Localization and structure of the ankyrin-binding site on beta2-spectrin

Abstract

Spectrins are tetrameric actin-cross-linking proteins that form an elastic network, termed the membrane skeleton, on the cytoplasmic surface of cellular membranes. At the plasma membrane, the membrane skeleton provides essential support, preventing loss of membrane material to environmental shear stresses. The skeleton also controls the location, abundance, and activity of membrane proteins that are critical to cell and tissue function. The ability of the skeleton to modulate membrane stability and function requires adaptor proteins that bind the skeleton to membranes. The principal adaptors are the ankyrin proteins, which bind to the beta-subunit of spectrin and to the cytoplasmic domains of numerous integral membrane proteins. Here, we present the crystal structure of the ankyrin-binding domain of human beta2-spectrin at 1.95 A resolution together with mutagenesis data identifying the binding surface for ankyrins on beta2-spectrin.

FIGURE 1.

B14–16 region of the spectrin tetramer. The diagram shows the arrangement of α₂- and β₂-spectrins in the spectrin tetramer along with the domain organization of each subunit. α-Spectrin is colored yellow, and β-spectrin is colored green. Spectrin repeats are indicated by number. Shown in red are the spectrin repeats of β-spectrin that were crystallized. The Src homology 3 (SH3) domain in α₂-spectrin is an insert in spectrin repeat 10. EF refers to the EF-hand domains at the C terminus of α₂-spectrin, which bind to calcium. In β-spectrin, the CH motifs bind to actin, whereas the pleckstrin homology (PH) domain binds to lipids. The end-to-end association of α- and β-spectrin is mediated by partial spectrin repeats that form a three-helix bundle consisting of one helix from α-spectrin and two helices from β-spectrin.

FIGURE 2.

Representative electron density. Shown is the representative electron density near Tyr¹⁸⁷⁴ with the final model shown as sticks. Carbons are colored gray; nitrogens, blue; and oxygens, red. Waters are shown as red pluses. Electron density at 1σ is shown as a green wire network.

FIGURE 3.

Crystal structure of B14–16. Shown are orthogonal ribbon representations of the crystal structure of B14–16. Repeat 14 is colored tan; repeat 15, green; and repeat 16, blue. The linkers connecting repeats are colored purple.

FIGURE 4.

Repeat 15 has a unique hydrophobic core near Arg¹⁸²⁰. Most spectrin repeats have a characteristic tryptophan at position 17 of the A-helix that forms part of the hydrophobic core as exemplified in repeat 14 by Trp¹⁷¹³ (A). In repeat 15, Arg¹⁸²⁰ resides at position 17, and the hydrophobic core is centered on Leu¹⁸⁵⁶ from helix-15B (B).

FIGURE 5.

Inter-repeat contacts control orientation between repeats. Shown are ribbon representations of the inter-repeat junctions between repeats 14/15 (A) and repeats 15/16 (B) of β₂-spectrin and the inter-repeat junctions between repeats 15/16 (C) and repeats 16/17 (D) of α₂-spectrin. Linkers are colored dark blue in β₂-spectrin and orange in α₂-spectrin. Terminal residues of the inter-repeat contacts are labeled and shown as sticks.

FIGURE 6.

Tyr¹⁸⁷⁴ of loop-15BC is required for ankyrin binding to repeats 14–16 of β₂-spectrin. β₂-Spectrin variants with alanine mutations at 23 surface-exposed residues from helix-15B, loop-15BC, and helix-15C were screened for the ability to bind to the spectrin-binding domain (SBD) of ankyrin-B and ankyrin-G by yeast two-hybrid analyses. Shown in A is growth of yeast strains expressing the indicated spectrin variants fused to the Gal4 activation domain and the ankyrin-B spectrin-binding domain fused to the Gal4 DNA-binding domain on plates deficient for adenosine, histidine, leucine, and tryptophan. Similar results were observed in yeast strains expressing the spectrin variants together with the ankyrin-G spectrin-binding domain (yeast growth not shown). The location of individual mutations relative to secondary structure elements is shown in B along-side relative growth ability as indicated by ++ for strong growth and - for no growth. The location of the proposed ankyrin-binding site is indicated on the B14–16 structure in C.