Mutational effects at the tetramerization site of nonerythroid alpha spectrin

Abstract

Spectrin, a prominent cytoskeletal protein, exerts its fundamental role in cellular function by forming a sub-membrane filamentous network. An essential aspect of spectrin network formation is the tetramerization of spectrin αβ heterodimers. We used laboratory methods, the yeast two-hybrid system and random mutagenesis, to investigate, for the first time, effects of amino acid mutations on tetramerization of nonerythroid (brain) spectrin (fodrin). Based on high sequence homology with erythroid spectrin, we assume the putative tetramerization region of nonerythroid α-spectrin at the N-terminal region. We introduced mutations in the region consisting of residues 1–45 and studied mutational effects on spectrin αβ association to form tetramers. We detected single, double, and triple mutations involving 24 residues in this region. These amino acid mutations of nonerythroid α-spectrin exhibit full, partial, or no effect on the association with nonerythroid β-spectrin. Single amino acid mutations in the region of residues 1–9 (D2Y, G5V, V6D, and V8M) did not affect the association. However, seven single mutations (I15F, I15N, R18G, V22D, R25P, Y26N, and R28P) affected the αβ association. These mutations were clustered in the region predicted by sequence alignment to be crucial in nonerythroid α-spectrin for tetramerization, a region that spanned residues 12–36, corresponding to the partial domain Helix C′ (residues 21–45) in erythroid α-spectrin. In addition, two other mutations, one upstream and one downstream of this region at positions 10 (E10D) and 37 (R37P), also affected the αβ association. Our results implied nonerythroid α-spectrin partial domain helix may be longer than Helix C′ (residues 21–45 and a total of 25 residues) in erythroid α-spectrin and spanned at least residues 10–37. It is interesting to note that seven out of these nine single mutations (I15F, I15N, R18G, V22D, R25P, Y26N, R37P) were at the a, d, e or g heptad positions based on sequence alignment with erythroid α-spectrin. Four of the mutated residues (I15, R18, V22, R25) are conserved in both erythroid and nonerythroid spectrin. These positions were previously identified as hot spots in erythroid α-spectrin that lead to severe hematological symptoms. This study clearly demonstrated that single mutation in a region predicted to be critical functionally in nonerythroid α-spectrin indeed leads to functional abnormalities and may lead to neurological disorders.