Structure of the Cytoplasmic Domain of Erythrocyte Band 3 Hereditary Spherocytosis Variant P327R: Band 3 Tuscaloosa †

Molecular Physiology and Biophysics, Vanderbilt University, Nashville, Tennessee 37232, USA.
Biochemistry (Impact Factor: 3.02). 10/2007; 46(36):10248-57. DOI: 10.1021/bi700948p
Source: PubMed


Previous studies have shown that a single P327R point mutation in the cytoplasmic domain of band 3 (cdb3) protein, known as band 3 Tuscaloosa, leads to a reduction in protein 4.2 content of the erythrocyte membrane and hemolytic anemia. Recent studies have shown that this point mutation does not dissociate the cdb3 dimer, nor does it lead to large-scale rearrangement of the protein structure (Bustos, S. P., and Reithmeier, R. A. F. (2006) Biochemistry 45, 1026-1034). To better define the structural changes in cdb3 that lead to the hemolytic anemia phenotype, site-directed spin labeling (SDSL), in combination with continuous wave electron paramagnetic resonance (EPR) and pulsed double electron-electron resonance (DEER) spectroscopies, has been employed in this study to compare the structure of the R327 variant with wild type P327 cdb3. It is confirmed that the P327R mutation does not dissociate the cdb3 dimer, nor does it change the spatial orientation of the two peripheral domains relative to the dimer interface. However, it does affect the packing of the C-terminal end of helix 10 of the dimerization arms in a subpopulation of cdb3 dimers, it leads to spectral changes at some residues in beta-strand 11 and in the N-terminal end of helix10, and it produces measurable spectral changes at other residues that are near the mutation site. The data indicate that the structural changes are subtle and are localized to one surface of the cdb3 dimer. The spectroscopic description of structural features of the P327R variant provides important clues about the location of one potential protein 4.2 binding surface on cdb3 as well as new insight into the structural basis of the membrane destabilization.

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Available from: Charles Cobb, Apr 18, 2014
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    • "Troponin C (TnC) cysteine mutant at positions 55 and 83 was expressed, purified, and spin labelled as previously described (Brown et al. 2002). Erythrocyte cytoplasmic domain of band 3 (cdb3) mutants were expressed and spin labelled as described by Zhou et al. (2007). T4 lysozyme (T4L) double cysteine mutants T4L_21C65C and T4L_65C89C were expressed in Escherichia coli K38 cells (McHaourab et al. 1996). "
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    ABSTRACT: To explore high-field EPR in biological applications we have compared measurements of dynamics with X-band (9 GHz) and W-band (94 GHz) saturation transfer EPR (ST-EPR) and distance determination by X and W-band DEER. A fourfold increase of sensitivity was observed for W-band ST-EPR compared with X-band. The distance measurements at both fields showed very good agreement in both the average distances and in the distance distributions. Multifrequency EPR thus provides an additional experimental dimension to facilitate extraction of distance populations. However, the expected orientational selectivity of W-band DEER to determine the relative orientation of spins has not been realized, most likely because of the large orientational disorder of spin labels on the protein surface.
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    • "However, these mutations do not localize to a common region of the 3D crystal structure of the cytoplasmic domain of B3. These mutations also do not grossly disturb the overall structure of the B3 N-terminus, as such, it is not currently understood precisely how they disturb the association of protein 4.2 with B3 [36] [48] [49]. "
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