Solution structure of neuronal bungarotoxin determined by two-dimensional NMR spectroscopy: sequence-specific assignments, secondary structure, and dimer formation

The solution structure of neuronal bungarotoxin (nBgt) has been studied by using two-dimensional 1H NMR spectroscopy. Sequence-specific assignments for over 95% of the backbone resonances and 85% of the side-chain resonances have been made by using a series of two-dimensional spectra at four temperatures. From these assignments over 75% of the NOESY spectrum has been assigned, which has in turn provided 582 distance constraints. Twenty-seven coupling constants (NH-alpha CH) were determined from the COSY spectra, which have provided dihedral angle constraints. In addition, hydrogen exchange experiments have suggested the probable position of hydrogen bonds. The NOE constraints, dihedral angle constraints, and the rates of amide proton exchange suggest that a triple-stranded antiparallel beta sheet is the major component of secondary structure, which includes 25% of the amino acid residues. A number of NOE peaks were observed that were inconsistent with the antiparallel beta-sheet structure. Because we have confirmed by sedimentation equilibrium that nBgt exists as a dimer, we have reinterpreted these NOE constraints as intermolecular interactions. These constraints suggest that the dimer consists of a six-stranded antiparallel beta sheet (three from each monomer), with residues 55-59 forming the dimer interface.     

The melanoma growth stimulatory activity receptor consists of two proteins. Ligand binding results in enhanced tyrosine phosphorylation.

The human melanoma growth-stimulatory activities (MGSA alpha, beta, gamma/GRO) are products of immediate early genes coding for cytokines that exhibit sequence similarity to platelet factor-4 and beta-thromboglobulin. MGSA/GRO alpha has been demonstrated to partially complete for binding to the approximately 58-kDa neutrophil receptor for another beta-thromboglobulin-related chemotactic protein, IL-8. We demonstrate that when [125I]MGSA/GRO alpha was cross-linked to receptors/binding proteins from human placenta, there were two major [125I]MGSA cross-linked bands of approximately 64,000 and approximately 84,000 Mr. Because [125I]MGSA exists primarily in monomer and dimer forms at the concentrations used here, it is not clear whether the receptor/binding proteins represented by the cross-linked bands are approximately 50,000 and approximately 70,000 or approximately 58,000 and approximately 78,000 Mr. Ligand binding to the receptor proteins is associated with enhanced tyrosine phosphorylation of a number of substrates, including proteins in the same Mr range as the MGSA/GRO receptor/binding proteins.     

Computational study of the intramolecular proton transfer reactions of 3-hydroxytropolone (2,7-dihydroxycyclohepta-2,4,6-trien-1-one) and its dimers

The proton transfer reaction and dimerization processes of 3-hydroxytropolone (3-OHTRN) have been investigated using density functional theory (DFT) at the B3LYP/6–31+G** level. The influence of the solvent on the proton transfer reaction of 3-OHTRN was examined using the self-consistent isodensity polarized continuum model (SCI-PCM) with different dielectric constants (ε?=?4.9, CHCI₃; ε?=?32.63, CH₃OH; ε?=?78.39, H₂O). The intramolecular proton transfer reaction occurs more readily in the gas phase than in solution. Results also show that the stability of 3-OHTRN dimers in the gas phase is directly affected by the hydrogen bond length in the dimer structure.     

Sequence-specific 1H NMR assignments and secondary structure in solution of Escherichia coli trp repressor

Sequence-specific 1H NMR assignments are reported for the active L-tryptophan-bound form of Escherichia coli trp repressor. The repressor is a symmetric dimer of 107 residues per monomer; thus at 25 kDa, this is the largest protein for which such detailed sequence-specific assignments have been made. At this molecular mass the broad line widths of the NMR resonances preclude the use of assignment methods based on 1H-1H scalar coupling. Our assignment strategy centers on two-dimensional nuclear Overhauser spectroscopy (NOESY) of a series of selectively deuterated repressor analogues. A new methodology was developed for analysis of the spectra on the basis of the effects of selective deuteration on cross-peak intensities in the NOESY spectra. A total of 90% of the backbone amide protons have been assigned, and 70% of the alpha and side-chain proton resonances are assigned. The local secondary structure was calculated from sequential and medium-range backbone NOEs with the double-iterated Kalman filter method [Altman, R. B., & Jardetzky, O. (1989) Methods Enzymol. 177, 218-246]. The secondary structure agrees with that of the crystal structure [Schevitz, R., Otwinowski, Z., Joachimiak, A., Lawson, C. L., & Sigler, P. B. (1985) Nature 317, 782], except that the solution state is somewhat more disordered in the DNA binding region and in the N-terminal region of the first alpha-helix. Since the repressor is a symmetric dimer, long-range intersubunit NOEs were distinguished from intrasubunit interactions by formation of heterodimers between two appropriate selectively deuterated proteins and comparison of the resulting NOESY spectrum with that of each selectively deuterated homodimer. Thus, from spectra of three heterodimers, long-range NOEs between eight pairs of residues were identified as intersubunit NOEs, and two additional long-range intrasubunits NOEs were assigned.     

The solution structures of Escherichia coli trp repressor and trp aporepressor at an intermediate resolution

We have determined the solution structures and examined the dynamics of the Escherichia coli trp repressor (a 25-kDa dimer), with and without the co-repressor L-tryptophan, from NMR data. This is the largest protein structure thus far determined by NMR. To obtain a set of data sufficient for a structure determination it was essential to resort to isotopic spectral editing. Line broadening observed in this molecular mass range precludes for the most part the measurement of coupling constants and stereospecific assignments, with the inevitable result that the attainable resolution of the final structure will be somewhat lower than the resolution reported for smaller proteins and peptides. Nevertheless the general topology of the protein can be deduced from the subsets of NOEs defining the secondary and tertiary structure, providing a basis for further refinement using the full set of NOEs and energy minimization. We report here (a) an intermediate resolution structure that can be deduced from NMR data, covalent, angular and van-der-Waals constraints only, without resort to detailed energy calculations, and (b) the limits of uncertainty within which this structure is valid. An examination of these structures combined with backbone amide exchange data shows that even at this resolution three important conclusions can be drawn: (a) the protein structure changes upon binding tryptophan; (b) the putative DNA binding region is much more flexible than the core of the molecule, with backbone amide proton exchange rates 1000 times faster than in the core; (c) the binding of tryptophan stabilizes the repressor molecule, which is reflected in both the appearance of additional NOEs, and in the slowing of backbone proton exchange rates by factors of 3-10. Sequence-specific 1H-NMR assignments and the secondary structure of the holopressor (L-tryptophan-bound form) have been reported previously [C. H. Arrowsmith, R. Pachter, R. B. Altman, S. B. Iyer & O. Jardetzky (1990) Biochemistry 29, 6332-6341]. Those for the trp aporepressor (L-tryptophan-free form), made using the same methods and conditions as described in the cited paper, are reported here. The secondary structure of the aporepressor was calculated from sequential and medium-range NOEs and is the same as reported for the holorepressor except that helix E is shorter. The tertiary solution structures for both forms of the repressor were calculated from long-range NOE data.(ABSTRACT TRUNCATED AT 400 WORDS)     

Hydrogen exchange kinetics of core peptide protons in Streptomyces subtilisin inhibitor

The hydrogen isotope exchange kinetics of the 10 slowest exchanging resonances in the 1H NMR spectrum of Streptomyces subtilisin inhibitor (SSI) have been determined at pH 7-11 and 30-60 degrees C. These resonances are assigned to peptide amide protons in the beta-sheet core that comprises the extensive protein-protein interface of the tightly bound SSI dimer. The core protons are atypical in that their exchange rates are orders of magnitude slower than those for all other SSI protons. When they do exchange at temperatures greater than 50 degrees C, they do so as a set and with a very high temperature coefficient. The pH dependence of the exchange rate constants is also atypical. Exchange rates are approximately first order in hydroxyl ion dependence at pH less than 8.5 and greater than 9.5 and pH independent between pH 8.5 and 9.5. The pH dependence and temperature dependence of the SSI proton exchange rates are interpreted by the two-process model [Woodward, C. K., & Hilton, B. D. (1980) Biophys. J. 32, 561-575]. The results suggest that in the average solution structure of SSI, an unusual mobility of secondary structural elements at the protein surface is, in a sense, compensated by an unusual rigidity and inaccessibility of the beta-sheet core at the dimer interface.     

Proton mobilities in water and in different stereoisomers of covalently linked gramicidin A channels

Proton conductivities in bulk solution (lambda(H)) and single-channel proton conductances (g(H)) in two different stereoisomers of the dioxolane-linked gramicidin A channel (the SS and RR dimers) were measured in a wide range of bulk proton concentrations ([H], 0.1-8000 mM). Proton mobilities (micro(H)) in water as well as in the SS and RR dimers were calculated from the conductivity data. In the concentration range of 0.1-2000 mM, a straight line with a slope of 0.75 describes the log (g(H))-log ([H]) relationship in the SS dimer. At [H] > 2000 mM, saturation is followed by a decline in g(H). The g(H)-[H] relationship in the SS dimer is qualitatively similar to the [H] dependence of lambda(H). However, the slope of the straight line in the log(lambda(H))-log([H]) plot is 0.96, indicating that the rate-limiting step for proton conduction through the SS dimer is not the diffusion of protons in bulk solution. The significant difference between the slopes of those linear relationships accounts for the faster decline of micro(H) as a function of [H] in the SS dimer in relation to bulk solution. In the high range of [H], saturation and decline of g(H) in the SS dimer can be accounted for by the significant decrease of micro(H) in bulk solution. At any given [H], g(H) in the RR dimer is significantly smaller than in the SS. Moreover, the g(H)-[H] relationship in the RR stereoisomer is qualitatively different from that in the SS. Between 1 and 50 mM [H], g(H) can be fitted with an adsorption isotherm, suggesting the presence of a proton-binding site inside the pore (pK(a) approximately 2), which limits proton exit from the channel. At 100 mM < [H] < 3000 mM, g(H) increases linearly with [H]. The distinctive shape of the g(H)-[H] relationship in the RR dimer suggests that the channel can be occupied simultaneously by more than one proton. At higher [H], the saturation and decline of g(H) in the RR dimer reflect the properties of micro(H) in bulk solution. In the entire range of [H], protons seem to cross the SS and RR channels via a Grotthuss-like mechanism. The rate-limiting step for proton transfer in the SS dimer is probably the membrane-channel/bulk solution interface. It is also proposed that the smaller g(H) in the RR dimer is the consequence of a different organization and dynamics of the H-bonded network of water molecules inside the pore of the channel, resulting in a slower proton transfer and multiple pore occupancy by protons.     

Solution conformation of brazzein by H nuclear magnetic resonance: resonance assignment and secondary structure

Brazzein is a sweet-tasting protein isolated from the fruit of the West African plant Pentadiplandra brazzeana Baillon. It is the smallest and the most water-soluble sweet protein discovered so far, it is also highly thermostable. The proton NMR study of brazzein at 600 MHz (pH 3.5, 300K) is presented. Complete sequence specific assignment of the individual backbone and sidechain proton resonances were achieved using through-bond and through-space connectivities obtained from standard two-dimensional NMR techniques. The secondary structure of brazzein contains one -helix (residues 21–29), one short 3₁₀-helix (residues 14–17), two strands of antiparallel β-sheet (residues 34–39, 44–50) and probably a third strand (residues 5–7) near the N-terminus.     

A molecular dynamics study of the C-terminal fragment of the L7/L12 ribosomal protein

The crystallographic dimer of the C-terminal fragment (CTF) of the L7/L12 ribosomal protein has been subjected to molecular dynamics (MD) simulations. A 90 picosecond (ps) trajectory for the protein dimer, 19 water molecules and two counter ions has been calculated at constant temperature. Effects of intermolecular interactions on the structure and dynamics have been studied. The exact crystallographic symmetry is lost and the atomic fluctuations differ from one monomer to the other. The average MD structure is more stable than the X-ray one, as judged by accessible surface area and energy calculations. Crystal (non-dimeric) interactions have been simulated in another 40 ps trajectory by using harmonic restraints to represent intermolecular hydrogen bonds. The conformational changes with respect ot the X-ray structure are then virtually suppressed.The unrestrained dimer trajectory has been scanned for cooperative motions involving secondary structure elements. The intrinsic collective motions of the monomer are transmitted via intermolecular contacts to the dimer structure.The existence of a stable dimeric form of CTF, resembling the crystallographic one, has been documented. At the cost of fairly small energy expenditure the dimer has considerable conformational flexibility. This flexibility may endow the dimer with some functional potential as an energy transducer.     

The effects of chain length on the secondary structure of oligoadenylates

The oligoadenylates (Ap)_2–4A have been studied by proton magnetic resonance (pmr) spectroscopy. All the exterior base protons and a number of the interior base proton resonance have been assigned. The results of this work showed that the adenine bases in these oligoadenylates are intramolecularly stacked at 20°C with their bases oriented preferentially in the anti conformation about their respective glycosidic bonds. The oligomers were found to associate extensively even at concentrations of 0.02 M, primarily via “end-to-end” stacking. With increasing temperature, the oligomer bases destack, but it is argued that this unfolding process cannot be described in terms of a two-state stacked versus unstacked model. Instead, the temperature dependences of the base proton chemical shifts support a base-oscillation model. The relationship between this model and the two-state model is discussed. Finally, on the basis of the chain-length dependence of the proton chemical shifts of the various adenine bases, it was concluded that subtle variations in the secondary structure of these oligomers exist with increasing chain length. Evidence is presented to show that the effects of distant base shielding are considerably smaller than what was previously estimated. The observed departures from the “extended dimer” model are attributed to differences in the relative orientations of the bases with respect to their neighbors in the oligomer.     

Sequence-specific 1H NMR assignments and secondary structure of porcine motilin

The solution structure of the 22-residue peptide hormone motilin has been studied by circular dichroism and two-dimensional 1H nuclear magnetic resonance spectroscopy. Circular dichroism spectra indicate the presence of alpha-helical secondary structure in aqueous solution, and the secondary structure can be stabilized with hexafluoro-2-propanol. Sequence-specific assignments of the proton NMR spectrum of porcine motilin in 30% hexafluoro-2-propanol have been made by using two-dimensional NMR techniques. All backbone proton resonances (NH and alpha CH) and most of the side-chain resonances have been assigned by using double-quantum-filtered COSY, RELAYED-COSY, and NOESY experiments. Simulations of NOESY cross-peak intensities as a function of mixing time indicate that spin diffusion has a relatively small effect in peptides the size of motilin, thereby allowing the use of long mixing times to confidently make assignments and delineate secondary structure. Sequential alpha CH-NH and NH-NH NOESY connectivities were observed over a significant portion of the length of the peptide. A number of medium-range NOESY cross-peaks indicate that the peptide is folded into alpha-helix from Glu9 to Lys20, which agrees favorably with the 50% helical content determined from CD measurements. The intensities of selected NOESY cross-peaks relative to corresponding diagonal peaks were used to estimate a rotational correlation time of approximately 2.5 ns for the peptide, indicating that the peptide exists as a monomer in solution under the conditions used here.     

Structural basis for the 14-3-3 protein-dependent inhibition of the regulator of G protein signaling 3 (RGS3) function

Regulator of G protein signaling (RGS) proteins function as GTPase-activating proteins for the α-subunit of heterotrimeric G proteins. The function of certain RGS proteins is negatively regulated by 14-3-3 proteins, a family of highly conserved regulatory molecules expressed in all eukaryotes. In this study, we provide a structural mechanism for 14-3-3-dependent inhibition of RGS3-Gα interaction. We have used small angle x-ray scattering, hydrogen/deuterium exchange kinetics, and Förster resonance energy transfer measurements to determine the low-resolution solution structure of the 14-3-3ζ·RGS3 complex. The structure shows the RGS domain of RGS3 bound to the 14-3-3ζ dimer in an as-yet-unrecognized manner interacting with less conserved regions on the outer surface of the 14-3-3 dimer outside its central channel. Our results suggest that the 14-3-3 protein binding affects the structure of the Gα interaction portion of RGS3 as well as sterically blocks the interaction between the RGS domain and the Gα subunit of heterotrimeric G proteins.     

Characterization of two high affinity human interleukin-8 receptors.

Interleukin 8 (IL-8) and melanocyte growth-stimulatory activity/gro (MGSA) are structurally related proinflammatory cytokines that are chemoattractants and activators of neutrophils. Recently, cDNA clones encoding a high affinity IL-8 receptor (IL-8R-A) and a "low affinity" IL-8 receptor (IL-8R-B) have been isolated from human cDNA libraries. These two receptors have 77% amino acid identity and are members of the G protein-coupled superfamily of receptors with seven transmembrane domains. We have expressed these two receptors in mammalian cells and find that in this system both receptors bind IL-8 with high affinity (Kd approximately 2 nM). The receptor affinities differ for MGSA, however. IL-8R-A binds MGSA with low affinity (Kd approximately 450 nM); IL-8R-B binds MGSA with high affinity (Kd approximately 2 nM). The transfected cells respond to ligand binding with a transient increase in the intracellular Ca2+ concentration. A Ca2+ response is found for IL-8R-A following the binding of IL-8; no response is found for MGSA. A Ca2+ response for IL-8R-B follows the binding of both ligands. Blot hybridization with oligonucleotide probes specific for the two receptors shows that mRNA for both receptors is present in human neutrophils. Analysis of IL-8 and MGSA binding data on neutrophils as well as Ca2+ response and desensitization data shows that the presence of these two IL-8 receptors on the cell surface can account for the profile of these two ligands on neutrophils.     

In situ IR and NMR study of the interactions between proton donors and the Re(I) hydride complex [{MeC(CH2PPh2)3}Re (CO)2H]. ReH…H bonding and proton-transfer pathways

The reactions of various proton donors (phenol, hexafluoro-2-propanol, perfluoro-2-methyl-2-propanol, monochloroacetic acid, and tetrafluoroboric acid) with the rhenium (I) hydride complex [(triphos)Re(CO)₂H] (1) have been studied in dichloromethane solution by in situ IR and NMR spectroscopy. The proton donors from [(triphos)Re(CO)₂H…HOR] adducts exhibiting rather strong H…H interactions. The enthalpy variations associated with the formation of the H-bonds (−ΔH = 4.4–6.0 kcal mol⁻¹) have been determined by IR spectroscopy, while the H…H distance in the adduct [(triphos)Re(CO)₂H…HOC(CF₃)₃] (1.83 Å) has been calculated by NMR spectroscopy through the determination of the T_1min relaxation time of the Re---H proton. It has been shown that the [(triphos)Re(CO)₂H…HOR] adducts are in equilibrium with the dihydrogen complex [(triphos)Re(CO)₂(η²-H₂)]⁺, which is thermodynamically more stable than any H-bond adduct.     

Structural constraints for the Crh protein from solid-state NMR experiments

We demonstrate that short, medium and long-range constraints can be extracted from proton mediated, rare-spin detected correlation solid-state NMR experiments for the microcrystalline 10.4 × 2 kDa dimeric model protein Crh. Magnetization build-up curves from cross signals in NHHC and CHHC spectra deliver detailed information on side chain conformers and secondary structure for interactions between spin pairs. A large number of medium and long-range correlations can be observed in the spectra, and an analysis of the resolved signals reveals that the constraints cover the entire sequence, also including inter-monomer contacts between the two molecules forming the domain-swapped Crh dimer. Dynamic behavior is shown to have an impact on cross signals intensities, as indicated for mobile residues or regions by contacts predicted from the crystal structure, but absent in the spectra. Our work validates strategies involving proton distance measurements for large and complex proteins as the Crh dimer, and confirms the magnetization transfer properties previously described for small molecules in solid protein samples. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Recombinant expression, biochemical characterization, and biological activities of the human MGSA/gro protein

Melanoma growth stimulatory activity (MGSA) is a mitogenic protein secreted by Hs294T melanoma cells that corresponds to the polypeptide encoded by the human gro gene. The MGSA/gro cDNA has been expressed in mammalian cells and the secreted recombinant factor has been purified. Biochemical and biological characterization shows that the recombinant protein is identical with the natural protein and is devoid of posttranslational glycosylation, sulfation, and phosphorylation. The two C-terminal amino acids are proteolytically removed from the mature recombinant MGSA, indicating a length of 71 instead of the predicted 73 amino acids. The recombinant MGSA is mitogenically active on the Hs294T melanoma cells. The purified MGSA competes with interleukin 8 for binding to neutrophil receptors and exhibits neutrophil chemotactic activity equivalent to that of interleukin 8.     

Biochemical, mutational and in silico structural evidence for a functional dimeric form of the ornithine decarboxylase from Entamoeba histolytica

Background

Entamoeba histolytica is responsible for causing amoebiasis. Polyamine biosynthesis pathway enzymes are potential drug targets in parasitic protozoan diseases. The first and rate-limiting step of this pathway is catalyzed by ornithine decarboxylase (ODC). ODC enzyme functions as an obligate dimer. However, partially purified ODC from E. histolytica (EhODC) is reported to exist in a pentameric state.

Methodology and Results

In present study, the oligomeric state of EhODC was re-investigated. The enzyme was over-expressed in Escherichia coli and purified. Pure protein was used for determination of secondary structure content using circular dichroism spectroscopy. The percentages of α-helix, β-sheets and random coils in EhODC were estimated to be 39%, 25% and 36% respectively. Size-exclusion chromatography and mass spectrophotometry analysis revealed that EhODC enzyme exists in dimeric form. Further, computational model of EhODC dimer was generated. The homodimer contains two separate active sites at the dimer interface with Lys57 and Cys334 residues of opposite monomers contributing to each active site. Molecular dynamic simulations were performed and the dimeric structure was found to be very stable with RMSD value ∼0.327 nm. To gain insight into the functional role, the interface residues critical for dimerization and active site formation were identified and mutated. Mutation of Lys57Ala or Cys334Ala completely abolished enzyme activity. Interestingly, partial restoration of the enzyme activity was observed when inactive Lys57Ala and Cys334Ala mutants were mixed confirming that the dimer is the active form. Furthermore, Gly361Tyr and Lys157Ala mutations at the dimer interface were found to abolish the enzyme activity and destabilize the dimer.

Conclusion

To our knowledge, this is the first report which demonstrates that EhODC is functional in the dimeric form. These findings and availability of 3D structure model of EhODC dimer opens up possibilities for alternate enzyme inhibition strategies by targeting the dimer disruption.     

Secondary Structure in Solution of the Hydrophobic Protein of Soybean (HPS) as Revealed by 1H NMR

Abstract

COSY, TOCSY and NOESY experiments have been used to assign sequentially the ¹H 500 MHz NMR spectra of the Hydrophobic Protein of Soybean (HPS). Spin systems identification combined with sequential assignment allowed to identify the proton resonances of this 80 residues protein. Analysis of medium range connectivities showed that its secondary structure involved four helical fragments similarly located as in the structure deduced from X-ray diffraction. This work set the basis for a further fine comparison between the crystal and the solution structures and a dynamical study of HPS in solution. In addition, search of secondary structure similarities showed that the global folding of HPS should be rather similar to that found for non specific Lipid Transfer Proteins (ns-LTP) from vegetal origin. Distributions of the helical fragments along the primary sequences of these two classes of proteins were compared.     

Structure and properties of a dimeric N-terminal fragment of human ubiquitin.

Previous peptide dissection and kinetic experiments have indicated that in vitro folding of ubiquitin may proceed via transient species in which native-like structure has been acquired in the first 45 residues. A peptide fragment, UQ(1-51), encompassing residues 1 to 51 of ubiquitin was produced in order to test whether this portion has propensity for independent self-assembly. Surprisingly, the construct formed a folded symmetrical dimer that was stabilised by 0.8 M sodium sulphate at 298 K (the S state). The solution structure of the UQ(1-51) dimer was determined by multinuclear NMR spectroscopy. Each subunit of UQ(1-51) consists of an N-terminal beta-hairpin followed by an alpha-helix and a final beta-strand, with orientations similar to intact ubiquitin. The dimer is formed by the third beta-strand of one subunit interleaving between the hairpin and third strand of the other to give a six-stranded beta-sheet, with the two alpha-helices sitting on top. The helix-helix and strand portions of the dimer interface also mimic related features in the structure of ubiquitin. The structural specificity of the UQ(1-51) peptide is tuneable: as the concentration of sodium sulphate is decreased, near-native alternative conformations are populated in slow chemical exchange. Magnetization transfer experiments were performed to characterize the various species present in 0.35 M sodium sulphate, namely the S state and two minor forms. Chemical shift differences suggest that one minor form is very similar to the S state, while the other experiences a significant conformational change in the third strand. A segmental rearrangement of the third strand in one subunit of the S state would render the dimer asymmetric, accounting for most of our results. Similar small-scale transitions in proteins are often invoked to explain solvent exchange at backbone amide proton sites that have an intermediate level of protection.     

Solution structure of the cytoplasmic domain of erythrocyte membrane band 3 determined by site-directed spin labeling

The cytoplasmic domain of the anion exchange protein (cdb3) serves as a critical organizing center for protein-protein interactions that stabilize the erythrocyte membrane. The structure of the central core of cdb3, determined by X-ray crystallography from crystals grown at pH 4.8, revealed a compact dimer for residues 55-356 and unresolved N- and C-termini on each monomer [Zhang et al. (2000) Blood 96, 2925-2933]. Given that previous studies had suggested a highly asymmetric structure for cdb3 and that pH dependent structural transitions of cdb3 have been reported, the structure of cdb3 in solution at neutral pH was investigated via site-directed spin labeling in combination with conventional electron paramagnetic resonance (EPR) and double electron electron resonance (DEER) spectroscopies. These studies show that the structure of the central compact dimer (residues 55-356) is indistinguishable from the crystal structure determined at pH 4.8. N-Terminal residues 1-54 and C-terminal residues 357-379 are dynamically disordered and show no indications of stable secondary structure. These results establish a structural model for cdb3 in solution at neutral pH which represents an important next step in characterizing structural details of the protein-protein interactions that stabilize the erythrocyte membrane.