Determining CD47 Structure and Discerning Function to Understand Signal Transduction Mechanism

Thrombospondin-1 (TSP-1), an ~450 kDa homotrimeric protein, was discovered as the first endogenous angiogenesis inhibitor. TSP-1 contains multiple domains and elicits numerous physiological responses. Many of these responses require binding to cell surface receptor CD47, an ~50 kDa plasma membrane protein with five transmembrane helices and an extracellular immunoglobulin-like domain. CD47 is central to immune evasion and is overexpressed in many cancers where binding to signal regulatory protein-α (SIRPα) on macrophages leads to immune escape. In endothelial cells, TSP-1 binding to CD47 leads to inhibition of nitric oxide (NO) signaling at multiple steps and is important for tumor growth inhibition. In some cellular contexts however, TSP-1 loses binding to CD47 and binding to the CD47 ectodomain alone in vitro is not detected. Understanding the mechanism behind TSP-1 binding as a step toward discerning CD47 signal transduction mechanism is central to our investigation. Presented herein are data describing the low resolution structure of full-length CD47 and its interaction with multiple binding partners, using cryo-electron microscopy (cryo-EM), surface plasmon resonance (SPR), and microscale thermophoresis (MST). CD47 alone is a challenging target for structure determination by cryo-EM due to its small size. To circumvent this, we pursued a CD47 structure in complex with a neutralizing Fab, which increases CD47-containing particle size and provides a known structural domain. Presented here are negative stain micrographs showing spherical micellar particles with three domains – the heavy and light chain domains of the Fab and the extracellular domain of CD47 – protruding from a micelle. Next, we resolved the CD47/Fab complex to ~6 Angstroms by cryo-EM, which will be highlighted in representative 2D classifications and a 3D model that includes the transmembrane helices. Also presented here are functional studies by SPR and MST, which show SIRPα binding to full-length CD47 with ~1 µM affinity, the first time binding has been measured with the full-length protein. We found TSP-1 binding to full-length CD47 to have nanomolar affinity as measured by MST, a binding event that was not detected for the extracellular domain alone in experiments by others. This suggests full-length CD47 is necessary for TSP-1 binding and signal transduction, perhaps due to a binding surface that includes the transmembrane domain.Release after 08/03/202