A comprehensive model of the spectrin divalent tetramer binding region deduced using homology modeling and chemical cross-linking of a mini-spectrin.
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.
Li
Donghai
D
Center for Systems and Computational Biology, The Wistar Institute, Philadelphia, Pennsylvania 19104, USA.
Harper
Sandra L
SL
Tang
Hsin-Yao
HY
Maksimova
Yelena
Y
Gallagher
Patrick G
PG
Speicher
David W
DW
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
D013049:Spectrin