type I collagen are common molecular causes of osteogenesis imperfecta. The structural and dynamic consequences of such mutations are addressed here by NMR studies on a peptide with a Gly-to-Ser substitution within an α1(I) sequence. Distances derived from nuclear Overhauser effects indicate that the three Ser residues are still packed in the center of the triple helix and that the standard 1-residue stagger is maintained. NMR dynamics using H-exchange and temperature-dependent amide chemical shifts indicate a greater disruption of hydrogen bonding and/or increased conformational flexibility C-terminal to the Ser site when compared with N terminal. This is consistent with recent suggestions relating clinical severity with an asymmetric effect of residues N- versus C-terminal to a mutation site. Dynamic studies also indicate that the relative position between a Gly in one chain and the mutation site in a neighboring staggered chain influences the disruption of the standard hydrogen-bonding pattern. The structural and dynamic alterations reported here may play a role in the etiology of osteogenesis imperfecta by affecting collagen secretion or interactions with other matrix molecules.Mutations in collagen result in a variety of connective tissue diseases (1, 2), with the clinical phenotype depending on the location and function of the collagen type. For instance, mutations in type I collagen, the major collagen in bone, lead to a bone disorder, osteogenesis imperfecta (OI),³ whereas mutations in type III collagen, which is present in high amounts in blood vessels, lead to aortic rupture in Ehlers-Danlos syndrome type IV (1, 2). All collagens have a triple helix motif composed of three polyproline II-like chains that are staggered by 1 residue and supercoiled about a common axis. The smallest residue Gly is typically present as every 3rd residue in each chain because of the tight packing of the chains, which generates the characteristic (Gly-Xaa-Yaa)_n repeating sequence. The Gly residues are all buried in the center, and the structure is stabilized by interchain N–H (Gly) … CO (Xaa) hydrogen bonds (3–5). The most common type of mutation leading to collagen disorders is a missense mutation that replaces 1 Gly in the repeating sequence by a larger residue.The best characterized collagen disease is OI, or brittle bone disease, which is distinguished by fragile bones due to mutations in type I collagen (2, 6). More than 400 Gly substitution missense mutations in the α1(I) and α2(I) chains of type I collagen have been reported to lead to OI (7). The severity of the disease varies widely from mild cases with multiple fractures to perinatal lethal cases (2, 6, 7). A single base change in a Gly codon can lead to one of 8 residues (Ser, Ala, Cys, Val, Arg, Asp, Glu, Trp) or a missense mutation. The smallest residue Ala is underrepresented in OI, suggesting that it may not always lead to pathology, whereas Ser mutations are overrepresented, corresponding to the most common substitutions observed. The 152 mutations leading to a Gly to Ser substitution account for ～39% of all missense mutations in the α1(I) of type I collagen (7), with 115 associated with mild phenotypes and 37 associated with lethal phenotypes.The identity of the residue replacing Gly may be a determinant in the clinical severity of OI. Model peptide studies indicate that the degree of triple helix destabilization depends on the residue replacing Gly, with a ranking of the least destabilizing to the most destabilizing Ala,Ser8). There is some correlation between clinical severity of OI cases and this destabilization scale, with the strongly destabilizing residues Val, Arg, Asp, and Glu associated largely with lethal phenotypes (8). 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).The standard triple helix conformation must undergo some structural perturbation as a result of a Gly replacement that is likely to relate to the development of the disorder. Thus it is important to define the structural consequences of a Gly substitution. It has not proved possible to obtain molecular information for the long collagen molecules themselves, but model collagen peptides have proved amenable to x-ray crystallography and NMR techniques (12, 13). The structure of a peptide containing a Gly to Ala substitution near the center of the peptide (Pro-Hyp-Gly)₁₀ has been solved by x-ray crystallography (5). This structure shows an overall straight molecule with standard triple helical structures at both ends and a localized conformational deformation at the Ala replacement site. The direct N–H (Gly) … CO (Xaa) hydrogen bond is replaced by a water-mediated hydrogen bond N–H (Ala) … H₂O … CO (Xaa).Here, NMR spectroscopy is used to define the structural and dynamic effect of a Gly to Ser replacement through the application of recently developed NMR methodology on selectively ¹³C/¹⁵N doubly labeled collagen peptides (14). This strategy includes chain assignments, measurement of NOEs, and scalar J-couplings to define the conformation of the peptide. These results combined with NMR hydrogen exchange experiments and temperature-dependent chemical shift data demonstrate the disturbed dynamic features and hydrogen bonding around the Ser substitution site. The NMR data of the Gly to Ser peptide are compared with the NMR and x-ray high resolution structure of the peptide containing a Gly to Ala substitution (5).