Structure and stability of hereditary spherocytosis mutants of the cytosolic domain of the erythrocyte anion exchanger 1 protein.


Abstract

Anion exchanger 1 (AE1, Band 3) is the predominant membrane protein of erythrocytes. Its 52 kDa C-terminal domain functions as a chloride-bicarbonate exchanger, while its 43 kDa N-terminal cytosolic domain (cdb3) anchors the cytoskeleton to the membrane. Several proteins bind to cdb3, including protein 4.2, a cytoskeletal protein. Three mutations in cdb3 are associated with hereditary spherocytosis (HS) and decreased levels of protein 4.2 in erythrocytes. In this study, these cdb3 mutants (E40K, G130R, and P327R) were expressed in and purified from Escherichia coli. Sedimentation experiments showed that the wild-type and mutant proteins are dimers. No difference in secondary structure between mutant and wild-type proteins was detected using circular dichroism (CD) analysis. The wild-type and mutant proteins underwent similar pH-dependent conformational changes when monitored by intrinsic tryptophan fluorescence. Urea denaturation of proteins monitored by intrinsic fluorescence showed no significant differences in the sensitivity of the proteins to this chemical denaturant. Thermal denaturation monitored by CD and by calorimetry revealed that only the P327R mutant had a significantly lower midpoint of transition (approximately 5 degrees C) than the wild-type protein, suggesting a modest decrease in stability. The results show that the HS mutant cdb3 proteins do not differ to any great extent in structure from the wild-type protein, suggesting that the HS mutations may directly affect protein 4.2 binding. Study holds ProTherm entries: 19788, 19789, 19790, 19791, 19792, 19793, 19794, 19795, 19796, 19797, 19798, 19799, 19800, 19801, 19802 Extra Details: 1 mM DTT was added in the experiment membrane protein; secondary structure; conformational changes; binding

Submission Details

ID: UM9SVFjh

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:52 p.m.

Version: 1

Publication Details
Bustos SP;Reithmeier RA,Biochemistry (2006) Structure and stability of hereditary spherocytosis mutants of the cytosolic domain of the erythrocyte anion exchanger 1 protein. PMID:16411779
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
1BTQ 1994-11-30 THE SOLUTION STRUCTURES OF THE FIRST AND SECOND TRANSMEMBRANE-SPANNING SEGMENTS OF BAND 3
3BTB 1998-01-28 NMR SOLUTION STRUCTURE OF A BAND 3 PEPTIDE INHIBITOR BOUND TO GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE, 20 STRUCTURES
1BNX 1998-08-05 STRUCTURAL STUDIES ON THE EFFECTS OF THE DELETION IN THE RED CELL ANION EXCHANGER (BAND3, AE1) ASSOCIATED WITH SOUTH EAST ASIAN OVALOCYTOSIS.
1BTS 1994-11-01 THE SOLUTION STRUCTURES OF THE FIRST AND SECOND TRANSMEMBRANE-SPANNING SEGMENTS OF BAND 3
2BTA 1996-06-10 NMR STUDY OF N-TERMINAL HUMAN BAND 3 PEPTIDE, RESIDUES 1-15
1BTR 1994-12-20 THE SOLUTION STRUCTURES OF THE FIRST AND SECOND TRANSMEMBRANE-SPANNING SEGMENTS OF BAND 3
1BH7 1998-11-04 A LOW ENERGY STRUCTURE FOR THE FINAL CYTOPLASMIC LOOP OF BAND 3, NMR, MINIMIZED AVERAGE STRUCTURE
2BTB 1996-06-10 NMR STUDY OF N-TERMINAL HUMAN BAND 3 PEPTIDE, RESIDUES 1-15
1BTT 1994-12-20 THE SOLUTION STRUCTURES OF THE FIRST AND SECOND TRANSMEMBRANE-SPANNING SEGMENTS OF BAND 3
1BZK 1999-06-01 STRUCTURAL STUDIES ON THE EFFECTS OF THE DELETION IN THE RED CELL ANION EXCHANGER (BAND3, AE1) ASSOCIATED WITH SOUTH EAST ASIAN OVALOCYTOSIS.
4KY9 2013-10-23 2.23 Structural and Functional Analysis of a Putative Substrate Access Tunnel in the Cytosolic Domain of Human Anion Exchanger 1
1HYN 2001-05-16 2.6 CRYSTAL STRUCTURE OF THE CYTOPLASMIC DOMAIN OF HUMAN ERYTHROCYTE BAND-3 PROTEIN
4YZF 2015-11-04 3.5 Crystal structure of the anion exchanger domain of human erythrocyte Band 3

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Band 3 anion transport protein P02730 B3AT_HUMAN