Essay: Cation Binding Sites in Chicken Annexin V – Literature Review

Cation Binding Sites in Chicken Liver Annexin V [CLAV]

A triclinic Annexin V crystal illuminated by an X-ray beam reveals its four α-helical domains and glowing calcium-binding sites. The artwork honours the precision and artistry of 1993 crystallographic studies at the University of Leeds.

Author: Adisha Kariyawasam BSc Molecular Biophysics, MScIT, PGCE (PCET), BCS

Originally written 1992–1993, BSc (Hons) Molecular Biophysics, University of Leeds
Republished and expanded 2025

Preface (2025 Edition)

This literature review was written in 1992–1993, prior to embarking on my final-year dissertation project, during the concluding phase of my BSc (Hons) Molecular Biophysics degree at the University of Leeds. It formed the preparatory groundwork for experimental work undertaken later that academic year within the Department of Biophysics.

At the time, the field of calcium-binding proteins was rapidly expanding. Researchers were discovering how divalent cations such as Ca²⁺ acted not merely as cofactors but as dynamic regulators of cellular communication, membrane fusion, and blood coagulation. This essay reflects the early stages of structural calcium biology that has since evolved into a major interdisciplinary research area, bridging molecular biophysics, physiology, and medical science.

Cation Binding Sites in Chicken Liver Annexin V [CLAV]

Introduction

Calcium ions (Ca²⁺) are essential to life, serving structural, catalytic, and regulatory functions. They stabilise extracellular matrices, act as intracellular messengers, and modulate enzyme activity. In vertebrates, cytosolic Ca²⁺ concentrations are tightly controlled at approximately 10⁻⁷ M, with transient spikes conveying signalling information.

Among the most intriguing Ca²⁺-binding proteins are the Annexins, a family of calcium-dependent phospholipid-binding proteins that play roles in membrane fusion, inflammation control, and anticoagulation.

Calcium Binding in General and in Proteins

In many biomolecules, Ca²⁺ is coordinated by oxygen atoms arranged in a pentagonal bipyramidal geometry with average Ca–O distances of 2.4 Å. Slight displacements of ligands can yield an octahedral coordination, accommodating other divalent cations such as Mg²⁺.

Most calcium-binding proteins exhibit a coordination number of seven, using oxygen atoms from carboxylate side chains (Asp, Glu), carbonyl groups, and water molecules. These binding geometries define the selectivity and flexibility of calcium interactions in biological systems.

Classes of Calcium-Binding Proteins

Three main classes of Ca²⁺-binding proteins can be distinguished:

1. Extracellular enzymes and structural proteins, where calcium enhances thermal stability or protects against proteolysis.

2. Intracellular regulatory proteins, which bind Ca²⁺ reversibly to modulate enzyme activity. These typically feature repeating motifs such as the EF-hand, a helix–loop–helix structure found in calmodulin, troponin C, and parvalbumin.

3. Annexins, a unique family of amphipathic proteins that bind phospholipids in a calcium-dependent manner but lack the EF-hand motif.

The EF-Hand Motif

The EF-hand, named for its E and F helices, consists of a 12-residue loop flanked by α-helices. Side-chain oxygens from residues such as Asp, Ser, and Thr coordinate a single Ca²⁺ ion, while backbone carbonyls contribute additional ligands. The motif usually occurs in pairs related by a pseudo-twofold symmetry axis, forming cooperative Ca²⁺-binding sites.

Although Annexins lack this structure, understanding the EF-hand provides a comparative framework for interpreting their calcium-binding behaviour.

Annexins: Structure and Characteristics

Annexins are acidic, calcium-dependent proteins that bind to negatively charged phospholipid membranes. They share a conserved structural core composed of four homologous domains, each containing five α-helices (A–E).

Their canonical calcium-binding motif is distinct from the EF-hand, typically represented as:
–K–G–X–G–T–(38 residues)–D/E–,
where X can be any amino acid.

Binding occurs predominantly at loop regions between helices, particularly within domains I, II, and IV. Upon calcium binding, the protein undergoes conformational changes that facilitate membrane association.

Chicken Annexin V

The crystal structure of chicken Annexin V (also known as anchorin CII) was solved to 2.54 Å resolution in 1992. The protein, with a molecular weight of 36 kDa and 320 amino acids, exhibits a bowl-shaped configuration with its convex surface exposed to solvent and the concave surface interacting with membranes.

Domains I & IV and II & III form tightly associated pairs, yet can slide relative to one another in the plane of the membrane. Both the N- and C-termini reside within domain IV. The overall molecular dimensions are approximately 64 Å × 40 Å.

Only the loop regions of domains I, II, and IV contain confirmed Ca²⁺-binding sites, primarily coordinated by carboxylate side chains from Asp and Glu residues.

Predicted Lanthanum Binding Sites

In 1990, Robert Huber and colleagues determined the structure of human Annexin V, revealing two additional cation-binding sites when crystals were soaked in lanthanum nitrate. Surprisingly, the crystals initially cracked but reformed within half an hour, suggesting reversible structural accommodation.

Given the high sequence homology (≈ 78 %) between human and chicken Annexin V, it was reasonable to predict analogous lanthanum-binding sites in the avian protein, located between helices A–B and C–D of domain I.

Unlike the canonical calcium-binding sites, these lanthanum positions lacked full coordination shielding, suggesting partial hydration and weaker binding affinity. Nonetheless, they hinted at potential regulatory or allosteric roles for non-physiological cations in crystal packing and structural flexibility.

Proposed Experimental Approach (1993 Project Plan)

The planned experimental work involved soaking trigonal crystals of chicken Annexin V in lanthanum nitrate and collecting X-ray diffraction data using a Xentronics Area Detector. Structural differences would be determined by calculating difference electron-density maps (Fₒ – Fₙ), phased with the native structure.

Each crystal measured only around 0.17 mm in width and had to be graded for optical clarity and absence of twinning before mounting. The manipulation of these fragile crystals demanded exceptional dexterity; in many cases, they were transferred into narrow 0.2mm quartz capillary tubes using a single human eyelash affixed to a matchstick - a traditional crystallographer’s tool of remarkable delicacy.

The resulting diffraction data were expected to reveal lanthanum-binding positions and any local conformational changes. It was hypothesised that crystal cracking and reformation would not permanently disrupt the protein’s tertiary architecture.

Computational Visualisation of 3D Structures (1992–1993 Context)

During the early 1990s, the interpretation of macromolecular structures relied heavily on stereoscopic computer graphics workstations. At the University of Leeds and other leading biophysics centres, crystallographers used Silicon Graphics (SGI) Indigo and Personal IRIS systems, as well as Evans & Sutherland PS300 vector graphics terminals, to visualise and manipulate protein models derived from X-ray diffraction data.

Molecular coordinates, refined using programs such as PROLSQ and X-PLOR, were rendered as wireframe or ribbon representations that could be viewed stereoscopically using polarised glasses or dual-screen mirror systems. These tools allowed researchers to inspect electron-density maps interactively, adjust atomic models in real time, and identify metal-binding geometries with unprecedented precision.

At Leeds, such systems were connected via VAX/VMS and DECstation networks, running early versions of FRODO, O, and TOM molecular modelling software. For many students, these platforms provided their first encounter with immersive molecular visualisation - a transformative experience that turned static diffraction data into tangible, three-dimensional molecular landscapes.

“To visualise a protein structure in three dimensions at that time required both patience and precision - each movement of a carbonyl group was adjusted manually with a trackball or dial box on a Silicon Graphics terminal, viewed through twin polarised displays that brought the molecular world to life.”

Significance of Cation Binding in Annexins

Cation binding in Annexins is crucial for their role in membrane dynamics. Calcium bridges acidic residues to phospholipid head groups, promoting adhesion and curvature stabilisation. This underlies functions such as exocytosis, endocytosis, and anticoagulant activity.

Lanthanum, with its larger ionic radius and higher charge density, serves as an experimental analogue that helps visualise these binding interactions crystalographically.

Such studies not only enhance understanding of Annexin function but also contribute to broader insights into calcium signalling, membrane repair, and protein–lipid interactions.

Conclusion

The study of cation binding in Annexin V highlights how small ions can govern large-scale biological phenomena. The interplay between metal coordination, protein conformation, and membrane interaction exemplifies structural biology’s power to unify chemical and physiological perspectives.

From these early crystallographic investigations emerged principles that now inform modern calcium-signalling biology and the development of biomimetic materials and medical diagnostics.

Afterword (2025 Reflection)

Since 1993, the Annexin family has grown to include over a dozen identified members, each with specific cellular functions ranging from apoptosis to vesicle trafficking. Advances in cryo-electron microscopy, molecular dynamics, and calcium imaging have validated many of the predictions first explored in this essay.

Annexin V is now widely used in medical diagnostics as a marker for early apoptosis through its selective binding to phosphatidylserine—a remarkable translation of biophysical insight into clinical practice.

The questions first posed in this Leeds project continue to resonate in modern structural biology: how do ions, proteins, and membranes coordinate to produce life’s most fundamental movements?

References

• Bewley, M.C., Boustead, C., Walker, J.H., Waller, D.A. and Huber, R. (1992) ‘Crystal structure of chicken Annexin V,’ Unpublished research paper, University of Leeds and Max-Planck Institute.

• Huber, R., et al. (1990) ‘Structure of human Annexin V and identification of lanthanum-binding sites,’ The EMBO Journal, 9(12), pp. 3867–3874.

• Huber, R., et al. (1990) ‘Lanthanum-binding and crystal rearrangement in Annexin V,’ FEBS Letters, 275(1–2), pp. 15–21.

• Huber, R., et al. (1992) ‘Annexin structures and functions,’ Journal of Molecular Biology, 223, pp. 683–704.

• Kretsinger, R.H. (1987) ‘Calcium-binding proteins,’ Cold Spring Harbor Symposia on Quantitative Biology, 52, pp. 499–510.

• Strynadka, N.C.J. and James, M.N.G. (1989) ‘Structural aspects of calcium binding in proteins,’ Annual Review of Biochemistry, 58, pp. 951–980.

• Swain, A.L., Kretsinger, R.H. and Amma, E.L. (1989) ‘Calcium coordination geometries,’ Journal of Biological Chemistry, 264(28), pp. 16620–16628.

• Voet, D. and Voet, J.G. (1990) Biochemistry. New York: Wiley.

• Walker, J.H., et al. (1992) ‘Phospholipid-binding properties of Annexin V,’ Biochemical Society Transactions, 20, pp. 828–833.

Appendix 1 – Glossary of Key Terms

Term	Definition
Annexin	A family of calcium-dependent phospholipid-binding proteins involved in membrane dynamics and signalling.
Cation	A positively charged ion, such as Ca²⁺ or La³⁺, which can form coordinate bonds with proteins.
Coordination Geometry	Spatial arrangement of atoms or ligands around a central metal ion.
EF-Hand	Helix–loop–helix motif common in calcium-binding proteins.
Electron-Density Map	A 3D representation of electron distribution used in crystallography to model atomic positions.
Lanthanum (La³⁺)	A trivalent rare-earth metal used as a calcium analogue in structural studies.
Phospholipid	A lipid containing a phosphate group, forming the bilayer of cell membranes.
Resolution (Ångström)	Measure of clarity in X-ray crystallographic data; 1 Å = 10⁻¹⁰ m.
Xentronics Area Detector	Early electronic imaging detector used to record X-ray diffraction patterns.
X-ray Crystallography	Technique used to determine atomic structures of macromolecules by analysing diffraction patterns from crystals.

Appendix 2 – Short Biographies of Scientists Mentioned (Alphabetical by Surname)

Amma, E.L.

American crystallographer known for co-authoring studies on calcium-binding coordination with Kretsinger and Swain. Her structural analyses provided foundational understanding of protein–ion interactions.

Bewley, Maria C.

British structural biologist and crystallographer based at the University of Leeds in the early 1990s. Dr Maria C. Bewley was the principal author of the 1992 study “Crystal Structure of Chicken Annexin V”, completed in collaboration with Christopher Boustead, John H. Walker, David A. Waller, and Robert Huber. Her work contributed significantly to understanding calcium-dependent phospholipid-binding proteins and provided one of the earliest high-resolution structures of an Annexin family member.

Huber, Robert (born 1937)

German biochemist awarded the 1988 Nobel Prize in Chemistry for work on protein crystallography. His research on Annexins provided some of the first structural insights into calcium-dependent membrane binding.

James, M.N.G. (born 1937)

Canadian structural biologist whose research focused on protease and calcium-binding enzyme mechanisms. Co-author of several key reviews on calcium-binding proteins.

Kretsinger, Robert H. (born 1937)

American biochemist who discovered the EF-hand motif, a defining structural feature of calcium-binding proteins. His research clarified the geometric basis of calcium coordination in biological systems.

Strynadka, Natalie C.J. (born 1966)

Canadian structural biologist who worked with James on the crystallographic elucidation of enzyme and calcium-binding structures, later pioneering structural studies of membrane proteins.

Swain, Alan L.

Co-researcher with Kretsinger and Amma, contributing to quantitative analyses of calcium-binding site geometries using X-ray diffraction.

Voet, Donald (born 1941) and Voet, Judith G. (born 1943)

American biochemists and co-authors of Biochemistry, one of the most influential textbooks integrating molecular structure with biological function.

Walker, John H.

British biochemist based at the University of Leeds whose research focused on membrane-associated proteins, especially Annexins and phospholipid-binding dynamics.

Waller, David A.

Leeds-based structural biologist and co-author of studies on Annexin V crystal structure and membrane-binding behaviour.

Disclaimer

The views, interpretations, and reflections expressed in this essay are those of the author and do not necessarily represent the positions of the University of Leeds or any affiliated institution.