Keywords

Spectrin, Coiled-Coil, Scaffold, Flexibility, Cytoskeleton

Reference

DOI: https://doi.org/10.1016/s0092-8674(00)81980-7

Abstract

Spectrin, a key component of the cytoskeleton, provides flexibility and scaffolding for various proteins. Composed of tandem, antiparallel coiled-coil repeats, spectrin shapes and stabilizes the cell membrane.
This study reports four related crystal structures (1.45 Å, 2.0 Å, 3.1 Å, 4.0 Å resolution) of two connected α-spectrin repeats. Notably, the linker region between repeats is α-helical without breaks, challenging prior assumptions about flexibility mechanisms.
Two main structural features underpin models for flexibility: (1) conformational rearrangement within repeats and (2) variable bending at the linker region. These findings yield atomic-level insights into spectrin flexibility, essential for its role in the reversible deformation of cell membranes.

Notes

1. General Summary

  • Spectrin consists of α (280 kDa) and β (246 kDa) subunits, assembling into tetramers via head-to-head and side-by-side interactions.
  • Each spectrin monomer contains ~20 α-repeats and 17 β-repeats, featuring triple-helical coiled-coil motifs with heptad patterns.
  • Linker regions between repeats were hypothesized to be flexible, but lacked structural confirmation.
  • Spectrin’s network provides elasticity to red blood cells, enabling deformation through capillaries.
  • PDB: 3F31 provides a structural reference for α-spectrin repeats.

2. Methodological Approach

  • Crystal structures were solved for two connected repeats of chicken brain α-spectrin, at resolutions from 1.45 Å to 4.0 Å.
  • 2U0 model partially refined due to poor diffraction, with polyalanine modeling for unclear regions.
  • Heptad patterns, loops, and linkers were analyzed to reveal coiled-coil architecture and flexibility mechanisms.

3. Core Findings and Insights

  • 2U0 contains two antiparallel coiled coils connected by a continuous α-helix linker, residues 1872–1876, previously predicted as non-helical.
  • Each repeat features helices A, B, C (or A’, B’, C’), with longer AB/A’B’ loops (9 residues) compared to BC loops (4–5 residues).
  • Heptad pattern disrupted at C–A’ linker, creating a boundary between repeats.
  • R16 includes a “stammer” in helix B, adjusting from three- to two-helix coiled coil.
  • Charged residues like Arg and Lys can fulfill hydrophobic roles in heptads.
  • Dimensions: width ≈ 20 Å, length ≈ 100 Å, with ~50 Å translation and 28°–52° rotation between repeats (R16 and R17).

4. Two Flexibility Models

A. Conformational Rearrangement Model

  • Shifting of the B’C’ loop in R17 elongates helix B’ and shortens helix C’.
  • Causes sliding of repeats past one another, enabling chain shortening/lengthening.
  • Although a single repeat’s change is minor, coordinated shifts across repeats could dramatically adjust spectrin length.


Figure: a pic from the paper to show the model. (Click to enlarge)

B. Linker Bending Model

  • Linker as ball-and-socket hinge, allowing multi-directional rotation.
  • Crystal structures show variable orientations, suggesting twists and tilts between repeats.
  • Inter-repeat alignments require complex, flexible interfaces, supporting membrane deformation capacity.

5. Stability of Repeats

  • Single repeat (R16+R17) can form stable units through coordinated linker and loop interactions.
  • Proper phasing of helices A and C allows contact between BC and AB loops, stabilizing single or double repeats.
  • Double repeats (like 2U0) feature four loop contacts, while single engineered repeats could achieve stability with two contacts.
  • Suggests possible engineering of highly stable spectrin modules for synthetic scaffolds.

6. RD’s Thoughts and Learnings

  • RD loves the writing style — especially phrasing like “as is customary…”, lending elegance and clarity to complex structural descriptions.
  • Fascinated by continuous α-helix linker, which defies prior predictions of flexible/disordered regions.
  • Models of flexibility (conformational rearrangement and linker bending) provide a mechanistic explanation for spectrin’s elastic properties.
  • Charged residues filling hydrophobic heptad roles — a novel insight into sequence-to-structure adaptation.
  • Engineering implications: Potential to design synthetic flexible scaffolds inspired by spectrin repeat modularity.
  • Intrigued by the ability to generate stable single repeats, mimicking double repeat stability — could this inform design of artificial cytoskeletal elements?

Take-home Messages

  • Spectrin’s flexibility arises from conformational rearrangement within repeats and linker bending between repeats.
  • Two connected repeats (R16 + R17) solved at high resolution reveal continuous α-helical linkers, challenging the notion of “unstructured” linkers.
  • Models of flexibility:
    • Conformational shift model: sliding repeats modulate spectrin length.
    • Linker hinge model: rotation/tilting between repeats.
  • Structural insights explain how spectrin forms elastic, reversible networks under the membrane, enabling cell shape changes.
  • The structural data redefine our understanding of spectrin’s mechanical properties, integrating atomic detail into models of cellular flexibility.

RD absolutely loves the elegance and depth of this paper — a masterclass in structural biology! 🧬✨