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 Fourier-transform ion cyclotron resonance detector. Six interchain cross-links distinguished between alternative docking models, and these distance constraints, as well as three intrachain cross-links, 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 alpha and beta repeats from among many very similar repeats in both subunits is driven primarily by long range electrostatic interactions.
Highlights
The identified cross-links are incompatible with the docking orientations of the A and B models (Fig. 5)
The Docking C model is highly consistent with both the cross-links identified in the current study
Our model of the complex, which used these cross-link distance constraints to refine the most plausible homology model, provides a reliable medium resolution conformational model for this critical heterodimer complex. Structural features of this model are consistent with prior protease protection analyses, thermodynamic properties of heterodimer assembly for wild type subunits, and inability of ␣ subunits containing the ␣LELY 6-residue deletion to heterodimerize
Summary
Covalently linked to the weaker repeats (12). This series of studies resulted in a “zipper” model of spectrin heterodimer assembly, which consisted of three discrete steps: 1) rapid, high affinity binding of the dimer initiation site; 2) rapid, low affinity lateral association of complementary repeats along the length of the monomer; and 3) latching the zipper by forming a closed dimer through a slow, moderate affinity, temperature-dependent association (14). Chemical cross-linking is a very old technique, it has historically been very challenging to identify crosslinked peptides, because they are usually substoichiometric and difficult to separate, detect, and identify (17) This situation has improved substantially over the past several years as high sensitivity, high speed mass spectrometers capable of very precise mass determinations have become available. The docking interfaces of the red cell spectrin heterodimer initiation site region were experimentally determined using a “zero length” cross-linker coupled with analysis of cross-linked tryptic peptides using liquid chromatographyMS/MS analysis on an LTQ FT-ICR mass spectrometer. Distance constraints from both intrachain and interchain crosslinks were used to further refine an initial homology-based. The final model correlates well with available biochemical and thermodynamic data, and it supports the hypothesis that initial alignment and pairing of correct ␣ and  repeats from among many very similar repeats in both subunits is driven primarily by long range electrostatic interactions
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