Calcium binding proteins in malaria

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

k_AB k_BA

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