2 - Calmodulin Flashcards
The EF hand
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.
motif
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.
Kd (M)
a measure of the affinity between two molecules, dissociation constant
P + L => PL (K1)
PL =>P + L (K2)
Kd = K2/K1
what is K1
association rate constant
What is K2
dissociation rate constant
Kd basics
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
EF hands bind calcium
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
Background to calcium ion concentrations
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
Key calcium binding residues
Aspartate and glutamate
Acidic amino acids have carboxylate oxygen ligands.
The basic structure
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
Residues that bind Ca2+
Acidic amino acids have carboxylate oxygen atoms that can ligate calcium.
Glycine found at position 15 permits a sharp bend.
Calmodulin
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
Ca2+ and EF hand motif
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.
Structure of calmodulin
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.
apo
An “apo” structure is missing its ligand or binding partner
Calcium binding triggers a conformational change
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.
what does the conformational change expose
Hydrophobic patches which allow interactions with other protein
What can hydrophobic patches do?
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.
Target enzymes
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.
