Phosphorylation-induced structural remodeling of band 3 as a driver of metabolic dysfunction in sickle cell disease

Abstract Red blood cells (RBCs), devoid of nuclei and organelles, rely on post-translational modifications and dynamic protein–protein interactions to adapt to changing oxygen tensions. Central to this adaptability is the cytoplasmic N-terminal domain of band 3 (AE1), an intrinsically disordered region (IDR, residues 1–56) that acts as a molecular scaffold. This region coordinates metabolic flux by reversibly binding glycolytic enzymes (GEs, e.g. GAPDH, aldolase) under normoxic conditions, thereby suppressing glycolysis and diverting glucose toward the pentose phosphate pathway to maintain antioxidant capacity. Upon hypoxia, deoxyhemoglobin competes for the same binding site and displaces these enzymes, restoring glycolysis to optimize ATP production and promoting oxygen off-loading (Campanella et al., PNAS 2005). This rapid, tunable mechanism allows RBCs to sense and respond to oxygen gradients in the absence of de novo protein synthesis. While immunochemical studiesdefined these functional interactions, the lack of structural information has long limited our mechanistic understanding. The challenge lies in the highly dynamic nature of the band 3 N-terminal IDR and its multi-protein complexes, compounded by regulatory phosphorylation of two key tyrosine residues (Y8, Y21). Hyperphosphorylation of these sites, especially by Syk/Lyn kinases, has been implicated in hemolytic anemias, including sickle cell disease (SCD), where persistent phosphorylation alters the band 3 interactome, increases 2,3-DPG production, decreases hemoglobin oxygen affinity, and promotes HbS polymerization (Rogers et al., Blood 2013). Here we present a recombinant expression platform for the band 3 N-terminal IDR and use in vitro phosphorylation by Lyn-primed Syk to generate mono- and di-phosphorylated peptides, confirmed by 2D 1H–15N HSQC NMR. Using NMR chemical shift perturbations, ITC, and cross-linking mass spectrometry, we dissect the structural basis of these interactions for the first time. Our results reveal that unphosphorylated band 3 displays a broad interaction surface with both GAPDH and aldolase; GAPDH binds in a distributed manner, whereas aldolase preferentially interacts with residues surrounding Y21. Phosphorylation markedly reshapes these interaction maps and changes the overall peptide conformation when bound to GEs. Aldolase binding contacts become restricted; however, its binding induces a marked perturbation of the overall peptide conformation spanning the entire IDR. In contrast to aldolase, GAPDH maintains multivalent contacts but with altered residue preferences and its induced IDR conformational changes are limited near the sites of phosphorylation. These distinct spectral signatures emphasize that phosphorylation does not simply abolish enzyme binding but fine-tunes the interactome specific to each GE, despite their occupation of identical binding sites. Crosslinking proteomics maps these contacts directly onto GAPDH and aldolase structures, demonstrating distinct and dynamic modes of interaction that could not be resolved by previous antibody-based approaches (Issaian et al., Haematologica 2021). These findings support a refined model in which phosphorylation of Y8/Y21 serves as a rheostat, modulating the competition between glycolytic enzymes and deoxyhemoglobin for access to the band 3 scaffold. Such structural plasticity ensures rapid metabolic reprogramming across the oxygenation gradient. Importantly, the novel structural data provides a mechanistic framework for how dysregulated phosphorylation in SCD disrupts this adaptive balance, promoting persistent glycolysis, excessive 2,3-DPG production, and reduced oxygen affinity, thereby exacerbating HbS polymerization and oxidative damage. By resolving the elusive residue-level details of the band 3 interactome, this work opens new avenues for targeting RBC metabolism in hypoxic stress, hemoglobinopathies, and storage biology. Our study highlights the central role of the band 3 N-terminal IDR as a tunable metabolic hub linking oxygen sensing to redox homeostasis. These new structural insights bridge decades of functional work and suggest that pharmacological modulation of band 3 phosphorylation may represent a therapeutic opportunity to restore homeostasis in sickle cell disease and other disorders of RBC metabolism.