Substrate binding and inhibition of the anion exchanger 1 transporter.

Anion exchanger 1 (AE1), a member of the solute carrier (SLC) family, is the primary bicarbonate transporter in erythrocytes, regulating pH levels and CO2 transport between lungs and tissues. Previous studies characterized its role in erythrocyte structure and provided insight into transport regulation. However, key questions remain regarding substrate binding and transport, mechanisms of drug inhibition and modulation by membrane components. Here we present seven cryo-EM structures in apo, bicarbonate-bound and inhibitor-bound states. These, combined with uptake and computational studies, reveal important molecular features of substrate recognition and transport, and illuminate sterol binding sites, to elucidate distinct inhibitory mechanisms of research chemicals and prescription drugs. We further probe the substrate binding site via structure-based ligand screening, identifying an AE1 inhibitor. Together, our findings provide insight into mechanisms of solute carrier transport and inhibition.

CryoEM structures of anion exchanger 1 capture multiple states of inward- and outward-facing conformations.

Anion exchanger 1 (AE1, band 3) is a major membrane protein of red blood cells and plays a key role in acid-base homeostasis, urine acidification, red blood cell shape regulation, and removal of carbon dioxide during respiration. Though structures of the transmembrane domain (TMD) of three SLC4 transporters, including AE1, have been resolved previously in their outward-facing (OF) state, no mammalian SLC4 structure has been reported in the inward-facing (IF) conformation. Here we present the cryoEM structures of full-length bovine AE1 with its TMD captured in both IF and OF conformations. Remarkably, both IF-IF homodimers and IF-OF heterodimers were detected. The IF structures feature downward movement in the core domain with significant unexpected elongation of TM11. Molecular modeling and structure guided mutagenesis confirmed the functional significance of residues involved in TM11 elongation. Our data provide direct evidence for an elevator-like mechanism of ion transport by an SLC4 family member.

Impaired trafficking and instability of mutant kidney anion exchanger 1 proteins associated with autosomal recessive distal renal tubular acidosis.

BACKGROUND: Mutations in solute carrier family 4 member 1 (SLC4A1) encoding anion exchanger 1 (AE1) are the most common cause of autosomal recessive distal renal tubular acidosis (AR dRTA) in Southeast Asians. To explain the molecular mechanism of this disease with hematological abnormalities in an affected family, we conducted a genetic analysis of SLC4A1 and studied wild-type and mutant AE1 proteins expressed in human embryonic kidney 293T (HEK293T) cells. METHODS: SLC4A1 mutations in the patient and family members were analyzed by molecular genetic techniques. Protein structure modeling was initially conducted to evaluate the effects of mutations on the three-dimensional structure of the AE1 protein. The mutant kidney anion exchanger 1 (kAE1) plasmid construct was created to study protein expression, localization, and stability in HEK293T cells. RESULTS: We discovered that the patient who had AR dRTA coexisting with mild hemolytic anemia carried a novel compound heterozygous SLC4A1 mutations containing c.1199_1225del (p.Ala400_Ala408del), resulting in Southeast Asian ovalocytosis (SAO), and c.1331C > A (p.Thr444Asn). Homologous modeling and in silico mutagenesis indicated that these two mutations affected the protein structure in the transmembrane regions of kAE1. We found the wild-type and mutant kAE1 T444N to be localized at the cell surface, whereas the mutants kAE1 SAO and SAO/T444N were intracellularly retained. The half-life of the kAE1 SAO, T444N, and SAO/T444N mutants was shorter than that of the wild-type protein. CONCLUSION: These results suggest impaired trafficking and instability of kAE1 SAO/T444N as the likely underlying molecular mechanism explaining the pathogenesis of the novel SLC4A1 compound heterozygous mutation identified in this patient.

Architecture of the human erythrocyte ankyrin-1 complex.

The stability and shape of the erythrocyte membrane is provided by the ankyrin-1 complex, but how it tethers the spectrin-actin cytoskeleton to the lipid bilayer and the nature of its association with the band 3 anion exchanger and the Rhesus glycoproteins remains unknown. Here we present structures of ankyrin-1 complexes purified from human erythrocytes. We reveal the architecture of a core complex of ankyrin-1, the Rhesus proteins RhAG and RhCE, the band 3 anion exchanger, protein 4.2, glycophorin A and glycophorin B. The distinct T-shaped conformation of membrane-bound ankyrin-1 facilitates recognition of RhCE and, unexpectedly, the water channel aquaporin-1. Together, our results uncover the molecular details of ankyrin-1 association with the erythrocyte membrane, and illustrate the mechanism of ankyrin-mediated membrane protein clustering.

The structural and functional workings of KEOPS.

KEOPS (Kinase, Endopeptidase and Other Proteins of Small size) is a five-subunit protein complex that is highly conserved in eukaryotes and archaea and is essential for the fitness of cells and for animal development. In humans, mutations in KEOPS genes underlie Galloway-Mowat syndrome, which manifests in severe microcephaly and renal dysfunction that lead to childhood death. The Kae1 subunit of KEOPS catalyzes the universal and essential tRNA modification N6-threonylcarbamoyl adenosine (t6A), while the auxiliary subunits Cgi121, the kinase/ATPase Bud32, Pcc1 and Gon7 play a supporting role. Kae1 orthologs are also present in bacteria and mitochondria but function in distinct complexes with proteins that are not related in structure or function to the auxiliary subunits of KEOPS. Over the past 15 years since its discovery, extensive study in the KEOPS field has provided many answers towards understanding the roles that KEOPS plays in cells and in human disease and how KEOPS carries out these functions. In this review, we provide an overview into recent advances in the study of KEOPS and illuminate exciting future directions.

Large conformational dynamics in Band 3 protein: Significance for erythrocyte senescence signalling.

Band 3 (Anion Exchanger 1, AE1), the predominant protein of erythrocyte membranes, facilitates Cl-/HCO3- exchange and anchors the plasma membrane to the cytoskeleton. The Band 3 crystal structure revealed the amino acid 812-830 region as intracellular, conflicting with protein chemical data that suggested extracellular disposition. Further, circulating senescent cell auto-antibody that cannot enter erythrocytes, binds two regions of Band 3: residues 538-554 and 812-830. To reconcile this discrepancy, we assessed localization of residues 812-830 with Band 3 expressed in HEK293 cells and human erythrocytes, using chemical labeling probes and an antibody against residues 812-830. Antibody and chemical probes revealed reorientation of 812-830 region between extracellular and intracellular. This dramatic conformational change is an intrinsic property of the Band 3 molecule, occurring when expressed in HEK293 cells and without the damage that occurs during erythrocyte circulation. Conditions used to crystallize Band 3 for structural determination did not alter conformational dynamics. Collectively, these data reveal large Band 3 conformational dynamics localized to a region previously identified as an erythrocyte senescence epitope. Surface exposure of the senescence epitope (812-830), limited by conformational dynamics, may act as the "molecular clock" in erythrocyte senescence.

The interactome of the N-terminus of band 3 regulates red blood cell metabolism and storage quality.

Band 3 (anion exchanger 1; AE1) is the most abundant membrane protein in red blood cells, which in turn are the most abundant cells in the human body. A compelling model posits that, at high oxygen saturation, the N-terminal cytosolic domain of AE1 binds to and inhibits glycolytic enzymes, thus diverting metabolic fluxes to the pentose phosphate pathway to generate reducing equivalents. Dysfunction of this mechanism occurs during red blood cell aging or storage under blood bank conditions, suggesting a role for AE1 in the regulation of the quality of stored blood and efficacy of transfusion, a life-saving intervention for millions of recipients worldwide. Here we leveraged two murine models carrying genetic ablations of AE1 to provide mechanistic evidence of the role of this protein in the regulation of erythrocyte metabolism and storage quality. Metabolic observations in mice recapitulated those in a human subject lacking expression of AE11-11 (band 3 Neapolis), while common polymorphisms in the region coding for AE11-56 correlate with increased susceptibility to osmotic hemolysis in healthy blood donors. Through thermal proteome profiling and crosslinking proteomics, we provide a map of the red blood cell interactome, with a focus on AE11-56 and validate recombinant AE1 interactions with glyceraldehyde 3-phosphate dehydrogenase. As a proof-of-principle and to provide further mechanistic evidence of the role of AE1 in the regulation of redox homeo stasis of stored red blood cells, we show that incubation with a cell-penetrating AE11-56 peptide can rescue the metabolic defect in glutathione recycling and boost post-transfusion recovery of stored red blood cells from healthy human donors and genetically ablated mice.

Molecular Simulations of Intact Anion Exchanger 1 Reveal Specific Domain and Lipid Interactions.

Anion exchanger 1 (AE1) is responsible for the exchange of bicarbonate and chloride across the erythrocyte plasma membrane. Human AE1 consists of a cytoplasmic and a membrane domain joined by a 33-residue flexible linker. Crystal structures of the individual domains have been determined, but the intact AE1 structure remains elusive. In this study, we use molecular dynamics simulations and modeling to build intact AE1 structures in a complex lipid bilayer that resembles the native erythrocyte plasma membrane. AE1 models were evaluated using available experimental data to provide an atomistic view of the interaction and dynamics of the cytoplasmic domain, the membrane domain, and the connecting linker in a complete model of AE1 in a lipid bilayer. Anionic lipids were found to interact strongly with AE1 at specific amino acid residues that are linked to diseases and blood group antigens. Cholesterol was found in the dimeric interface of AE1, suggesting that it may regulate subunit interactions and anion transport.

Crystallization of Human Erythrocyte Band 3, the anion exchanger, at the International Space Station "KIBO".

Band 3 mediates the Cl- and HCO3- exchange across the red blood cell membrane and plays a pivotal role for delivering oxygen appropriately to metabolically active tissues. For understanding molecular mechanisms, it is essential to know the structure and function relationship. In terrestrial environments, however, nobody could make good quality crystals of Band 3 for the X-ray crystallographic study. In this study, we purified the transmembrane domain of Band 3 from human red blood cells and crystallized the purified Band 3 without the Fab fragment at the International Space Station "KIBO" under microgravity environments.

Interaction of the human erythrocyte Band 3 anion exchanger 1 (AE1, SLC4A1) with lipids and glycophorin A: Molecular organization of the Wright (Wr) blood group antigen.

The Band 3 (AE1, SLC4A1) membrane protein is found in red blood cells and in kidney where it functions as an electro-neutral chloride/bicarbonate exchanger. In this study, we have used molecular dynamics simulations to provide the first realistic model of the dimeric membrane domain of human Band 3 in an asymmetric lipid bilayer containing a full complement of phospholipids, including phosphatidylinositol 4,5-bisphosphate (PIP2) and cholesterol, and its partner membrane protein Glycophorin A (GPA). The simulations show that the annular layer in the inner leaflet surrounding Band 3 was enriched in phosphatidylserine and PIP2 molecules. Cholesterol was also enriched around Band 3 but also at the dimer interface. The interaction of these lipids with specific sites on Band 3 may play a role in the folding and function of this anion transport membrane protein. GPA associates with Band 3 to form the Wright (Wr) blood group antigen, an interaction that involves an ionic bond between Glu658 in Band 3 and Arg61 in GPA. We were able to recreate this complex by performing simulations to allow the dimeric transmembrane portion of GPA to interact with Band 3 in a model membrane. Large-scale simulations showed that the GPA dimer can bridge Band 3 dimers resulting in the dynamic formation of long strands of alternating Band 3 and GPA dimers.

Insights into the morphological pattern of erythrocytes' aging: Coupling quantitative AFM data to microcalorimetry and Raman spectroscopy.

Erythrocytes (RBCs) constitute a very interesting class of cells both for their physiological function and for a variety of peculiarities. Due to their exceptionally strong relationship with the environment, the morphology and nanoscale characteristics of these cells can reveal their biochemical status and structural integrity. Among the possible subjects of investigations, the RBCs' ageing is of the utmost importance. This is a fundamental phenomenon that, in physiological conditions, triggers the cell turnover and ensures the blood homeostasis. With these premises, in recent years, we have presented an atomic force microscopy-based methodology to characterize the patterns of RBC ageing from the morphological point of view. In the present work, we used an ageing protocol more similar to the physiological conditions and we used differential scanning calorimetry and atomic force microscopy to probe the cross correlation between important structural and functional proteins. We also assessed the role played by fundamental structural and membrane proteins in the development of the most relevant morphological intermediates observed along the ageing. Furthermore, we coupled the morphological ageing patterns to the (bio)chemical alterations detected by Raman spectroscopy. This allowed identifying the chronology of the ageing morphologies and the metabolic pathways most involved in their development. As a whole, the present study provides the base to correlate specific molecular alterations to the development of structural anomalies, and these latter to the functional status of blood cells.

Band 3 function and dysfunction in a structural context.

PURPOSE OF REVIEW: Current research on the human band 3 glycoprotein, the red cell chloride/bicarbonate anion exchanger (AE1), is highlighted and placed within a structural context. RECENT FINDINGS: The determination of the crystal structure of the membrane domain of human band 3, the founding member of the solute carrier 4 (SLC4) family of bicarbonate transporters, is a major breakthrough toward understanding the mechanism of action of this membrane transport protein, its interaction with partner proteins, and how mutations linked to disease affect its ability to fold and function. SUMMARY: Band 3 contains 14 transmembrane segments arranged in a 7+7 transmembrane inverted repeat topology common to all members of the SLC4 family and the unrelated SLC26 anion transporter family. A functional feature of this fold is the presence of a core and a gate domain: the core domain contains two short transmembrane helices (TM3 and 10) that face each other in the middle of the membrane with the positive N-terminal helix dipoles creating the anion-binding site, whereas the gate domain forms the dimer interface. During transport, the movement of these two domains relative to each other provides the intracellular and extracellular compartments with alternating access to the central anion-binding site.

Molecular mechanism for the red blood cell senescence clock.

Lacking protein synthesis machinery and organelles necessary for autophagy or apoptosis, aged red blood cells (RBCs) are marked by circulating auto-antibodies for macrophage-mediated clearance. The antigen recognized by these auto-antibodies is the major protein of the RBC membrane, Band 3. To ensure regulation and specificity in clearance, the molecular "clock" must mark senescent cells in a way that differentiates them from younger cells, to prevent premature clearance. Predominant models of Band 3 senescence signaling are reviewed, and merits are discussed in light of the recently published crystal structure of the Band 3 membrane domain. © 2017 IUBMB Life, 70(1):32-40, 2018.

Asymmetry of inverted-topology repeats in the AE1 anion exchanger suggests an elevator-like mechanism.

The membrane transporter anion exchanger 1 (AE1), or band 3, is a key component in the processes of carbon-dioxide transport in the blood and urinary acidification in the renal collecting duct. In both erythrocytes and the basolateral membrane of the collecting-duct α-intercalated cells, the role of AE1 is to catalyze a one-for-one exchange of chloride for bicarbonate. After decades of biochemical and functional studies, the structure of the transmembrane region of AE1, which catalyzes the anion-exchange reaction, has finally been determined. Each protomer of the AE1 dimer comprises two repeats with inverted transmembrane topologies, but the structures of these repeats differ. This asymmetry causes the putative substrate-binding site to be exposed only to the extracellular space, consistent with the expectation that anion exchange occurs via an alternating-access mechanism. Here, we hypothesize that the unknown, inward-facing conformation results from inversion of this asymmetry, and we propose a model of this state constructed using repeat-swap homology modeling. By comparing this inward-facing model with the outward-facing experimental structure, we predict that the mechanism of AE1 involves an elevator-like motion of the substrate-binding domain relative to the nearly stationary dimerization domain and to the membrane plane. This hypothesis is in qualitative agreement with a wide range of biochemical and functional data, which we review in detail, and suggests new avenues of experimentation.

Papain cleavage of the 38,000-dalton fragment inhibits the binding of 4, 4'-diisothiocyanostilbene-2, 2'-disulfonate to lys-539 on the 60,000-dalton fragment in human band 3.

Human band 3 is a 98-kDa transmembrane (TM) protein comprising 14 TM segments. Papain cleavages band 3 into 38- and 60-kDa fragments. Under vigorous conditions, the cleavage of the loop region between the TM 7 of gate domain and the TM 8 of core domain in the 38-kDa fragment produces 7- and 31-kDa fragments. Conformational changes of the TM 5 segment containing Lys-539 by cleavage of the 38-kDa fragment remain unclear. Pressure-induced haemolysis of erythrocytes was suppressed by binding of 4, 4'-diisothiocyanostilbene-2, 2'-disulfonate (DIDS) to Lys-539. Such effect of DIDS was not observed upon cleavage of the 38-kDa fragment, because of inhibition of DIDS binding to Lys-539. Using fluorescence of DIDS labelled to Lys-539, conformational changes of band 3 were examined. Fluorescence spectra demonstrated that the molecular motion of DIDS is more restricted upon digestion of the 38-kDa fragment. Interestingly, the quenching of DIDS fluorescence showed that Hg2+ is less accessible to DIDS upon digestion of the 38-kDa fragment. Taken together, we propose that the conformational changes of the TM 5 segment characterized by the sequestration and restricted motion of Lys-539 are induced by the cleavage of the loop region between the TM 7 and the TM 8.

Effect of the Southeast Asian Ovalocytosis Deletion on the Conformational Dynamics of Signal-Anchor Transmembrane Segment 1 of Red Cell Anion Exchanger 1 (AE1, Band 3, or SLC4A1).

The first transmembrane (TM1) helix in the red cell anion exchanger (AE1, Band 3, or SLC4A1) acts as an internal signal anchor that binds the signal recognition particle and directs the nascent polypeptide chain to the endoplasmic reticulum (ER) membrane where it moves from the translocon laterally into the lipid bilayer. The sequence N-terminal to TM1 forms an amphipathic helix that lies at the membrane interface and is connected to TM1 by a bend at Pro403. Southeast Asian ovalocytosis (SAO) is a red cell abnormality caused by a nine-amino acid deletion (Ala400-Ala408) at the N-terminus of TM1. Here we demonstrate, by extensive (∼4.5 µs) molecular dynamics simulations of TM1 in a model 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine membrane, that the isolated TM1 peptide is highly dynamic and samples the structure of TM1 seen in the crystal structure of the membrane domain of AE1. The SAO deletion not only removes the proline-induced bend but also causes a "pulling in" of the part of the amphipathic helix into the hydrophobic phase of the bilayer, as well as the C-terminal of the peptide. The dynamics of the SAO peptide very infrequently resembles the structure of TM1 in AE1, demonstrating the disruptive effect the SAO deletion has on AE1 folding. These results provide a precise molecular view of the disposition and dynamics of wild-type and SAO TM1 in a lipid bilayer, an important early biosynthetic intermediate in the insertion of AE1 into the ER membrane, and extend earlier results of cell-free translation experiments.

Full-Length Anion Exchanger 1 Structure and Interactions with Ankyrin-1 Determined by Zero Length Crosslinking of Erythrocyte Membranes.

Anion exchanger 1 (AE1) is a critical transporter and the primary structural scaffold for large macromolecular complexes responsible for erythrocyte membrane flexibility and integrity. We used zero-length crosslinking and mass spectrometry to probe AE1 structures and interactions in intact erythrocyte membranes. An experimentally verified full-length model of AE1 dimers was developed by combining crosslink-defined distance constraints with homology modeling. Previously unresolved cytoplasmic loops in the AE1 C-terminal domain are packed at the domain-domain interface on the cytoplasmic face of the membrane where they anchor the N-terminal domain's location and prevent it from occluding the ion channel. Crosslinks between AE1 dimers and ankyrin-1 indicate the likely topology for AE1 tetramers and suggest that ankyrin-1 wraps around AE1 tetramers, which may stabilize this oligomer state. This interaction and interactions of AE1 with other major erythrocyte membrane proteins show that protein-protein contacts are often substantially more extensive than previously reported.

Global transformation of erythrocyte properties via engagement of an SH2-like sequence in band 3.

Src homology 2 (SH2) domains are composed of weakly conserved sequences of ∼100 aa that bind phosphotyrosines in signaling proteins and thereby mediate intra- and intermolecular protein-protein interactions. In exploring the mechanism whereby tyrosine phosphorylation of the erythrocyte anion transporter, band 3, triggers membrane destabilization, vesiculation, and fragmentation, we discovered a SH2 signature motif positioned between membrane-spanning helices 4 and 5. Evidence that this exposed cytoplasmic sequence contributes to a functional SH2-like domain is provided by observations that: (i) it contains the most conserved sequence of SH2 domains, GSFLVR; (ii) it binds the tyrosine phosphorylated cytoplasmic domain of band 3 (cdb3-PO4) with Kd = 14 nM; (iii) binding of cdb3-PO4 to erythrocyte membranes is inhibited both by antibodies against the SH2 signature sequence and dephosphorylation of cdb3-PO4; (iv) label transfer experiments demonstrate the covalent transfer of photoactivatable biotin from isolated cdb3-PO4 (but not cdb3) to band 3 in erythrocyte membranes; and (v) phosphorylation-induced binding of cdb3-PO4 to the membrane-spanning domain of band 3 in intact cells causes global changes in membrane properties, including (i) displacement of a glycolytic enzyme complex from the membrane, (ii) inhibition of anion transport, and (iii) rupture of the band 3-ankyrin bridge connecting the spectrin-based cytoskeleton to the membrane. Because SH2-like motifs are not retrieved by normal homology searches for SH2 domains, but can be found in many tyrosine kinase-regulated transport proteins using modified search programs, we suggest that related cases of membrane transport proteins containing similar motifs are widespread in nature where they participate in regulation of cell properties.

Single Molecule Studies of the Diffusion of Band 3 in Sickle Cell Erythrocytes.

Sickle cell disease (SCD) is caused by an inherited mutation in hemoglobin that leads to sickle hemoglobin (HbS) polymerization and premature HbS denaturation. Previous publications have shown that HbS denaturation is followed by binding of denatured HbS (a.k.a. hemichromes) to band 3, the consequent clustering of band 3 in the plane of the erythrocyte membrane that in turn promotes binding of autologous antibodies to the clustered band 3, and removal of the antibody-coated erythrocytes from circulation. Although each step of the above process has been individually demonstrated, the fraction of band 3 that is altered by association with denatured HbS has never been determined. For this purpose, we evaluated the lateral diffusion of band 3 in normal cells, reversibly sickled cells (RSC), irreversibly sickled cells (ISC), and hemoglobin SC erythrocytes (HbSC) in order to estimate the fraction of band 3 that was diffusing more slowly due to hemichrome-induced clustering. We labeled fewer than ten band 3 molecules per intact erythrocyte with a quantum dot to avoid perturbing membrane structure and we then monitored band 3 lateral diffusion by single particle tracking. We report here that the size of the slowly diffusing population of band 3 increases in the sequence: normal cells

Multiple Plasmodium vivax proteins of Pv-fam-a family interact with human erythrocyte receptor Band 3 and have a role in red cell invasion.

Elucidation of molecular mechanisms of receptor-ligand biology during host-parasite interaction helps in developing therapeutic targets. Several Pv-fam-a family proteins of Plasmodium vivax bind to host erythrocytes but their erythrocyte receptors remains to be explored. Here, we show that three merozoite proteins (PvTRAg36, PvATRAg74, and PvTRAg38) of this family interact with Band 3 on human erythrocytes through its three exofacial loops (loop 1, loop 3, and loop 6). These parasite proteins also interfered with the parasite growth in in-vitro, and the inhibition rate seems to be associated with their binding affinity to Band 3. This redundancy in receptor-ligand interaction could be one of the probable mechanism parasite utilizes to invade the host erythrocyte more efficiently.