Alphafold2-Assisted Structure-Function Analysis of Human Erythroferrone Relevant for Its Iron-Regulatory Function

• Published in: Blood 2023-11, Vol.142 (Supplement 1), p.921-921
• Main Authors: Srole, Daniel, Jung, Chun-Ling, Waring, Alan J, Nemeth, Elizabeta, Ganz, Tomas
• Format: Article
• Language: English
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood-2023-187670

Erythroferrone (ERFE), an erythrokine that helps mobilize iron for developing erythrocytes, acts by decreasing the production of hepcidin, through sequestration of bone morphogenetic proteins (BMPs) that bind to BMP receptors to activate hepcidin transcription. We used Alphafold2 (AF2) to model the conformation of human erythroferrone monomers and various multimers, with or without interactions with BMPs, with emphasis on the principal ligand, the BMP2/6 heterodimer. We then tested the models by targeted mutations to disrupt key interactions suggested by the models, and assessed the effect of the mutations on bioactivity and BMP binding. ERFE is a member of the C1q/TNFa family of proteins known for containing N-terminal unstructured regions and C-terminal TNFa-like globular heads that promote the formation of a spectrum of biologically significant multimers. After cleavage by a prohormone convertase, the ERFE molecule consists of an unstructured N-terminal segment residues 43-185 and a globular TNF-like head 186-354. We found that a segment of the N-terminal region of the molecule-segment 43-148 of the 354 amino acid protein-was sufficient to bind BMPs and suppress hepcidin; the larger C-terminal region does neither. For bioassays, we prepared ERFE or its mutants in HEK293 mammalian cells and tested these without purification, so as not to disrupt their mixture of multimeric conformations. The Hep3B human liver cell line was used for bioactivity assays with the readout of hepcidin mRNA concentrations, measured by qRT-PCR. To avoid the confounding effects of ERFE multimerization driven by the TNFa-like globular regions on avidity, we purified untagged ERFE N-terminal (43-148) segments or their mutants from bacteria, and measured the binding between these and BMPs using surface plasmon resonance. These forms lacked TNFa-like heads and, unlike full length ERFE, did not form multimers in nondenaturing PAGE. We identified and explored several structural elements within the ERFE N-terminus. All models of BMP interaction with ERFE predicted the binding of a tryptophan-containing hydrophobic helix of ERFE to a deep groove formed at the interface of all BMP dimers (BMP2/6 in cyan/green, ERFE in magenta; interaction in callout box, left panel). The highly evolutionarily conserved hydrophobic region (residues 81-86) was required for bioactivity and for tight binding to BMPs. Deletion of the hydrophobic segment or W82A substitution of the strictly conserved tryptophan