Interactive Protein Tutorial

Sequence and structure of EF hands

The EF hand motif is present in a many proteins and it commonly bestows the ability to bind Ca²⁺ ions. It was first identified in parvalbumin, a muscle protein. Here we shall study the Ca²⁺-binding protein calmodulin which possesses four EF hands. Calmodulin and its isoform troponinC are important intracellular Ca²⁺-binding proteins.

The ball and stick structure on the right, obtained by X-ray crystallography, represents the Ca²⁺-binding protein calmodulin. It has a dumbell-shaped structure with two identical lobes connected by a central α helix. Each lobe comprises three a helices joined by loops. α helix-loop-helix motif forms the basis of each EF hand. Click on the the buttons below to examine this molecule in more detail.

Here are some questions that you should try to answer.

What forms of secondary structure does this protein possess?
Where are the EF hands located in calmodulin
Where are the amino acid residues that bind each Ca²⁺ ion?
What are the consequences of Ca²⁺-binding?

BACKBONE

Press the button to simplify this ball and stick model to show the backbone structure. Notice that helical regions can now be seen.

SECONDARY STRUCTURE

This is shown more clearly by a ribbon diagram. The computer calculates where regions of secondary structure occur and draws them as ribbons. The α-helical region is now clearly defined and there are also regions of β-structure.

The short anti-parallel β sheet between the adjacent EF hand loops are observed in calmodulins from various species.

The terminal helices are collapsed down concealing their hydrophobic surfaces and the central chain, which is not now a single α helix along its whole length, but is broken and bent, is not exposed.

CALCIUM IONS

In each EF hand loop the Ca²⁺ ions are bound by residues in and near the loops.

The structure shown has 4 Ca²⁺ ions bound. In this condition the protein adopts the extended structure shown. The EF hand-forming helices are bent away from the long linking helix, revealing hydrophobic residues and exposing the linking chain.

CO-ORDINATING RESIDUES

To illustrate how Ca²⁺ is bound, this display shows the residues that take part in binding one of the Ca²⁺ ions.

ZOOM

To see this more clearly.

CO-ORDINATING ATOMS

To highlight the atoms that co-ordinate the Ca²⁺ ion, the program now enlarges those that are close (within 2.7 Å). This shows that seven oxygen atoms form the calcium co-ordination shell. Five are contributed by side chains carboxyl groups of Asp and Glu and the sixth by the peptide carbonyl of Gln. The seventh is provided by a water molecule.

Notice the irregular structure of the co-ordination shell.This favours calcium binding over magnesium.

INACTIVE CALMODULIN

At resting levels of cytosol Ca²⁺ (~100 nM) calmodulin exists predominantly in the Ca²⁺ -free form. This is called apo-calmodulin and its structure is more compact.

The terminal helices are folded down concealing their hydrophobic surfaces and the central chain, which is not a helical along its whole length, is not exposed.

CALMODULIN INTERACTS WITH ITS TARGET

The Ca²⁺-bound form of calmodulin with its exposed hydrophobic surfaces that you have already observed can interact with a target protein. It does this by wrapping around a specific sequence on the target molecule, forcing it to adopt an a helical structure.

The target molecule here is the calmodulin-regulated enzyme myosin light chain kinase. Only a short sequence, the calmodulin binding domain, is shown.

MENUS

These displays were created by providing Jmol with a series of scripts (lists of commands). Many of these commands can be applied directly. Press the button under the JMol window to reset to ball and stick mode, then place the cursor over the display and press the right mouse button. See if you can display calmodulin as a ribbon structure with all its hetero-atoms in the spacefilled format.