Structure of the human marker of self 5-transmembrane receptor CD47

Gustavo Fenalti,Sirish Kaushik Lakkaraju,Alok Sharma,Nicolas Villanueva,Kandasamy Hariharan,Barbra Pagarigan,Jeffrey Johnson,Nadia Ladygina,Mahan Abbasian,Philip P Chamberlain,Mark Griffith,Dan Zhu,David Mikolon,Haralambos Hadjivassiliou,Ho Cho,Richard Y.-C Huang

doi:10.1038/s41467-021-25475-w

Gustavo Fenalti, Sirish Kaushik Lakkaraju + Show 14 more

Open Access

https://doi.org/10.1038/s41467-021-25475-w

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Journal: Nature Communications	Publication Date: Sep 1, 2021
Citations: 24	License type: open-access

Affiliation: Bristol-Myers Squibb (United States)

Abstract

CD47 is the only 5-transmembrane (5-TM) spanning receptor of the immune system. Its extracellular domain (ECD) is a cell surface marker of self that binds SIRPα and inhibits macrophage phagocytosis, and cancer immuno-therapy approaches in clinical trials are focused on blocking CD47/SIRPα interaction. We present the crystal structure of full length CD47 bound to the function-blocking antibody B6H12. CD47 ECD is tethered to the TM domain via a six-residue peptide linker (114RVVSWF119) that forms an extended loop (SWF loop), with the fundamental role of inserting the side chains of W118 and F119 into the core of CD47 extracellular loop region (ECLR). Using hydrogen-deuterium exchange and molecular dynamics simulations we show that CD47’s ECLR architecture, comprised of two extracellular loops and the SWF loop, creates a molecular environment stabilizing the ECD for presentation on the cell surface. These findings provide insights into CD47 immune recognition, signaling and therapeutic intervention.

Highlights

Cluster of Differentiation 47 (CD47) is the only 5-transmembrane (5-TM) spanning receptor of the immune system
To facilitate crystallization of CD47 in lipidic mesophases[24] we engineered a construct consisting of the full-length human CD47 with a thermostabilized (M7W, H102I, and R106L) apocytochrome b562RIL from Escherichia coli (BRIL)[25] fusion protein inserted in the intracellular loop (ICL) 1 of the receptor, namely CD47BRIL (Methods)
The crystallographic asymmetric unit contains a dimer of the CD47BRILB6H12 assembly, and interactions between the two CD47BRILB6H12 units are mediated entirely through the Fragment antigen binding (Fab)’s from each unit (Supplementary Fig. 1b)

Summary

Introduction

Its extracellular domain (ECD) is a cell surface marker of self that binds SIRPα and inhibits macrophage phagocytosis, and cancer immuno-therapy approaches in clinical trials are focused on blocking CD47/SIRPα interaction. Using hydrogen-deuterium exchange and molecular dynamics simulations we show that CD47’s ECLR architecture, comprised of two extracellular loops and the SWF loop, creates a molecular environment stabilizing the ECD for presentation on the cell surface These findings provide insights into CD47 immune recognition, signaling and therapeutic intervention. CD47 is an essential component of the innate immune system, and binding of the ECD to signal regulatory protein alpha (SIRPα)[5,6,7], abundant in myeloid cells and on macrophages, activates a signaling response that inhibits cell phagocytosis (‘don’t eat me’ signal). Members of the Poxviridea family of viruses, which devote numerous genes to the expression of molecules for evasion of the host immune system, express

Methods

Results

Conclusion