Solution Structure of the Cytoplasmic Domain of Erythrocyte Membrane Band 3 Determined by Site-Directed Spin Labeling

Zhou, Zheng; DeSensi, Susan C.; Stein, Richard A.; Brandon, Suzanne; Dixit, Mrinalini; Mcardle, Erin J.; Warren, Eric M.; Kroh, Heather K.; Song, Likai; Cobb, Charles E.; Hustedt, Eric J.; Beth, Albert H.

doi:10.1021/bi050931t

Cited by 55 publications

(74 citation statements)

References 54 publications

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“…10 do not indicate any direct interactions between the peripheral domain of cdb3 and ankyrin repeats 22 and 23, although previous studies have shown that these repeats are important for high affinity binding (6,44). In previous work, it has been shown that the N-terminal domain of cdb3, which is composed of residues 1-54, is dynamically disordered in solution with no observable interactions between this domain and the central folded domain of cdb3 (17). However, when spin label side chains are placed at sequential positions along the central portion of the N-terminal domain, a restricted motion signal is observed when cdb3 is bound to AnkD34 (data not shown).…”

Section: Discussionmentioning

confidence: 79%

“…Protein Preparation and Spin Labeling-The preparation and spin labeling of wild-type and single or double Cys mutants of cdb3 have been described in previous work (17). The cDNA encoding ankyrin repeats 13-24 plus 12 residues from the spectrin binding domain (AnkD34; residues 403-827) of ankyrin-R were provided by Dr. Peter Michaely (University of Texas, Southwestern).…”

Section: Methodsmentioning

confidence: 99%

“…For the measurement of spin label side chain accessibilities, a singly labeled protein mixed with a 4 M excess amount of its non-labeled binding partner was used to ensure saturation of binding sites. NiEDDA accessibility of individual sites on each protein was measured using a gas-permeable TPX (a tradename for polymethylpentene) capillary and an ER4123D resonator (BrukerBiospin) as described previously (17). Accessibilities are expressed numerically as dimensionless ⌸ values, which were calculated from the following equation…”

Section: Methodsmentioning

confidence: 99%

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Determination of Structural Models of the Complex between the Cytoplasmic Domain of Erythrocyte Band 3 and Ankyrin-R Repeats 13–24

Kim

Brandon

Zhou

et al. 2011

Journal of Biological Chemistry

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The adaptor protein ankyrin-R interacts via its membrane binding domain with the cytoplasmic domain of the anion exchange protein (AE1) and via its spectrin binding domain with the spectrin-based membrane skeleton in human erythrocytes. This set of interactions provides a bridge between the lipid bilayer and the membrane skeleton, thereby stabilizing the membrane. Crystal structures for the dimeric cytoplasmic domain of AE1 (cdb3) and for a 12-ankyrin repeat segment (repeats 13-24) from the membrane binding domain of ankyrin-R (AnkD34) have been reported. However, structural data on how these proteins assemble to form a stable complex have not been reported. In the current studies, site-directed spin labeling, in combination with electron paramagnetic resonance (EPR) and double electron-electron resonance, has been utilized to map the binding interfaces of the two proteins in the complex and to obtain inter-protein distance constraints. These data have been utilized to construct a family of structural models that are consistent with the full range of experimental data. These models indicate that an extensive area on the peripheral domain of cdb3 binds to ankyrin repeats 18 -20 on the top loop surface of AnkD34 primarily through hydrophobic interactions. This is a previously uncharacterized surface for binding of cdb3 to AnkD34. Because a second dimer of cdb3 is known to bind to ankyrin repeats 7-12 of the membrane binding domain of ankyrin-R, the current models have significant implications regarding the structural nature of a tetrameric form of AE1 that is hypothesized to be involved in binding to full-length ankyrin-R in the erythrocyte membrane.Human erythrocytes exhibit an unusual biconcave disc shape and remarkable plasma membrane mechanical stability and deformability, all of which are necessary for their survival in the circulatory system. It is now well established that the unusual cell shape and membrane mechanical properties are due in large part to the presence of an extensive membrane skeleton, composed primarily of the proteins spectrin and actin, that lines the inner membrane surface and to specific bridging interactions between this membrane skeleton and intrinsic membrane proteins in the lipid bilayer. The spectrin-actin skeleton associates with the membrane bilayer via two types of contacts, one involving short actin protofilaments and protein 4.1, which interact with the cytoplasmic domain of glycophorin C, and the second involving the bridging protein ankyrin-R and protein 4.2, which interact with the cytoplasmic domain of the anion exchange protein (AE1) 3 also known as band 3. Alterations in this second class of interactions often result in spherical erythrocytes with decreased cell size and increased fragility, a condition known clinically as hereditary spherocytosis. Hereditary spherocytosis is a spectrum of inherited diseases, occurring in one family out of 2,000 -3,000, which present clinically as varying degrees of hemolytic anemia resulting from hemolysis of the spherical erythrocytes ...

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Section: Discussionmentioning

confidence: 79%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Determination of Structural Models of the Complex between the Cytoplasmic Domain of Erythrocyte Band 3 and Ankyrin-R Repeats 13–24

Kim

Brandon

Zhou

et al. 2011

Journal of Biological Chemistry

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“…Within the past decade, distance distribution measurements for pairs of spin labels with pulsed dipolar spectroscopy methods have been performed for a number of proteins with known structures [60,100,[128][129][130][131][132][133][134][135][136][137][138][139][140]. From these studies, a few were selected where mean distances were given and where the duration of the dipolar evolution was sufficient [21] for these mean distances to be reliable.…”

Section: Effects On Measurements Of Distance Distributionsmentioning

confidence: 99%

Conformational dynamics and distribution of nitroxide spin labels

Jeschke

2013

Progress in Nuclear Magnetic Resonance Spectroscopy

137

115

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Long-range distance measurements based on paramagnetic relaxation enhancement (PRE) in NMR, quantification of surface water dynamics near biomacromolecules by Overhauser dynamic nuclear polarization (DNP) and sensitivity enhancement by solid-state DNP all depend on introducing paramagnetic species into an otherwise diamagnetic NMR sample. The species can be introduced by site-directed spin labeling, which offers precise control for positioning the label in the sequence of a biopolymer. However, internal flexibility of the spin label gives rise to dynamic processes that potentially influence PRE and DNP behavior and leads to a spatial distribution of the electron spin even in solid samples. Internal dynamics of spin labels and their static conformational distributions have been studied mainly by electron paramagnetic resonance spectroscopy and molecular dynamics simulations, with a large body of results for the most widely applied methanethiosulfonate spin label MTSL. These results are critically discussed in a unifying picture based on rotameric states of the group that carries the spin label. Deficiencies in our current understanding of dynamics and conformations of spin labeled groups and of their influence on NMR observables are highlighted and directions for further research suggested.

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“…As a flexible arm would in essence be an unstructured, disordered segment, we chose an experimental approach that would provide specific measurements for any residue of interest, whether in a structured or unstructured region and that could be applied to intact chemoreceptors in their native or reconstituted membrane environment. Thus, we chose site-directed spin labeling and electron paramagnetic resonance (EPR) spectroscopy, a combination that has already provided useful information about disordered protein regions, [27][28][29][30][31][32] and Freeman, reproduced by permission). The ratio of the low-field to central peak amplitudes (top spectrum) can be used to derive a semiquantitative mobility parameter h(þ1)/h(0).…”

Section: Introductionmentioning

confidence: 99%

Direct evidence that the carboxyl‐terminal sequence of a bacterial chemoreceptor is an unstructured linker and enzyme tether

Bartelli¹,

Hazelbauer²

2011

Protein Science

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Sensory adaptation in bacterial chemotaxis involves reversible methylation of specific glutamyl residues on chemoreceptors. The reactions are catalyzed by a dedicated methyltransferase and dedicated methylesterase. In Escherichia coli and related organisms, control of these enzymes includes an evolutionarily recent addition of interaction with a pentapeptide activator located at the carboxyl terminus of the receptor polypeptide chain. Effective enzyme activation requires not only the pentapeptide but also a segment of the receptor polypeptide chain between that sequence and the coiled-coil body of the chemoreceptor. This segment has features consistent with a role as a flexible and presumably unstructured linker and enzyme tether, but there has been no direct information about its structure. We used site-directed spin labeling and electron paramagnetic resonance spectroscopy to characterize structural features of the carboxyl-terminal 40 residues of E. coli chemoreceptor Tar. Beginning~35 residues from the carboxyl terminus and continuing to the end of the protein, spectra of spin-labeled Tar embedded in native membranes or in reconstituted proteoliposomes, exhibited mobilities characteristic of unstructured, disordered segments. Binding of methyltransferase substantially reduced mobility for positions in or near the pentapeptide but mobility for the linker sequence remained high, being only modestly reduced in a gradient of decreasing effects for 10-15 residues, a pattern consistent with the linker providing a flexible arm that would allow enzyme diffusion within defined limits. Thus, our data identify that the carboxyl-terminal linker between the receptor body and the pentapeptide is an unstructured, disordered segment that can serve as a flexible arm and enzyme tether.

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Solution Structure of the Cytoplasmic Domain of Erythrocyte Membrane Band 3 Determined by Site-Directed Spin Labeling

Cited by 55 publications

References 54 publications

Determination of Structural Models of the Complex between the Cytoplasmic Domain of Erythrocyte Band 3 and Ankyrin-R Repeats 13–24

Determination of Structural Models of the Complex between the Cytoplasmic Domain of Erythrocyte Band 3 and Ankyrin-R Repeats 13–24

Conformational dynamics and distribution of nitroxide spin labels

Direct evidence that the carboxyl‐terminal sequence of a bacterial chemoreceptor is an unstructured linker and enzyme tether

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