Conformational Stabilities of the Structural Repeats of Erythroid Spectrin and Their Functional Implications

Xiuli An,Xinhua Guo,Xihui Zhang,Anthony J Baines,Gargi Debnath,Damali Moyo,Marcela Salomao,Nishant Bhasin,Colin Johnson,Dennis Discher,Walter B Gratzer,Narla Mohandas

doi:10.1074/jbc.m513725200

Abstract

The two polypeptide chains of the erythroid spectrin heterodimer contain between them 36 structural repeating modules, which can function as independently folding units. We have expressed all 36 and determined their thermal stabilities. These vary widely, with unfolding transition mid-points (T(m)) ranging from 21 to 72 degrees C. Eight of the isolated repeats are largely unfolded at physiological temperature. Constructs comprising two or more adjacent repeats show inter-repeat coupling with coupling free energies of several kcal mol(-1). Constructs comprising five successive repeats from the beta-chain displayed cooperativity and strong temperature dependence in forced unfolding by atomic force microscopy. Analysis of aligned sequences and molecular modeling suggests that high stability is conferred by large hydrophobic side chains at position e of the heptad hydrophobic repeats in the first helix of the three-helix bundle that makes up each repeat. This inference was borne out by the properties of mutants in which the critical residues have been replaced. The marginal stability of the tertiary structure at several points in the spectrin chains is moderated by energetic coupling with adjoining structural elements but may be expected to permit adaptation of the membrane to the large distortions that the red cell experiences in the circulation.

Highlights

Spectrin, an elongated flexible tetrameric molecule, defines the unusual shear resistance and elasticity of the red cell membrane [1, 2]
Successive spectrin repeats are joined by a continuous ␣-helix, of which the N-terminal half forms the C-terminal helix (C-helix) of the three-helix bundle of one repeat and the other half forms the first helix (A-helix) of the adjoining repeat; 5 residues in the center of the helix are not part of the repeating structure and are referred to as a “linker” region (9 –11)
To establish how repeats of low stability are distributed through the spectrin molecule, we have examined the thermal unfolding of all of the 36 repeats that make up the two chains of erythroid spectrin

Summary

Introduction

An elongated flexible tetrameric molecule, defines the unusual shear resistance and elasticity of the red cell membrane [1, 2]. The tetramers are made up of two ␣I␤I heterodimers, associated headto-head [3] Both the ␣ and the ␤ chain are characterized by a series of homologous repeating units of about 106 amino acids, each with the structure of a loose triple-stranded ␣-helical coiled-coil [4, 5]. Successive spectrin repeats are joined by a continuous ␣-helix, of which the N-terminal half forms the C-terminal helix (C-helix) of the three-helix bundle of one repeat and the other half forms the first helix (A-helix) of the adjoining repeat; 5 residues in the center of the helix are not part of the repeating structure and are referred to as a “linker” region (9 –11) It has been suggested [10, 11] that the flexibility of the spectrin chain under physiological conditions may be governed by kinks result-. Implications for the mechanical function of spectrin in the cell are considered

Methods

Results

Conclusion