Abstrakt

Structural studies of interferon-gamma and extracellular portions of its two
receptors R1 and R2
Mikulecký Pavel (1) , Zahradník Jiří (1) , Kolenko Petr (1) , Klumpler Tomáš (2) ,
Černý Jiří (1) , Charnavets Tatsiana (1) , Kolářová Lucie (1) , Kukačka Zdeněk (3) ,
Phuong Ngoc Pham (1) , and Schneider Bohdan (1)
(1) Institute of Biotechnology CAS, BIOCEV, Prague West, Czech Republic
(2) CEITEC, Masaryk University, Brno, Czech Republic
(3) Institute of Microbiology CAS, Prague, Czech Republic
E-mail: [email protected]
Signaling of interferon-gamma (IFNg), cytokine important in innate and adaptive immune
response, depends on forming a ternary complex with two receptors, ubiquitously expressed
R1, and tightly regulated R2. We will present our structural studies of interactions between
IFNg, and the extracellular portions of its two receptors. Firstly, we will briefly discuss our
computer-aided modulation of R1 affinity to IFNg [1, 2]. Designed variations of Receptor 1
residues directly interacting with IFNg and in Receptor 2 cavities led to the seven-fold
increase of interaction affinity (measured as Kd). Secondly, we will concentrate on so far
poorly described R2 receptor. The successful expression of R2 allowed us to solve its crystal
structure (PDB 5eh1) and, perhaps most importantly, for the first time characterize the ternary
complex between IFNg and receptors R1 and R2. The ternary complex was studied by small
angle X-ray scattering (SAXS) and other biophysical techniques. Binding between IFNg and
its receptors measured by microscale thermophoresis (MST) confirmed the high-affinity
binding between IFNg and R1 but much weaker one between the binary IFNg/R1 complex
and R2. The weaker binding of R2 to IFNg/R1 is supported by the results of SAXS
experiments. Their interpretation provided a few possible topologies of the ternary complex
none of which confirms the expected symmetrical binding between IFNg homodimer with
two molecules of R1 and two molecules of R2 with 2:2:2 stoichiometry. Instead, both SAXS
and mass spectrometry of fragments after chemical crosslinking point to surprising 2:2:1
stoichiometry and highly asymmetric binding of R2 to the binary IFNg/R1 complex.
Acknowledgements. This study was supported by institutional support to IBT, RVO
86652036, by the ERDF project CZ.1.05/1.1.00/02.0109 to BIOCEV, and by Czech Science
foundation project 16-20507S.
[1] Pavel Mikulecký, Jiří Černý, Lada Biedermannová, Hana Petroková, Milan Kuchař, Jiří
Vondrášek, Petr Malý, Peter Šebo & Bohdan Schneider: Increasing affinity of interferon-γ
receptor 1 to interferon-γ by computer-aided design. BioMed Research International 2013:
752514 (2013). doi: 10.1155/2013/752514.
[2] Jiří Černý, Lada Biedermannová, Pavel Mikulecký, Jiří Zahradník, Tatsiana Charnavets,
Peter Šebo & Bohdan Schneider: Redesigning protein cavities as a strategy for increasing
affinity in protein-protein interaction. Interferon-γ receptor 1 as a model. BioMed Research
International 2015: 716945 (2015). doi: 10.1155/2015/716945.