The 22.5 kDa spectrin-binding domain of ankyrinR binds spectrin with high affinity and changes the spectrin distribution in cells in vivo.

It was previously shown that ankyrins play a crucial role in the membrane skeleton arrangement. Purifying ankyrinR obtained from erythrocytes is a time-consuming process. Therefore, cloned and bacterially expressed ankyrinR-spectrin-binding domain (AnkSBD) is a demanded tool for studying spectrin-ankyrin interactions. In this communication, we report on the cloning and purification of AnkSBD and describe the results of binding experiments, in which we showed high-affinity interactions between the AnkSBD construct and isolated erythrocyte or non-erythroid spectrins. pEGFP-AnkSBD-transfected cells co-localised with non-erythroid spectrin in HeLa cells. The functional interactions of the AnkSBD construct in vivo and in vitro open many possibilities to study the structure and function of this domain, which has not yet been as extensively studied when compared to the aminoterminal domain of this protein.

Initiation and propagation of spectrin heterodimer assembly involves distinct energetic processes.

Red cell spectrin alpha and beta subunits consist primarily of many tandem homologous motifs with very similar three- helix-bundle structures and similar dimer interfaces. Although misassembled homodimers can form under some conditions, correctly aligned heterodimers consistently assemble provided a small "dimer initiation" site near the actin binding domain is present. The dimer initiation site has been characterized to some extent, but little is known about the subsequent, low-affinity lateral interactions of the remaining motifs along the length of this ropelike molecule or the forces involved in these two steps of the dimerization process. In this study, we used isothermal titration calorimetry to deduce the mechanism and energetics of the two heterodimer assembly phases. The high-affinity initiation of dimerization is primarily enthalpically driven, which is consistent with initial alignment and docking of specific complementary alpha and beta motifs in the dimer initiation site driven by long-range electrostatic interactions followed by tight binding stabilized by hydrogen bonds and other hydrophilic interactions. In contrast, the subsequent weak lateral associations of additional motifs are primarily entropically driven, suggesting binding primarily involves weak hydrophobic interactions. Although initial docking is largely electrostatic, the only lateral interaction within the first four pairs of motifs that involves a net change in protons is the interaction of the alpha18 and beta4 repeats. This substoichiometric uptake of protons could be due to a pKa shift of a histidine in the alpha18 motif located near the dimer interface in a proposed homology-based model. On the basis of this analysis of heterodimer thermodynamics, a detailed model of spectrin dimer assembly is proposed.

Enhanced oxidative cross-linking of hemoglobin E with spectrin and loss of erythrocyte membrane asymmetry in hemoglobin Ebeta-thalassemia.

Oxidative stress to the erythrocytes is associated with formation of large molecular complexes of hemoglobin and the skeletal protein, spectrin. In this work, such complexes are formed with hemoglobin mixtures isolated from patients suffering from HbEbeta-thalassemia with elevated levels of the HbE and purified erythroid spectrin in the presence of hydrogen peroxide. The complexes are separated on 4% SDS-PAGE and analyzed by densitometry. The results indicate enhanced formation of complexes with higher amounts of HbE, the most common hemoglobin variant prevalent in Southeast Asia. The binding affinity of spectrin with hemoglobin, in the absence of hydrogen peroxide, was found to increase with hemoglobin mixtures enriched with HbE. The presence of ATP was also found to decrease the overall yield of such complexes. Flow cytometric measurements of phosphatidylserine on the red cell surface also showed a lower degree of membrane asymmetry in HbEbeta-thalassemic patients than in normal subjects. The present work shows enhanced formation of high molecular weight cross-linked complexes of hemoglobin derivatives with erythroid spectrin in HbEbeta-thalassemia.

Conformational studies of the tetramerization site of human erythroid spectrin by cysteine-scanning spin-labeling EPR methods.

We used cysteine-scanning and spin-labeling methods to prepare singly spin labeled recombinant peptides for electron paramagnetic resonance studies of the partial domain regions at the tetramerization site (N-terminal end of alpha and C-terminal end of beta) of erythroid spectrin. The values of the inverse line width parameter (deltaH0(-1)) from a family of Sp alphaI-1-368delta peptides scanning residues 21-30 exhibited a periodicity of approximately 3.5-4. We used molecular dynamics calculations to show that the asymmetric mobility of this helix is not necessarily due to tertiary contacts, but is likely due to intrinsic properties of helix C', a helix with a heptad pattern sequence. The residues with low deltaH0(-1) values (residues at positions 21, 25, and 28/29) were those on the hydrophobic side of this amphipathic helix. Native gel electrophoresis results showed that these residues were functionally important and are involved in the tetramerization process. Thus, EPR results readily identified functionally important residues in the alpha spectrin partial domain region. Mutations at these positions may lead to clinical symptoms. Similarly, the deltaH0(-1) values from a family of spin-labeled Sp betaI-1898-2083delta peptides also exhibited a periodicity of approximately 3.5-4, indicating a helical conformation in the two scanned regions (residues 2008-2018 and residues 2060-2070). However, the region consisting of residues 2071-2076 was in a disordered conformation. Both helical regions include a hydrophilic side with high deltaH0(-1) values and a hydrophobic side with low deltaH0(-1) values, demonstrating the amphipathic nature of the helical regions. Residues 2008, 2011, 2014, and 2018 in the first scanned region and residues 2061, 2065, and 2068 in the second scanned region were on the hydrophobic side. These residues were critical in alphabeta spectrin association at the tetramerization site. Mutations at some of these positions have been reported to be detrimental in clinical studies.

Effect of ionic strength on the organization and dynamics of tryptophan residues in erythroid spectrin: a fluorescence approach.

The ionic strength of the medium plays an important role in the structure and conformation of erythroid spectrin. The spectrin dimer is a flexible rod at physiological ionic strength. However, lower ionic strength results in elongation and rigidification (stiffening) of spectrin as shown earlier by electron microscopy and hydrodynamic studies. The ionic strength induced structural transition does not involve any specific secondary structural changes. In this article, we have used a combination of fluorescence spectroscopic approaches that include red edge excitation shift (REES), fluorescence quenching, time-resolved fluorescence measurements, and chemical modification of the spectrin tryptophans to assess the environment and dynamics of tryptophan residues of spectrin under different ionic strength conditions. Our results show that while REES, fluorescence anisotropy, lifetime, and chemical modification of spectrin tryptophans remain unaltered in low and high ionic strength conditions, quenching of tryptophan fluorescence by the aqueous quencher acrylamide (but not the hydrophobic quencher trichloroethanol) and resonance energy transfer to a dansyl-labeled fatty acid show differences in tryptophan environment. These results, which report tertiary structural changes in spectrin upon change in ionic strength, are relevant in understanding the molecular details underlying the conformational flexibility of spectrin.

Independent movement, dimerization and stability of tandem repeats of chicken brain alpha-spectrin.

Previous X-ray crystal structures have shown that linkers of five amino acid residues connecting pairs of chicken brain alpha-spectrin and human erythroid beta-spectrin repeats can undergo bending without losing their alpha-helical structure. To test whether bending at one linker can influence bending at an adjacent linker, the structures of two and three repeat fragments of chicken brain alpha-spectrin have been determined by X-ray crystallography. The structure of the three-repeat fragment clearly shows that bending at one linker can occur independently of bending at an adjacent linker. This observation increases the possible trajectories of modeled chains of spectrin repeats. Furthermore, the three-repeat molecule crystallized as an antiparallel dimer with a significantly smaller buried interfacial area than that of alpha-actinin, a spectrin-related molecule, but large enough and of a type indicating biological specificity. Comparison of the structures of the spectrin and alpha-actinin dimers supports weak association of the former, which could not be detected by analytical ultracentrifugation, versus strong association of the latter, which has been observed by others. To correlate features of the structure with solution properties and to test a previous model of stable spectrin and dystrophin repeats, the number of inter-helical interactions in each repeat of several spectrin structures were counted and compared to their thermal stabilities. Inter-helical interactions, but not all interactions, increased in parallel with measured thermal stabilities of each repeat and in agreement with the thermal stabilities of two and three repeats and also partial repeats of spectrin.

In vivo phosphorylation of human erythrocyte spectrin occurs in a sequential manner.

Spectrin is the major component of the erythrocyte membrane skeleton and exists as a 526 kDa alphabeta heterodimer. The 246 kDa beta-chain of human spectrin is phosphorylated near the C-terminus, but the exact phosphorylation sites are unknown and the role of this phosphorylation is not fully characterized. In this study, we produced a monoclonal antibody, Sp316, capable of recognizing the C-terminal region of beta-spectrin regardless of its phosphorylation state and used it to purify the phosphorylated region after 2-nitro-5-thiocyanobenzoic acid cleavage of spectrin. Two-dimensional gels, mass spectrometry, and reversed-phase high-performance liquid chromatography were used to characterize these phosphorylation states. Only about 1.5% of spectrin isolated from fresh blood is unphosphorylated, about 9% has more than four phosphates per molecule, and the majority of the protein has one to four phosphates per molecule. A total of six phosphorylation sites were identified by tandem mass spectrometry. Quantitative analysis of the phosphorylation states by reversed-phase high-performance liquid chromatography revealed that phosphorylation of beta-spectrin occurs in a sequential manner where each specific site is completely phosphorylated before the next site is modified. The first phosphorylation event occurs on Ser-2114, followed by Ser-2125, Ser-2123, Ser-2128, Ser-2117, and Thr-2110. The identification of the specific phosphorylated beta-spectrin residues and the ordered sequence of phosphorylation events in vivo should provide an invaluable basis for further studies of the role of these posttranslational modifications in spectrin function in situ.

Ankyrin is a target of spectrin's E2/E3 ubiquitin-conjugating/ligating activity.

Ubiquitin is a small protein of 8.6 kDa molecular weight. When polyubiquitin is attached to target proteins, they are tagged for destruction by cytoplasmic organelles called proteasomes. We now know that ubiquitination of target proteins also regulates functions as diverse as the sorting of proteins to different intracellular destinations, cell signaling, cell division, gene transcription, and protein-protein interactions. The ubiquitination of target proteins requires a cascade of enzymes: E1 ubiquitin activating enzyme, E2 ubiquitin conjugating enzyme and E3 ubiquitin ligating enzyme. Recently we have demonstrated that the red blood cell (RBC) membrane skeletal protein, spectrin, has E2/E3 enzymatic activities in its alpha-subunit, that can transfer ubiquitin to itself. We have now created a cell free assay using biotinylated ubiquitin that allows detection of target proteins by streptavidin peroxidase. This approach coupled with immunoprecipitation, purification and micro liquid chromatography coupled to tandem mass spectrometry has identified ankyrin as a target of spectrin's E2/E3 activity. Western blotting, with ubiquitin antibody, of purified ankyrin and its well characterized functional domains, has demonstrated that both the spectrin and band 3 binding domains are ubiquitinated in vivo.

Proteins with spectrin motifs which do not belong to the spectrin-alpha-actinin-dystrophin family.

Using several consensus sequences for the 106 amino acid residue alpha-spectrin repeat segment as probes we searched animal sequence databases using the BLAST program in order to find proteins revealing limited, but significant similarity to spectrin. Among many spectrins and proteins from the spectrin-alpha-actinin-dystrophin family as well as sequences showing a rather high degree of similarity in very short stretches, we found seven homologous animal sequences of low overall similarity to spectrin but showing the presence of one or more spectrin-repeat motifs. The homology relationship of these sequences to alpha-spectrin was further analysed using the SEMIHOM program. Depending on the probe, these segments showed the presence of 6 to 26 identical amino acid residues and a variable number of semihomologous residues. Moreover, we found six protein sequences, which contained a sequence fragment sharing the SH3 (sarc homology region 3) domain homology of 42-59% similarity. Our data indicate the occurrence of motifs of significant homology to alpha-spectrin repeat segments among animal proteins, which are not classical members of the spectrin-alpha-actinin-dystrophin family. This might indicate that these segments together with the SH3 domain motif are conserved in proteins which possibly at the early stage of evolution were close cognates of spectrin-alpha-actinin-dystrophin progenitors but then evolved separately.

Rapid purification and crystal structure analysis of a small protein carrying two terminal affinity tags.

Small peptide tags are often fused to proteins to allow their affinity purification in high-throughput structure analysis schemes. To assess the compatibility of small peptide tags with protein crystallization and to examine if the tags alter the three-dimensional structure, the N-terminus of the chicken alpha-spectrin SH3 domain was labeled with a His6 tag and the C-terminus with a StrepII tag. The resulting protein, His6-SH3-StrepII, consists of 83 amino-acid residues, 23 of which originate from the tags. His6-SH3-StrepII is readily purified by dual affinity chromatography, has very similar biophysical characteristics as the untagged protein domain and crystallizes readily from a number of sparse-matrix screen conditions. The crystal structure analysis at 2.3 A resolution proves native-like structure of His6-SH3-StrepII and shows the entire His6 tag and part of the StrepII tag to be disordered in the crystal. Obviously, the fused affinity tags did not interfere with crystallization and structure analysis and did not change the protein structure. From the extreme case of His6-SH3-StrepII, where affinity tags represent 27% of the total fusion protein mass, we extrapolate that protein constructs with N- and C-terminal peptide tags may lend themselves to biophysical and structural investigations in high-throughput regimes.

T-cell specificity in murine autoimmune haemolytic anaemia induced by rat red blood cells.

Autoimmune haemolytic anaemia (AIHA) can be induced in mice by repeated injections with rat red blood cells (RBC). Here we describe the identification of rat and murine RBC antigens recognized by T-cells from mice with this disease. Splenic T-cells from mice with AIHA proliferated in response to multiple murine RBC membrane components, each of which is recognized by rat RBC induced autoantibodies. Thus, there were responses to murine autoantigen fractions that correspond in apparent molecular mass with the anion channel Band 3, with spectrin from the membrane skeleton and with the high and low molecular mass glycophorins, and the equivalent fractions from rat RBC also stimulated proliferation by T-cells. It was confirmed that purified Band 3 from murine and rat RBC also elicited responses. In contrast with the results in AIHA, T-cells from healthy control mice failed to respond to the antigens from either species, with the exception of proliferation induced by murine spectrin in one experiment and weak responses elicited by rat Band 3. It is suggested that T-cells activated by multiple cross-reactions between rat and murine RBC proteins, and by epitope spreading, are necessary to drive autoantibody production in this model of AIHA.

Brain spectrin exerts much stronger effect on anionic phospholipid monolayers than erythroid spectrin.

Red blood cell spectrin and its nonerythroid analogues are linked to integral proteins of the membrane by several skeletal protein receptors, such as ankyrin and protein 4.1 together with p55. However, there are also many reasons for believing that they are insufficient to engender all the properties that characterise the native membrane. Therefore, we are concerned with the mechanism by which brain spectrin interacts with phospholipids of the membrane bilayer. Brain and erythrocyte spectrin were shown previously to bind phospholipid vesicles as well as monolayers prepared from aminophospholipids: phosphatidylethanolamine and phosphatidylserine and their mixtures with phosphatidylcholine (PC). In the present study, it is shown that brain spectrin binds to monolayers prepared from anionic phospholipids, such as phosphatidylinositol (PI), phosphatidic acid (PA), phosphatidyl glycerol, diphosphatidylglycerol, and their mixtures with PC. Brain spectrin injected into the subphase to reach nanomolar concentration induced a substantial increase in the surface pressure of monolayers prepared from the phospholipids and their mixtures mentioned above, possibly by penetrating them. This effect is stronger in the case of monolayers prepared from anionic phospholipids alone and weaker when monolayers were prepared from mixtures with PC. The weakest effect was observed in the case of phosphatidylinositol-4,5-bisphosphate monolayers. An interaction of brain spectrin with monolayers prepared from anionic phospholipids (PI/PC 7:3 and PA/PC 7:3) was inhibited (PI/PC much stronger than PA/PC) by purified erythrocyte ankyrin, which indicates that the binding site for those lipids is located in the beta-subunit, possibly in, or in close proximity of, the ankyrin-binding site. In contrast, erythrocyte spectrin injected into the subphase induced a change in the surface pressure of monolayers prepared from anionic phospholipids, which was equal or smaller than the value of surface pressure change induced by protein without a monolayer. This effect was different from what had been observed previously for monolayers prepared from aminophospholipids and their mixtures with PC, and from the data for nonerythroid spectrin presented here.

Isolation of spectrin subunits by reverse-phase high-performance liquid chromatography.

We present a one-step uncomplicated method of separation of spectrin subunits. The method is based on reverse-phase HPLC employing an analytical C4 column. Reverse-phase HPLC combines the steps of dissociation and separation of spectrin subunits. The method can be applied to different spectrin isoforms. It can be used for analytical purposes, as well as for small-scale (<0.4 mg) isolation of spectrin subunits.

Erythrocyte spectrin is an E2 ubiquitin conjugating enzyme.

The involvement of red blood cell spectrin in the ubiquitination process was studied. Spectrin was found to form two ubiquitin-associated derivatives, a DTT-sensitive ubiquitin adduct and a DTT-insensitive conjugate, characteristic intermediate and final products of the ubiquitination reaction cascade. In addition to spectrin and ubiquitin, ubiquitin-activating enzyme (E1) and ATP were necessary and sufficient to form both the spectrin-ubiquitin adduct and conjugate. No exogenous ubiquitin-conjugating (E2) or ligase (E3) activities were required, suggesting that erythrocyte spectrin is an E2 ubiquitin-conjugating enzyme able to target itself. Both ubiquitin adduct and conjugate were linked to the alpha subunit of spectrin, suggesting that the ubiquitin-conjugating (UBC) domain and its target regions reside on the same subunit.

Computer-assisted re-design of spectrin SH3 residue clusters.

We have developed a protein design computer program, called Perla, which performs searches in sequence space to uncover optimal amino acid sequences for desired protein three-dimensional structures. Optimal sequences are localised at the minima of a sequence-structure energy landscape defined using a complex scoring function (an all-atom molecular mechanics force field plus statistical terms including entropy and solvation) measured with respect to a reference state simulating a denatured protein. Sequence choices eventually optimise side chain packing, secondary structure propensities, and hydrogen bonding and electrostatics interactions. Perla was used to re-design clusters of residues of the SH3 domain of alpha-spectrin. Several mutant proteins were produced and characterised. Some of our designed proteins have significantly higher stabilities (stability enhancements about 0.25, 0.70 and 1.0 kcal mol(-1)) than the wild-type protein. These successful protein re-designs, and similar examples found in the literature, establish the quality of the structure-based computational approach to protein design.

Human alpha spectrin II and the FANCA, FANCC, and FANCG proteins bind to DNA containing psoralen interstrand cross-links.

Repair of DNA interstrand cross-links is a complex process critical to which is the identification of sites of damage by specific proteins. We have recently identified the structural protein nonerythroid alpha spectrin (alphaSpIISigma) as a component of a nuclear protein complex in normal human cells which is involved in the repair of DNA interstrand cross-links and have shown that it forms a complex with the Fanconi anemia proteins FANCA, FANCC, and FANCG. Using DNA affinity chromatography, we now show that alphaSpIISigma, present in HeLa cell nuclei, specifically binds to DNA containing psoralen interstrand cross-links and that the FANCA, FANCC, and FANCG proteins are bound to this damaged DNA as well. That spectrin binds directly to the cross-linked DNA has been shown using purified bovine brain spectrin (alphaSpIISigma1/betaSpIISigma1)2. Binding of the Fanconi anemia (FA) proteins to the damaged DNA may be either direct or indirect via their association with alphaSpIISigma. These results demonstrate a role for alpha spectrin in the nucleus as well as a new function for this protein in the cell, an involvement in DNA repair. alphaSpIISigma may bind to cross-linked DNA and act as a scaffold to help in the recruitment of repair proteins to the site of damage and aid in their alignment and interaction with each other, thus enhancing the efficiency of the repair process.

Atomic resolution structure of a mutant of the spectrin SH3 domain.

The crystal structure of an alpha-spectrin Src-homology 3 (SH3) domain mutant has been refined at 1.12 A resolution. This X-ray structure is at the highest resolution achieved so far for an SH3 domain. The structure allows the identification of a complete set of specific interactions and is useful for the elucidation of relations between structure and pH-dependent thermodynamic stability in a series of SH3 domain mutants.

Atomic force microscopy of the erythrocyte membrane skeleton.

The atomic force microscope was used to examine the cytoplasmic surface of untreated as well as fixed human erythrocyte membranes that had been continuously maintained under aqueous solutions. To assess the effects of drying, some membranes were examined in air. Erythrocytes attached to mica or glass were sheared open with a stream of isotonic buffer, which allowed access to the cytoplasmic membrane face without exposing cells to non-physiological ionic strength solutions. Under these conditions of examination, the unfixed cytoplasmic membrane face revealed an irregular meshwork that appeared to be a mixture largely of triangular and rectilinear openings with mesh sizes that varied from 35 to 100 nm, although few were at the upper limit. Fixed ghosts were similar, but slightly more contracted. These features represent the membrane skeleton, as when the ghosts were treated to extract spectrin and actin, these meshworks were largely removed. Direct measurements of the thickness of the membrane skeleton and of the lateral dimensions of features in the images suggested that, especially when air dried, spectrin can cluster into large, quite regularly distributed aggregates. Aggregation of cytoskeletal components was also favoured when the cells were attached to a polylysine-treated substrate. In contrast, the membrane skeletons of cells attached to substrates rendered positively charged by chemical derivatization with a cationic silane were much more resistant to aggregation. As steps were taken to reduce the possibility of change of the skeleton after opening the cells, the aggregates and voids were eliminated, and the observed structures became shorter and thinner. Ghosts treated with Triton X-100 solutions to remove the bilayer revealed a meshwork having aggregated components resembling those seen in air. These findings support the proposition that the end-to-end distance of spectrin tetramers in the cell in the equilibrium state is much shorter than the contour length of the molecule and that substantial rearrangements of the spectrin-actin network occur when it is expanded by low ionic strength extraction from the cell. This study demonstrates the applicability of AFM for imaging the erythrocyte membrane skeleton at a resolution that appears adequate to identify major components of the membrane skeleton under near-physiological conditions.

A novel brain-specific isoform of beta spectrin: isolation and its interaction with Munc13.

Munc13 is a component of the neurotransmitter release machinery which is specifically expressed in brain. Munc13 interacts with Doc2 and syntaxin which are also implicated in the neurotransmitter release process. Here we isolated another Munc13-interacting molecule from a rat brain cDNA library by use of the yeast two-hybrid system, identified it to be a novel type of beta spectrin, and named it beta SpIII sigma 1. beta SpIII sigma 1 was specifically expressed in brain, where it was enriched in the synaptic vesicle and plasma membrane fractions. Because spectrin has been shown to interact with the actin cytoskeleton which is involved in the exocytotic process, the present results suggest that the Munc13-beta SpIII sigma 1 interactions play a role in neurotransmitter release.

Purification of erythrocyte spectrin alpha- and beta-subunits at alkaline pH and structural and hydrodynamic properties of the isolated subunits.

A new method for the isolation of the alpha- and beta-subunits of human erythrocyte spectrin was developed, and structural properties and association behavior of the isolated subunits were studied by means of CD, nondenaturing gel electrophoresis, and analytical ultracentrifugation. The alpha- and beta-subunits were isolated using ion-exchange FPLC (pH 11) followed by size-exclusion FPLC (pH 7.5), having shown that alkaline pH dissociates spectrin polymers to their monomers [see Fujita et al. (1998) Biochemistry 37, 264-271]. The isolated subunits had alpha-helical content and thermal stability almost equivalent to those of native spectrin and reassembled to form heterodimers and tetramers which were indistinguishable from native spectrin with respect to secondary structure content, thermal stability, migration pattern on nondenaturing gels, and sedimentation coefficients. Thus, our data show that the increase in the structural stability of a heterodimer by association of the two monomers is very small. Sedimentation coefficients for the isolated alpha- and beta-subunits were 6.3 and 5.7 S, respectively. The similar frictional ratios (f/f0) of the isolated alpha-subunit (2.42) and the beta-subunit (2.45) indicate that the flexibility of both these wormlike chains and the range of shapes they can adopt in solution are very similar. The f/f0 value for spectrin dimer (2.41) indicates that its flexibility is somewhat, but not grossly, reduced compared to that of the individual subunits. Consequently, the folded repeat units of the subunits and the flexible connections between them are probably "in register" along the length of the dimer.