Solution Structural Studies on Human Erythrocyte α-Spectrin Tetramerization Site

Abstract

We have determined the solution NMR structure of a recombinant peptide that consists of the first 156 residues of erythroid ␣-spectrin. The first 20 residues preceding the first helix (helix C) are in a disordered conformation. The subsequent three helices (helices A 1 , B 1 , and C 1 ) form a triple helical bundle structural domain that is similar, but not identical, to previously published structures for spectrin from Drosophila and chicken brain. Paramagnetic spin label-induced NMR resonance broadening shows that helix C, the partial domain involved in ␣-and ␤-spectrin association, exhibits little interaction with the structural domain. Surprisingly, helix C is connected to helix A 1 of the structural domain by a segment of 7 residues (the junction region) that exhibits a flexible disordered conformation, in contrast to the predicted rigid helical structure. We suggest that the flexibility of this particular junction region may play an important role in modulating the association affinity of ␣-and ␤-spectrin at the tetramerization site of different isoforms, such as erythroid spectrin and brain spectrin. These findings may provide insight for explaining various physiological and pathological conditions that are a consequence of varying ␣-and ␤-subunit self-association affinities in their formation of the various spectrin tetramers.Spectrin, a member of the spectrin superfamily and a major protein in the membrane (cyto)skeleton, is ubiquitous among vertebrate tissues, as well as in simple metazoans, implying that spectrin plays a fundamental role in cells (1-3). After first being identified in erythrocytes (4), many distinct spectrin isoforms have since been discovered. In humans, two ␣-spectrin subunits (␣I and ␣II), four ␤-spectrin subunits (␤I, ␤II, ␤III, and ␤IV), and a ␤-H subunit have been sequenced (1). Similar isoforms in mice have also been studied (5). Alternative splicing (e.g. see Ref. 6) adds additional diversity among ␣-and ␤-spectrin isoforms. Many functions of different spectrin isoforms involve interactions with other molecules such as spectrinactin interaction, spectrin-membrane interaction, spectrin-ion channel interaction, etc. (1). Yet some of the most fundamental functions of spectrin involve spectrin "self-association." The activity of spectrin in stabilizing cell-cell contacts and in achieving normal columnar epithelial cell shape requires the formation of tetramers (1,7,8). Spectrin tetramers have been suggested to be cooperatively coupled to membrane assembly (9). Many hereditary hemolytic anemias involve single amino acid mutations in erythrocyte spectrin that destabilize its tetramers, resulting in low levels of spectrin tetramers and high levels of dimers (9 -11). Thus, the tetramerization site is an important functional site for most spectrins.In erythrocyte spectrin (␣I/␤I), ␣-and ␤-spectrin associate with relatively high affinity (nM K d values) at the N-terminal end of the ␤-spectrin and the C-terminal end of the ␣-spectrin (dimer nucleation site) to give ␣␤ heterodimers (12, 1...

Apparent structural differences at the tetramerization region of erythroid and nonerythroid beta spectrin as discriminated by phage displayed scFvs

“…[32][33][34]41,49 Spin label EPR studies provide the first experimental information on a local region of Helix B 0 in free bI-spectrin (not associated with aI-spectrin), with residues before 2071P in helical conformation (Helix B 0 ), and residues after 2071P in an unstructured conformation. 33,34 The predicted structure of free Helix B 0 of bI in this study also showed that 42 and bound 15,30,36 forms of a-spectrin have been observed experimentally, with the N-terminal junction region in aI-spectrin (residues 46-52) undergoing conformational changes, from unstructured to helical conformation, upon binding to the C-terminal fragment of b1-spectrin. 30,32 The conformation of the bI-Helix B 0 bound to G5 was predicted to consist of residues 2044-2068, a conformation different from the free form and a form in the presence of aI [ Fig.…”

“…15,16 Thus, the affinity differences in tetramer formation between spectrin I and II are mostly attributable to the conformational differences between aI and aII. 17,18,42 The bI and bII C-terminal partial domains (residues 2008-2083 in bI and 2016-2091 in bII) are considered to be similar both in function and in conformation. These two regions exhibit high sequence homology ($70% identity and 80% similarity).…”

“…Yet, the majority of the structural studies of spectrin are of its structural domains (e.g., 1AJ3 43 ; 2SPC 44 ; 3EDV 45 ; 3KBT/ 3KBU 32 ) or SH3 domain (2RMO 46 ; 2OAW 47 ; 1U06 48 ). For the tetramerization regions, many studies have focused on the N-terminal region of aI-and aII-spectrin, [15][16][17][18]30,31,42,48 but only a few have studied the Cterminal region of b-spectrin. [32][33][34]41,49 Spin label EPR studies provide the first experimental information on a local region of Helix B 0 in free bI-spectrin (not associated with aI-spectrin), with residues before 2071P in helical conformation (Helix B 0 ), and residues after 2071P in an unstructured conformation.…”

Important residue (G46) in erythroid spectrin tetramer formation

“…The sequences of all plasmids were confirmed by DNA sequencing (DNA Services Facility, Research Resources Center, University of Illinois at Chicago). Recombinant proteins were expressed and purified as before [6,9]. The protein molecular masses were determined with high resolution LTQ-FT mass spectrometry (Proteomics and Informatics Services Facility, Research Resources Center, University of Illinois at Chicago).…”

“…Several hereditary hemolytic anemia diseases, such as hereditary elliptocytosis and hereditary pyropoikilocytosis, are found to be related to mutations in this region to give lower levels of spectrin tetramers in erythrocytes [7,8]. High resolution solution NMR studies of αI-N1 show that the region commonly referred to as the N-terminal partial domain of SpαI consists of an unstructured region (residues [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] and Helix C' (residue 21-45) [9]. The first triple helical bundle structural domain consists of residues 52-156.…”