Calcium binding proteins in malaria and heart disease
Patrik Lundström
Department of Physics, Chemistry and Biology, Linköping University
patlu@ifm.liu.se
Vetenskapsdag 2016-10-06
Outline
• Nuclear magnetic resonance (NMR) spectroscopy as a method for detailed studies of proteins
• Regulation of the malaria protein CDPK3
• Involvement of calmodulin in long QT syndrome (LQTS)
Atomic nuclei outside of a magnetic field
Atomic nuclei in a magnetic field
Magnetization vector
Magnetization vector after 90° pulse
U The rotating magneti- zation induces a vol- tage in a coil
Fourier transformation
The NMR signal and the corresponding spectrum:
The NMR spectrum of a protein reports on its structure
Each peak represents one pair of atoms!
The positions of the peaks report on protein structure!
Examples of protein structures
Protein function depends on structure!
Interaction with drugs
• Movement of peaks reports on
binding of drugs to particular regions
• The affinity can be determined
Binding of the drug MTX (used in cancer therapy) to the protein TPMT
Information content
Thermodynamics: populations Kinetics: Rate constants
Structure: Chemical shifts
A B
kAB kBA
visible ground state invisible “excited” state
Line shapes report on protein dynamics
Analysis of line shapes can be used to obtain information on a protein
exchanging between a ground state (A) and a low-populated state (B)
The NMR spectrometer & sample
The NMR spectrometer comprises a very strong magnet and equipment for delivering radio frequency radiation
The NMR sample consists of a few mg protein dissolved in 0.5 ml buffer
Malaria
The malaria parasite P. falciparum
Distribution
The mosquito A. stephensi
The principal victim
The life cycle of the malaria parasite
The malaria parasite requires both a human and a mosquito host!
The life cycle consists of both
asexual and sexual stages.
Calcium dependent protein kinases (CDPKs)
Unstructured Kinase domain Autoinhibitory CLD(A) CLD(B)
• Are restricted to plants and certain parasites
• Comprise a kinase domain, an autoinhibitory helix and two calcium ligating EF- hand domains
• Are activated by calcium
Inactive Active
CLD(A) CLD(B) CLD(A) CLD(B) The focus of my study
Expression and purification of CLD(A) and CLD(B)
Expression in E. coli IMAC
Purification based on Ni2+ affinity
Gel filtration
Purification based on size
SDS-PAGE
Check of purity
CLD(A) and CLD(B) are well-folded and are able to bind calcium
CLD(A) without Ca2+ CLD(A) with Ca2+ CLD(B)* with Ca2+
*It was impossible to obtain Ca2+-free CLD(B)
The structures of CLD(A) and CLD(B)
Calcium binding to CLD(A) is extremely weak
Increasing [Ca2+]
Kd = 350 μM (extremely weak)
CLD(B) forms dimers in solution
Ca2+ -CLD(A)(Ca2+ ) 2-CLD(B)
Analysis of how fast magnetization decays allows calculation of effective molecular size
CLD(A) also has an alternative structure
Blue areas indicate regions with alterna- tive structures
The dynamics and alternative structure may be important for substrate recognition Distribution of rates
Conclusions CDPK3
• CLD(B) does not tolerate absence of calcium always calcium bound
• Binding of calcium to CLD(A) activates CDPK3
• Extreme levels of calcium are necessary for activation
• CLD(B) interacts with the regulatory helix even at low calcium levels
• CLD(A) adopts alternative conformations that may be important for interactions
Inactive Active
Inactive
Long QT syndrome
LQTS: LQ period >400 ms
• Life-threatening arrhythmias
• Median lifespan: 34 years
• Triggered by exercise, emotional upset and sleep
The underlying cause of LQTS
Common ion channels Regulation ion channels by calmodulin
Mutations in calmodulin may explain dysregulation of ion channels
Calmodulin changes structure when binding calcium
L: Calmodulin without calcium M: Calmodulin with calcium
R: Calmodulin activating a protein
Ca2+-bound calmodulin without and with substrate
Details of calcium binding to calmodulin and variants
Several conserved amino acids are required to bind calcium properly.
Calcium binding to variants F141L reduced 50 %
D95V reduced 70 % D129G reduced 90 % We are studying the following calmodulin mutants involved in LQTS: D95V, D129G and F141L
NMR spectra are similar but not identical for variants
Without calcium With calcium
WT D95V WT D95V
D129G F141L D129G F141L
Structures of variants show interesting differences
Correlation of peak positions between variant and native calmodulin.
without calcium
with calcium Correlation of peak positions between
calcium-free and calcium-bound calmodulin.
WT D95V
D129G F141L
D95V D129G F141L
Mutated loops are not able to bind calcium
Expected peaks in this area:
G98 & G134
Observed peaks:
F141L: G98 & G134 D95V: G134
D129G: G98
Variant calmodulin remains monomeric and highly dynamic at both low and high calcium levels
Without calcium With calcium
WT
D95V
D129G
F141L
Dynamics differ between variants
Without calcium With calcium
Structure and dynamics at the surface for
F141L differs from native calmodulin
Conclusions: Calmodulin in LQTS
• Calcium binding is severely impaired for variants D95V and D129G
• Variant D129G does not change its structure in response to calcium
• Dimerization of variants is not the cause of LQTS
• Variants are more dynamic both with and without calcium
• D95V “freezes” at intermediate calcium levels
• The surface of F141L is has different structure and dynamics than native calmodulin
WT
D129G
D95V
F141L
Without calcium With calcium Without calcium With some calcium With calcium
Without calcium With calcium Without calcium With calcium
Dynamic Less dynamic
Very dynamic Less dynamic
Surface is more dynamic
Very dynamic Not dynamic Very dynamic
Very dynamic
Unchanged structure Very dynamic
Same but different…
Thanks you:
Cecilia Andresen Markus Niklasson Sofie Cassman-Eklöf
Björn Wallner
Christine Dyrager
Magdalena Svenssen
Cecilia Markus