Solution Structure of an ABC Collagen Heterotrimer Reveals a Single-register Helix Stabilized by Electrostatic Interactions

Abstract

Collagen, known for its structural role in tissues and also for its participation in the regulation of homeostatic and pathological processes in mammals, is assembled from triple helices that can be either homotrimers or heterotrimers. High resolution structural information for natural collagens has been difficult to obtain because of their size and the heterogeneity of their native environment. For this reason, peptides that self-assemble into collagen-like triple helices are used to gain insight into the structure, stability, and biochemistry of this important protein family. Although many of the most common collagens in humans are heterotrimers, almost all studies of collagen helices have been on homotrimers. Here we report the first structure of a collagen heterotrimer. Our structure, obtained by solution NMR, highlights the role of electrostatic interactions as stabilizing factors within the triple helical folding motif. This addresses an issue that has been actively researched because of the predominance of charged residues in the collagen family. We also find that it is possible to selectively form a collagen heterotrimer with a well defined composition and register of the peptide chains within the helix, based on information encoded solely in the collagenous domain. Globular domains are implicated in determining the composition of several collagen types, but it is unclear what their role in controlling register may be. We show that is possible to design peptides that not only selectively choose a composition but also a specific register without the assistance of other protein constructs. This mechanism may be used in nature as well.Collagens constitute an important structural protein family. They are found in the extracellular matrix and undergo a hierarchical self-assembly into large supramolecular structures with specific morphologies carefully crafted by nature to fulfill diverse structural and functional roles in a wide variety of tissues. In total, there are 28 known isoforms of collagen in humans arranged in a variety of structures and in a wide range of tissues. The feature defining this protein family is the presence of domains with uninterrupted Xaa-Yaa-Gly sequence repeats. These domains adopt a left-handed polyproline type II conformation because of the predominance of proline in the X position and hydroxyproline (Hyp ϭ O), a post-translationally modified amino acid with a hydroxyl group on the ␥-carbon of the proline side chain, in the Y position. Three such domains associate to form tightly packed right-handed triple helices in a folding motif commonly known as the collagen triple helix.Collagens are also implicated in pivotal homeostatic events in mammals such as the production of new vascular tissue and pathological conditions such as cancer metastasis (1). These processes are notoriously governed by interactions at the molecular level between cell surface proteins and the collagen triple helix and not by the morphology of the collagen aggregates (2, 3). Thus, an understanding of the colla...

Structural Insights into Charge Pair Interactions in Triple Helical Collagen-like Proteins

“…Given that no increased thermal stability is observed for the sequence containing only lateral interactions we propose that they contribute marginally, if at all, to the stability of triple helical proteins. In contrast, the KGE and KGD peptides have thermal stabilities comparable with unsubstituted (POG) 8 triple helices which can only be explained by invoking a pair-wise inter-amino acid contribution that does not exist for peptides with only lateral interactions. By this logic, the increased thermal stability observed in the KGE/D sequences comes from the two axial interactions that we observe in the both NMR ensembles as well as the x-ray structure of both peptides.…”

Anti-citrullinated protein antibodies cause arthritis by cross-reactivity to joint cartilage

“…Citrullination may alter triple-helical stability if this modification disrupts favorable interchain and/or interhelical electrostatic interactions (23)(24)(25)(26)(27). The citrulline appears to be distinguished from its unmodified predecessor arginine via electrostatic-controlled interactions, which also allow cross-reactivity with other collagen epitopes containing fewer polar amino acids at the equivalent position.…”

The NC2 Domain of Type IX Collagen Determines the Chain Register of the Triple Helix

“…Another approach in achieving the determined stagger is a system of three artificial collagen-like chains bearing compensating charges that allow a specific assembly of hetero-trimeric molecules (16). Surprisingly, the highly preferred stagger was detected using NMR technique (17). Despite successful development of the artificial hetero-trimeric collagen system with the stagger control, its application for natural sequences seems to be impractical due to unpredictable effects of the loaded natural sequence on composition and chain register.…”