Dynamic molecular modeling of pathogenic mutations in the spectrin self-association domain

Abstract

Disruption of spectrin self-association underlies many inherited hemolytic disorders. Using dynamic modeling and energy minimization, the 3-dimensional structure of the self-association domain has been estimated in human erythrocyte spectrin and the structural consequences of 17 elliptogenic mutations determined. The predicted structure of the normal self-association domain was remarkably similar to the crystal structure of the Drosophila ␣-spectrin 14th repeat unit, despite replacement in the human sequence of over 70% of the amino acids relative to fly spectrin, including 2 prolines in the human sequence that appear in helical regions of the fly structure. The predicted structure placed all hydrophilic residues at the surface and identified 4 salt bridges, 9 hydrophobic interactions, and 4 H-bonds that stabilize the native self-association unit. Remarkably, every pathologic point mutation, including seemingly conservative substitutions such as G for A, A for V, or K for R (single-letter amino acid codes), led to conformational rearrangements in the predicted structure. The degree of structural disruption, as measured by root-meansquare deviation of the predicted backbone structure from the Drosophila structure, correlated strongly with the severity of clinical disease associated with each mutation. This approach thus enables an accurate prediction, from the primary sequence, of the clinical consequences of specific point mutations in spectrin.

Interaction between protein 4.1R and spectrin heterodimers

“…Thus, spectrins play a central role in establishing the organization of the membrane skeleton (2,12). Mutations in spectrins lead to clinically significant forms of hereditary elliptocytosis and hereditary pyropoikilocytosis (13). Spectrin tetramers associated in a hexagonal array connect the membrane via junctional complexes by interacting with actin filaments, protein 4.1R, and glycophorin C, among which 4.1R plays a crucial role in regulating the junctional complexes by stabilizing the spectrin-actin network and anchoring it to the membrane (14).…”

Mutation of a highly conserved isoleucine disrupts hydrophobic interactions in the αβ spectrin self-association binding site

“…As noted by Zhang et al, 18 even though heterozygous a-spectrin self-association binding site mutations are among those most commonly found in HE and HPP patients, homozygous mutations of the a-spectrin self-association site have not been found, perhaps because complete disruption of spectrin self-association is incompatible with life. The significant disruption of hydrophobic interactions in the core of the ab spectrin repeat caused by the spectrin Los Angeles mutation Figure 3 Dynamic molecular modeling of the structural effects of the spectrin Los Angeles mutation.…”

“…28 In general, interactions of helix C with helices A and B provide stability to the spectrin self-association unit. 18 In previous studies, comparison of the degree of predicted structural disruption in the self-association site with clinical severity revealed a strong correlationr. 18 The degree of structural deviation, as derived by the root-mean-square deviation from the predicted spectrin backbone, for the Ile24Thr spectrin Los Angeles mutation was 3.296 Angstroms (normal 2.846 Å ).…”

“…18 In previous studies, comparison of the degree of predicted structural disruption in the self-association site with clinical severity revealed a strong correlationr. 18 The degree of structural deviation, as derived by the root-mean-square deviation from the predicted spectrin backbone, for the Ile24Thr spectrin Los Angeles mutation was 3.296 Angstroms (normal 2.846 Å ). Thus, the predicted structural disruption of the spectrin Los Angeles mutation correlates with the clinical severity observed.…”

“…18 These computations place the starting structure of the spectrin self-association domain in a 6-Å water shell that contains B11 1000 water molecules. The application of this approach and its fidelity has been described.…”