Calcium binding proteins in malaria and heart disease

Transcription

1 Calcium binding proteins in malaria and heart disease Patrik Lundström Department of Physics, Chemistry and Biology, Linköping University Vetenskapsdag

2 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)

3 Atomic nuclei outside of a magnetic field Atomic nuclei in a magnetic field Magnetization vector Magnetization vector after 90 pulse The rotating magnetization induces a voltage in a coil U Fourier transformation The NMR signal and the corresponding spectrum:

4 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!

5 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

6 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) visible ground state A k AB k BA invisible excited state B Information content Thermodynamics: populations Kinetics: Rate constants Structure: Chemical shifts

7 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

8 Malaria The malaria parasite P. falciparum The mosquito A. stephensi The principal victim Distribution

9 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.

10 Calcium dependent protein kinases (CDPKs) Are restricted to plants and certain parasites Comprise a kinase domain, an autoinhibitory helix and two calcium ligating EFhand domains Inactive Active Are activated by calcium Unstructured Kinase domain Autoinhibitory CLD(A) CLD(B) CLD(A) CLD(B) CLD(A) CLD(B) The focus of my study

11 Expression and purification of CLD(A) and CLD(B) Expression in E. coli IMAC Purification based on Ni 2+ affinity Gel filtration Purification based on size SDS-PAGE Check of purity

12 CLD(A) and CLD(B) are well-folded and are able to bind calcium CLD(A) without Ca 2+ CLD(A) with Ca 2+ CLD(B)* with Ca 2+ *It was impossible to obtain Ca 2+ -free CLD(B)

13 The structures of CLD(A) and CLD(B)

14 Calcium binding to CLD(A) is extremely weak Kd = 350 μm (extremely weak) Increasing [Ca 2+ ]

15 (Ca 2+ ) 2 -CLD(B) Ca 2+ -CLD(A) CLD(B) forms dimers in solution Analysis of how fast magnetization decays allows calculation of effective molecular size

16 CLD(A) also has an alternative structure Blue areas indicate regions with alternative structures Distribution of rates The dynamics and alternative structure may be important for substrate recognition

17 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 Inactive Active

18 Long QT syndrome LQTS: LQ period >400 ms Life-threatening arrhythmias Median lifespan: 34 years Triggered by exercise, emotional upset and sleep

19 The underlying cause of LQTS Common ion channels Regulation ion channels by calmodulin Mutations in calmodulin may explain dysregulation of ion channels

20 Calmodulin changes structure when binding calcium L: Calmodulin without calcium M: Calmodulin with calcium R: Calmodulin activating a protein Ca 2+ -bound calmodulin without and with substrate

21 Details of calcium binding to calmodulin and variants We are studying the following calmodulin mutants involved in LQTS: D95V, D129G and F141L Several conserved amino acids are required to bind calcium properly. Calcium binding to variants F141L reduced 50 % D95V reduced 70 % D129G reduced 90 %

22 NMR spectra are similar but not identical for variants WT D95V WT D95V D129G F141L D129G F141L Without calcium With calcium

23 Structures of variants show interesting differences D95V D129G F141L WT D95V Correlation of peak positions between variant and native calmodulin. without calcium D129G F141L with calcium Correlation of peak positions between calcium-free and calcium-bound calmodulin.

24 Mutated loops are not able to bind calcium Expected peaks in this area: G98 & G134 Observed peaks: F141L: G98 & G134 D95V: G134 D129G: G98

25 Variant calmodulin remains monomeric and highly dynamic at both low and high calcium levels WT D95V D129G F141L Without calcium With calcium

26 Dynamics differ between variants Without calcium With calcium

27 Structure and dynamics at the surface for F141L differs from native calmodulin

28 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

29 Same but different WT D95V Without calcium With calcium Without calcium With some calcium With calcium Dynamic Less dynamic Very dynamic Not dynamic Very dynamic D129G F141L Without calcium With calcium Without calcium With calcium Very dynamic Very dynamic Unchanged structure Very dynamic Less dynamic Surface is more dynamic

30 Thanks you: Cecilia Andresen Markus Niklasson Sofie Cassman-Eklöf Cecilia Markus Björn Wallner Christine Dyrager Magdalena Svenssen