Collagen VI, Conformation of A-domain Arrays and Microfibril Architecture

Abstract

Collagen VI is a ubiquitous extracellular matrix protein that assembles into beaded microfibrils that form networks linking cells to the matrix. Collagen VI microfibrils are typically formed from a heterotrimer of the α1, α2, and α3 chains. The α3 chain is distinct as it contains an extended N terminus with up to 10 consecutive von Willebrand factor type A-domains (VWA). Here, we use solution small angle x-ray scattering (SAXS) and single particle analysis EM to determine the nanostructure of nine of these contiguous A-domains. Both techniques reveal a tight C-shape conformation for the A-domains. Furthermore, using biophysical approaches, we demonstrate that the N-terminal region undergoes a conformational change and a proportion forms dimers in the presence of Zn2+. This is the first indication that divalent cations interact with collagen VI A-domains. A three-dimensional reconstruction of tissue-purified collagen VI microfibrils was generated using EM and single particle image analysis. The reconstruction showed the intricate architecture of the collagen VI globular regions, in particular the highly structurally conserved C-terminal region and variations in the appearance of the N-terminal region. The N-terminal domains project out from the globular beaded region like angled radial spokes. These could potentially provide interactive surfaces for other cell matrix molecules.

A Structure of a Collagen VI VWA Domain Displays N and C Termini at Opposite Sides of the Protein

“…This is in good agreement with the domain arrangement in collagen VI (Figure 1) and the observed extended structure of the N9-N1 fragment of collagen VI α3 (Beecher et al., 2011). Analysis of the Cα atom B factors shows that the C-terminal extension is less ordered than the core of VWA domain (data not shown), suggesting a degree of plasticity in the arrangement of the N5-N4 domain pair.…”

Defining the hierarchical organisation of collagen VI microfibrils at nanometre to micrometre length scales

“…The tail region is likely formed from the N1 domains from α1, 2 and 3 chains and these regions could accommodate six VWA domains consistent with a collagen VI triple-helical dimer in the half-bead structure. This new model is largely in agreement with the previous negative stain derived model [34]. A distinct difference though is the four lobed head structure, which was not seen previously.…”

“…Purified corneal microfibrils were imaged using cryo-TEM; a representative micrograph is shown in Fig.2C. Microfibrils had the characteristic globular bead structures separated by the collagen helical region as has been seen previously using negative stain TEM [34] and rotary shadowing [52].…”

“…This region was also shown to be poorly defined in the negative stain TEM model of the half-bead of collagen VI [34]. To determine if the heterogeneity observed in the N-terminal tail regions is due to flexibility in the α3 chain N-terminal VWA domains or potentially due to different compositions of α-chains, the volume of collagen VI beads was measured using AFM.…”

The expanded collagen VI family: new chains and new questions

“…The function of these N-terminal domains are poorly understood although the N-terminal globular domains of α3(VI) appear to function in the extracellular space, probably as potential binding sites for other ECM molecules. Studies on recombinantly produced α3(VI) suggest that portions of the N-terminus project away from the microfibril and are well-positioned to interact with other ECM components (13). Data from experiments in transfected SaOs-2 cells indicate that the N5 domain of α3(VI) plays an active role in microfibril formation in the extracellular space (14).…”