NMR spectroscopy and molecular dynamics simulation studies of EMAP II cytokine and
its nanocomposite complexes
Alexander Kornelyuka, Dmytro Lozhkoa, Alena Malinaa and Igor Zhukovb,c
Institute of Molecular Biology and Genetics, Department of
Protein Engineering and Bioinformatics, National Academy of Sciences of Ukraine, 150 Acad. Zabolotnogo Str.,
Kyiv, Ukraine
b
Institute of Biochemistry and Biophysics, Polish Academy of Sciences
5a, Pawinskiego, 02-106 Warsaw, Poland
cNanoBioMedical Centre, Adam Mickiewicz University, Umultowska 85, 61-614 Poznań, Poland
a
Endothelial and monocyte-activating polypeptide II (EMAPII) is a proinflammatory
cytokine, which induces endothelial cells apoptosis and reveals strong antitumor activity in
vivo. Recently we have determined the 3D structure of EMAP II in solution by
multidimensional NMR spectroscopy. In order to make all sequence-specific assignments of
backbone 1H, 13C, and 15N nuclei more reliable, several multidimensional (4D and 5D)
NMR experiments were performed. According to NMR data, the cytokine motif in EMAP II
(Val6-Thr18) is much higher accessible in solution in comparison with crystal structure.
Solution structure of EMAP II also supports the existence of extended α-helix Pro120–
Pro130, which was observed in our MD simulations. This flexible region of EMAP II is
proposed to be involved in the formation of RNA binding site.
In order to evaluate conformational flexibility of EMAP II in solution, the MD
simulations were performed using GROMACS program. MD simulations revealed the
rearrangement of Lys116-Lys123 loop structure and stabilization of the adjucent α-helix.
Conformational mobility of flexible fragment Lys116-Pro130 may increase upon
temperature increase and resulted in the reversible refolding and exposure of Trp125 from
hydrophobic environment at 42 оС. Analysis of NMR data revealed the most temperaturesensitive regions of EMAP II in solution: Leu9-Lys21, S27-G36, Arg41-Arg59, Cys65-Lys68,
Val75-Met82, Ser85-Gln129, G153-Lys166. In the case of N-terminal subdomain, the high
csp values caused by temperature observed for N-terminal cytokine motif and two β-turns
(β1-β2 and β1'-β2') served as additional place for tRNA binding.
Cyclodextrins are widely used in biotechnological applications due to their ability to
sequester hydrophobic moieties on protein surface stabilizing its conformation. We have
studied the interactions of
EMAP II with β-cyclodextrin and 2-hydroxypropyl-βcyclodextrin in nanocomposite complexes. Cyclodextrins were bound at the surface of
EMAP II in the cavity of partially exposed Trp125. It was found that cyclodextrin binding
prevents the local conformational change of Trp125 site at 42 oC , thus stabilizing protein
conformation. HSQC-1H/15N NMR spectra revealed the changes of chemical shifts of
Thr106, Phe110, Gly115, Lys123 and Trp125 in the complex of EMAP II with 2hydroxypropyl-β-cyclodextrin. Hence, we have demonstrated that the binding of
cyclodextrins with EMAP II cytokine take place at specific location on the protein surface
resulted in the protein stabilization in nanocomposite complex.
Acknowledgment
The project was supported by East-NMR inside of transnational access program.