A Structural Model of the Erythrocyte Spectrin Heterodimer Initiation Site Determined Using Homology Modeling and Chemical Cross-linking

Abstract

Spectrin assembles into an anti-parallel heterodimeric flexible rod-like molecule through a multistep process initiated by a high affinity interaction between discrete complementary homologous motifs or "repeats" near the actin binding domain. Attempts to determine crystallographic structures of this critical dimer initiation complex have so far been unsuccessful. Therefore, in this study we determined the subunit-subunit docking interface and a plausible medium resolution structure of the heterodimer initiation site using homology modeling coupled with structural refinement based on experimentally determined distance constraints. Intramolecular and intermolecular cross-links formed by the "zero length" cross-linking reagent, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide were identified after trypsin digestion of cross-linked heterodimer complex using liquid chromatography-tandem mass spectrometry analysis. High confidence assignment of cross-linked peptides was facilitated by determination of cross-linked peptide masses with an uncertainty of a few parts per million using a high sensitivity linear ion trap mass spectrometer equipped with a Fouriertransform ion cyclotron resonance detector. Six interchain cross-links distinguished between alternative docking models, and these distance constraints, as well as three intrachain crosslinks, were used to further refine an initial homology-based structure. The final model is consistent with all available physical data, including protease protection experiments, isothermal titration calorimetry analyses, and location of a common polymorphism that destabilizes dimerization. This model supports the hypothesis that initial docking of the correct ␣ and ␤ repeats from among many very similar repeats in both subunits is driven primarily by long range electrostatic interactions.Spectrin is an actin cross-linking protein that lines the intracellular side of the plasma membrane of most cell types where it is a major component of a submembraneous scaffold. This membrane skeleton plays important roles in maintaining membrane integrity and organization, particularly in red cells. Red cell spectrin is composed of a 281-kDa ␣ subunit and a 246-kDa ␤ subunit that associate side-to-side in an anti-parallel orientation, and two heterodimers associate head-to-head to form a tetramer, which cross-links actin oligomers. The spectrin-actin membrane skeleton is linked to the membrane through several indirect linkages to transmembrane proteins as well as direct interactions with lipid head groups (1-6).Most hereditary hemolytic anemias, which are quite common in the human population, destabilize the red cell membrane by disrupting either the spectrin-actin scaffold or its linkages to the plasma membrane. For example, most hereditary elliptocytosis and hereditary pyropoikilocytosis patients have mutations in one of the spectrin genes, and many of these mutations disrupt spectrin self-assembly into actin cross-linking tetramers (7-9). Spectrin mutations are usually heterozygous, and clinical s...

Cell organization, growth, and neural and cardiac development require αII-spectrin

“…1A). The exontrapped gene generates a spectrin b-gal fusion message that truncates the aII-spectrin gene product at codon 1153, corresponding to a polypeptide terminating within spectrin repeat ten, lacking the C-terminal site responsible for heterodimer formation with b-spectrin (Li et al, 2008). In tissues or cells homozygous for this insertion (Fig.…”

Major Vault Protein Regulates Class A Scavenger Receptor-mediated Tumor Necrosis Factor-α Synthesis and Apoptosis in Macrophages

“…The reaction mixtures were gently incubated at room temperature, and the reactions were quenched by the addition of 10 l of aqueous dithiothreitol (DTT); the final concentration of DTT was 20 mM. Finally, cells were centrifuged at 3000 ϫ g and 4°C for 10 min, and the precipitation was used for the following Western blotting assays (15).…”

“…The extract was adjusted to 40% sucrose by adding 2 ml of 80% sucrose, and a total volume of 4 ml was transferred to an ultracentrifuge tube and overlaid with 1 ml of 35,30,25,20,15, and 5% sucrose in MES buffer as above but minus the Triton X-100. All of the materials were kept at 0°C.…”

Anatomy of the red cell membrane skeleton: unanswered questions

“…16 The isolated a-and b-spectrin chains join to form spectrin heterodimers at a nucleation site near the tail end of spectrin (repeats a-21 pairs with b-1, and a-20 with b-2) and then zip together in a cooperative manner. 17 Actin and protein 4.1R bind to CH domains at the N-terminal end of b-spectrin, just beyond the nucleation site. 18 Binding to the CH2 domain is activated by phosphatidylinositol-4,5-bisphosphate (PIP 2 ).…”