Extracellular Regulation of Bone Morphogenetic Protein Activity by the Microfibril Component Fibrillin-1

Clair Baldock,Gerhard Sengle,Richard F Collins,Helen Troilo,Alexander P Wohl

doi:10.1074/jbc.m115.704734

Abstract

Since the discovery of bone morphogenetic proteins (BMPs) as pluripotent cytokines extractable from bone matrix, it has been speculated how targeting of BMPs to the extracellular matrix (ECM) modulates their bioavailability. Understanding these processes is crucial for elucidating pathomechanisms of connective tissue disorders characterized by ECM deficiency and growth factor dysregulation. Here, we provide evidence for a new BMP targeting and sequestration mechanism that is controlled by the ECM molecule fibrillin-1. We present the nanoscale structure of the BMP-7 prodomain-growth factor complex using electron microscopy, small angle x-ray scattering, and circular dichroism spectroscopy, showing that it assumes an open V-like structure when it is bioactive. However, upon binding to fibrillin-1, the BMP-7 complex is rendered into a closed ring shape, which also confers latency to the growth factor, as demonstrated by bioactivity measurements. BMP-7 prodomain variants were used to map the critical epitopes for prodomain-growth factor and prodomain-prodomain binding. Together, these data show that upon prodomain binding to fibrillin-1, the BMP-7 complex undergoes a conformational change, which denies access of BMP receptors to the growth factor.

Highlights

Since the discovery of bone morphogenetic proteins (BMPs) as pluripotent cytokines extractable from bone matrix, it has been speculated how targeting of BMPs to the extracellular matrix (ECM) modulates their bioavailability
We present the nanoscale structure of the BMP-7 prodomain-growth factor complex using electron microscopy, small angle x-ray scattering, and circular dichroism spectroscopy, showing that it assumes an open V-like structure when it is bioactive
We determined for the first time the nanoscale structure of the BMP-7 complex, a PD-growth factor (GF) complex that is bioactive in solution, by using electron microscopy, small angle x-ray scattering (SAXS), and other biophysical approaches

Summary

Experimental Procedures

Protein Expression and Purification—A stably transfected BMP-7 complex expressing the HEK293 EBNA cell line and an expression plasmid coding for the N-terminal half of fibrillin-1 (rF11) were kindly provided by Dr Lynn Sakai (Shriners Hospital for Children Portland, Oregon Health and Science University, Portland, OR) [20, 21]. Transmission Electron Microscopy (TEM) and Single Particle Analysis—BMP-7 complex alone or dialyzed together with the fibrillin-1 N terminus at a 1:4 molar ratio (total protein concentration 10 –20 ␮g/ml) was negatively stained as described previously [24]. BMP-7 PD variants were dialyzed together with the GF in TBS containing 1 M urea overnight followed by incubation with the coated capture antibody and detection of the GF. For competition experiments with the BMP type II receptor, PD variants were immobilized at 500 – 800 RUs, and 100 nM BMP-7 GF was injected in the presence of 0 –500 nM of soluble extracellular domain of BMPRII receptor (R&D Systems) followed by a subsequent 100 nM injection (“coinject” mode) of a monoclonal anti-BMP-7 GF antibody (MAB3542, R&D Systems) to detect bound GF. The BMP-9 complex structure (Protein Data Bank code 4YCI) and the BMP-7 complex EM map were used to guide rotation of the PD into an open conformation

Results

KD nM

Discussion