A structural model of the erythrocyte spectrin heterodimer initiation site determined using homology modeling and chemical cross-linking.
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.
Li
Donghai
D
Wistar Institute, Philadelphia, Pennsylvania 19104.
Tang
Hsin-Yao
HY
Wistar Institute, Philadelphia, Pennsylvania 19104.
Speicher
David W
DW
Wistar Institute, Philadelphia, Pennsylvania 19104. Electronic address: speicher@wistar.org.
D000595:Amino Acid Sequence; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D019281:Dimerization; D004912:Erythrocytes / Q000737:chemistry*; D005022:Ethyldimethylaminopropyl Carbodiimide / Q000494:pharmacology; D006801:Humans; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D020449:Repetitive Sequences, Amino Acid; D016415:Sequence Alignment; D013049:Spectrin / Q000737:chemistry*; D055672:Static Electricity; D040681:Structural Homology, Protein; D013388:Succinimides / Q000494:pharmacology
D003432:Cross-Linking Reagents; C465543:N-hydroxysulfosuccimide; D010455:Peptides; D013388:Succinimides; D013049:Spectrin; D005022:Ethyldimethylaminopropyl Carbodiimide