2 - Calmodulin

Question 1

The EF hand

Answer 1

A protein motif that is associated with Ca2+ binding.
It was named from the Ca2+ binding site occurring between the E
and F a-helices in parvalbumin.
The EF hand is characterised by a HELIX-LOOP-HELIX configuration.
The name is derived from the resemblance of the motif to a
clenched right hand.

Question 2

motif

Answer 2

a conserved amino acid sequence alignment: it is a local alignment corresponding to a region whose function or structure is known, or its significance may be unknown.

Question 3

Kd (M)

Answer 3

a measure of the affinity between two molecules, dissociation constant
P + L => PL (K1)
PL =>P + L (K2)
Kd = K2/K1

Question 4

what is K1

Answer 4

association rate constant

Question 5

What is K2

Answer 5

dissociation rate constant

Question 6

Kd basics

Answer 6

The Kd is the concentration at which 50% of the ligand is free and 50% is bound to a receptor.
Ligand conc > Kd = most of the ligand is bound to the receptor
Ligand conc < Kd = most of the ligand is free
If the Kd is low then the affinity of the ligand for the receptor is high

Question 7

EF hands bind calcium

Answer 7

There are a very large number of EF-hand containing proteins and members of this superfamily are found solely in the cytosol.
They bind calcium with an affinity of Kd~ 10-6 M

Question 8

Background to calcium ion concentrations

Answer 8

There are free Ca2+ ions at a low concentration in an unstimulated cell, as Ca2+ concentration is lower than EF hand Kd.
Following cell stimulation Ca2+ concentration rises to reach the EF hand Kd and so is now bound by EF-hand containing proteins

Question 9

Key calcium binding residues

Answer 9

Aspartate and glutamate

Acidic amino acids have carboxylate oxygen ligands.

Question 10

The basic structure

Answer 10

The EF-hand contains 29 residues.
The first a-helix has a Glu at position 1.
It also has hydrophobic residues facing the core of the molecule at positions 2, 5, 6 & 9
The second a-helix has a Glu at position 21 and residues 22, 25, 26 and 29 are hydrophobic

Question 11

Residues that bind Ca2+

Answer 11

Acidic amino acids have carboxylate oxygen atoms that can ligate calcium.
Glycine found at position 15 permits a sharp bend.

Question 12

Calmodulin

Answer 12

17 kDa protein with a highly conserved amino acid sequence which contains 4 EF hands.
Following calcium binding it changes shape and is able to bind to and activate kinases

Question 13

Ca2+ and EF hand motif

Answer 13

Ca2+ is an important secondary messenger involved in muscle
contraction
It controls release of hormones and neurotransmitters.
It is involved in binding of carbohydrates by lectins.
Many Ca2+-binding proteins contain an EF hand.
Calmodulin is one of the main effectors of Ca2+ signalling.

Question 14

Structure of calmodulin

Answer 14

The molecule has a dumbbell shape.
The N- and C-terminal domains each have 2 EF hands (EF1–4) separated by a unique 6-turn single a-helix.
EF hands generally occur in pairs within globular domains and there is cooperative calcium binding.

Question 15

apo

Answer 15

An “apo” structure is missing its ligand or binding partner

Question 16

Calcium binding triggers a conformational change

Answer 16

When 4 calcium ions bind to calmodulin, a conformational change is triggered that exposes a hydrophobic patch in each globular domain.
The molecule folds at this central region (Ser81) to expose this hydrophobic patch.

Question 17

what does the conformational change expose

Answer 17

Hydrophobic patches which allow interactions with other protein

Question 18

What can hydrophobic patches do?

Answer 18

bind target peptides
The long helix of the dumb-bell unwinds and the two lobes of calmodulin swing around to enfold the a-helical target peptide.
A hydrophobic patch on each lobe of the calmodulin contacts the hydrophobic side of the helical target peptide.
Thus, the target peptide sits in a hydrophobic channel.

Question 19

Target enzymes

Answer 19

Phosphorylase kinase, myosin light chain kinase, adenylate cyclases and the Ca2+ ATP-ase.
Target enzymes have a mixture of basic and hydrophobic amino acids which are capable of adopting an a-helix.