Dataset Information

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

ABSTRACT: 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.

SUBMITTER: Rivera-Santiago R

PROVIDER: S-EPMC5214978 | biostudies-literature | 2017 Jan

REPOSITORIES: biostudies-literature

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Publications

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

Rivera-Santiago Roland R Harper Sandra L SL Sriswasdi Sira S Hembach Peter P Speicher David W DW

Structure (London, England : 1993) 20161215 1

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 loo ...[more]

PMID: 27989623

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Project description:The human erythrocyte anion exchanger (AE1, Band 3) contains up to 14 transmembrane segments, with a single site of N-glycosylation at Asn642 in extracellular (EC) loop 4. Scanning and insertional N-glycosylation mutagenesis were used to determine the folding pattern of AE1 in the membrane. Full-length AE1, when expressed in transfected human embryonic kidney (HEK)-293 or COS-7 cells, retained a high-mannose oligosaccharide structure. Scanning N-glycosylation mutagenesis of EC loop 4 showed that N-glycosylation acceptor sites (Asn-Xaa-Ser/Thr) spaced 12 residues from the ends of adjacent transmembrane segments could be N-glycosylated. An acceptor site introduced at position 743 in intracellular (IC) loop 5 that could be N-glycosylated in a cell-free translation system was not N-glycosylated in transfected cells. Mutations designed to disrupt the folding of this loop enhanced the level of N-glycosylation at Asn743 in vitro. The results suggest that this loop might be transiently exposed to the lumen of the endoplasmic reticulum during biosynthesis but normally folds rapidly, precluding N-glycosylation. EC loop 4 insertions into positions 428, 484, 754 and 854 in EC loops 1, 2, 6 and 7 respectively were efficiently N-glycosylated, showing that these regions were extracellular. EC loop 4 insertions into positions 731 or 785 were poorly N-glycosylated, which was inconsistent with an extracellular disposition for these regions of AE1. Insertion of EC loop 4 into positions 599 and 820 in IC loops 3 and 6 respectively were not N-glycosylated in cells, which was consistent with a cytosolic disposition for these loops. Inhibitor-affinity chromatography with 4-acetamido-4'-isothiocyanostilbene-2,2'-disulphonate (SITS)-Affi-Gel was used to assess whether the AE1 mutants were in a native state. Mutants with insertions at positions 428, 484, 599, 731 and 785 showed impaired inhibitor binding, whereas insertions at positions 754, 820 and 854 retained binding. The results indicate that the folding of the C-terminal region of AE1 is more complex than originally proposed and that this region of the transporter might have a dynamic aspect.

Dataset Information

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

Publications

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

Similar Datasets

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