NMR spectroscopic and bioinformatic analyses of the LTBP1 C-terminus reveal a highly dynamic domain organisation

Abstract

Proteins from the LTBP/fibrillin family perform key structural and functional roles in connective tissues. LTBP1 forms the large latent complex with TGFβ and its propeptide LAP, and sequesters the latent growth factor to the extracellular matrix. Bioinformatics studies suggest the main structural features of the LTBP1 C-terminus are conserved through evolution. NMR studies were carried out on three overlapping C-terminal fragments of LTBP1, comprising four domains with characterised homologues, cbEGF14, TB3, EGF3 and cbEGF15, and three regions with no homology to known structures. The NMR data reveal that the four domains adopt canonical folds, but largely lack the interdomain interactions observed with homologous fibrillin domains; the exception is the EGF3-cbEGF15 domain pair which has a well-defined interdomain interface. (15)N relaxation studies further demonstrate that the three interdomain regions act as flexible linkers, allowing a wide range of motion between the well-structured domains. This work is consistent with the LTBP1 C-terminus adopting a flexible "knotted rope" structure, which may facilitate cell matrix interactions, and the accessibility to proteases or other factors that could contribute to TGFβ activation.

Figure 1. LTBP1 constructs.

Three overlapping constructs, cbEGF14-TB3 (LTBP1L residues 1468–1582), TB3-EGF3 (LTBP1L residues 1509–1662) and EGF3-cbEGF15 (LTBP1L residues 1617–1722), were generated which span the fibrillin-1 binding region. The numbering of selected domains is also shown for clarity. Fragments were expressed prokaryotically and refolded in vitro. Some of the activities attributed to different regions of the LTBP1 molecule are highlighted by black lines.

Figure 2. Alignment of cbEGF14-TB3-EGF3-cbEGF15 domains of LTBP1 and LTBP-like proteins from a variety of organisms.

Cysteine residues coloured yellow are found in 100% of the aligned sequences. Residues coloured blue are found in 70% or more of the aligned sequences. Numbers at the top of the alignment refer to residue numbers of human LTBP1. Black boxes highlight sites where interdomain packing G(Y/F) motifs would be expected in cbEGF14, TB3, and EGF3 domains (for packing with the C-terminal domain). A black arrow labelled “BMP1?” highlights a potential BMP1 cleavage site.

Figure 3. Residues in cbEGF14-TB3 and EGF3-cbEGF15 affected by calcium binding.

Calcium-dependant chemical shifts mapped onto homology models of A) cbEGF14-TB3 and B) EGF3-cbEGF15. Residues with calcium-dependant chemical shifts are shown in stick representation and coloured blue. Calcium is also shown as a red sphere in both structures.

Figure 4. Dynamics and packing interactions of the LTBP1 C-terminus, as determined by heteronuclear NOE experiments and comparison of HSQC peak positions in overlapping constructs.

A) Heteronuclear NOE ratios observed for residues in the cbEGF14-TB3 (dark blue), TB3-EGF3 (green) and EGF3-cbEGF15 (purple) constructs. A summary of the domain organisation is shown below. Error bars give the standard error for each value based on peak intensity relative to background noise. The heteronuclear NOE values for EGF3 are higher in the EGF3-cbEGF15 construct than in the TB3-EGF3 construct. In EGF3-cbEGF15, the presence of a well-defined packing interface means that the pair of domains tumbles as a single unit. In TB3-EGF3, where no interface is observed for the two domains, EGF3 tumbles as a smaller independent domain. B) Heteronuclear NOE values from A) plotted on a homology model of the complete LTBP1 C-terminus, with linker regions placed randomly using the PyMOL manual sculpting tool. Colour code provided at the bottom left, for overlapping residues 1488–1578 values from cbEGF14-TB3 are used, for residues 1579–1648 values from TB3-EGF3 are used, and for residues 1649–1722 values from EGF3-cbEGF15 pair are used. C) Differences in HSQC peak positions between pairs of constructs are plotted for the TB3 and EGF3 domains and inform on the presence or absence of inter-domain packing sites. Generally, only small differences in peak position are seen, the exception is in the GY packing motif of EGF3 (highlighted with **). This demonstrates the absence of inter-domain packing sites in TB3 and supports a packing interaction between EGF3 and cbEGF15.

Figure 5. RDC analysis of the LTBP1 EGF3-cbEGF15 domain pair.

A) Experimentally measured RDC values for EGF3-cbEGF15 are plotted as a function of sequence. Filled circles indicate the RDC values that were used in the fitting procedure to determine the inter-domain orientation of EGF3 and cbEGF15. The dotted line shows the boundary between EGF3 and cbEGF15. B) Plot of experimental RDCs versus RDCs obtained from the fitting procedure. Residues from EGF3 are shown as open triangles and those from cbEGF15 as filled triangles. The good correlation between experimental and calculated RDC values (R = 0.86) indicates that the RDC data are consistent with a rigid structure for EGF3-cbEGF15 with a well-defined inter-domain orientation.

Figure 6. Current model for the structure of the LTBP1 C-terminus.

A) Homology model of the LTBP1 C-terminus (coloured as in Figure 1A), and the structure of TGFβ1 (coloured dark red) in complex with its propeptide (coloured orange) , both shown on the same scale (rendered using PyMOL). For context the LTBP1 TB2 domain and cbEGF13 domain are also included, along with the four domains studied here, but rendered semi transparently. B) Cartoon representation of the LTBP1 C-terminus using arrows to highlight regions of significant flexibility.