title: A comprehensive model of the spectrin divalent tetramer binding region deduced using homology modeling and chemical cross-linking of a mini-spectrin.
abstract: Spectrin dimer-tetramer interconversion is a critical contributor to red cell membrane stability, but some properties of spectrin tetramer formation cannot be studied effectively using monomeric recombinant domains. To address these limitations, a fused αβ mini-spectrin was produced that forms wild-type divalent tetramer complexes. Using this mini-spectrin, a medium-resolution structure of a seven-repeat bivalent tetramer was produced using homology modeling coupled with chemical cross-linking. Inter- and intramolecular cross-links provided critical distance constraints for evaluating and optimizing the best conformational model and appropriate docking interfaces. The two strands twist around each other to form a super-coiled, rope-like structure with the AB helix face of one strand associating with the opposing AC helix face. Interestingly, two tetramer site hereditary anemia mutations that exhibit wild-type binding in univalent head-to-head assays are located in the interstrand region. This suggests that perturbations of the interstrand region can destabilize spectrin tetramers and the membrane skeleton. The α subunit N-terminal cross-links to multiple sites on both strands, demonstrating that this non-homologous tail remains flexible and forms heterogeneous structures in the tetramer complex. Although no cross-links were observed involving the β subunit non-homologous C-terminal tail, several cross-links were observed only when this domain was present, suggesting it induces subtle conformational changes to the tetramer site region. This medium-resolution model provides a basis for further studies of the bivalent spectrin tetramer site, including analysis of functional consequences of interstrand interactions and mutations located at substantial molecular distances from the tetramer site.
authors.affiliation: Center for Systems and Computational Biology, The Wistar Institute, Philadelphia, Pennsylvania 19104, USA.
authors.forename: Donghai
authors.initials: D
authors.lastname: Li
authors.forename: Sandra L
authors.initials: SL
authors.lastname: Harper
authors.forename: Hsin-Yao
authors.initials: HY
authors.lastname: Tang
authors.forename: Yelena
authors.initials: Y
authors.lastname: Maksimova
authors.forename: Patrick G
authors.initials: PG
authors.lastname: Gallagher
authors.forename: David W
authors.initials: DW
authors.lastname: Speicher
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D055503:Protein Multimerization / Q000235:genetics / Q000502:physiology; D017386:Sequence Homology, Amino Acid; D013049:Spectrin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D053719:Tandem Mass Spectrometry
chemical_list: D013049:Spectrin