Human cardiac ryanodine receptor: Structural
study of the N-terminal region
Lubomir Borko1, Julius Kostan2,
Vladimir Pevala1 , Lubica Urbanikova3, Juraj Gasperik1, Eva Hostinova1, Alexandra Zahradnikova1, Vladena
Bauerová-Hlinková1 and Jozef Sevcik1
1Biochemistry
and Structural Biologydepartment, Institute of
Molecular Biology SAS, Dubravska cesta
21, Bratislava, 84551, Slovak Republic,
2Biochemistry
& Biophysics, Structural & Computational Biology department, Max F. Perutz Laboratories, Dr. Bohr-Gasse
9 (VBC 5), Vienna, 1030, Austria,
3Genomics
and Biotechnology department, Institute of Molecular Biology SAS, Dubravska cesta 21, Bratislava,
84551, Slovak Republic
Human ryanodine receptor (hRyR2) is a
cardiac calcium ion channel present in sarcoplasmatic
membrane [1, 2]. It mediates the calcium ions release in
response to electrical stimulation during excitation-contraction coupling. Human RyR2 is large
homotetramer, composed of four subunits with a
molecular weight of ≈ 560 kDa [3]. N-terminal (aa. ≈ 1-655) and central (aa. ≈ 2100-2500) region
are believed to be involved in channel gating regulation [4]. Mutations located in
these two regions are linked to several heart diseases [5]. To understand hRyR2 gating mechanism and
prevent its malfunction, it is of high importance to know the atomic structure
of the key regions as well as the entire molecule.
This contribution
reports structural study of hRyR2 1-606 region. This region was analysed by
x-ray crystallography as well as by small angle x-ray scattering analysis (SAXS).
Samples for crystallization and SAXS analysis were prepared through IMAC and
size exclusion chromatography. Quality of the samples was tested via SDS and native PAGE and stability
was analysed with DLS. After a successful crystallization, diffraction quality crystals
and diffraction data to 2.5Å were obtained. Model of the hRyR2 1-606
structure was created using software package CCP4 and the phase problem was
solved by molecular replacement using homolog rabbit
RyR1 structure (PDB ID: 2XOA). The structure model has revealed conserved three
domain structure, similar to rabbit RyR1 (2XOA) [6] and inositol
1,4,5-trisphosphate receptor (3UJ0) [7]. SAXS analysis led to low resolution model
of hRyR2 1-606 showing regions missing in x-ray model. Docking of x-ray model
into SAXS envelope was done and modelling of missing parts is yet to be done.
Acknowledgement: This work was
supported by the research grants from the Slovak Grant Agency VEGA No.
2/0131/10 and Slovak Research and development agency APVV-0628-10.The authors
thank to Dr. Jacob A. Bauer for helpful discussion.