Late Abstracts

LA1

Theoretical studies on conformational changes imposed by phosphorylation and ATP binding in serine/threonine kinase from Bacillus subtilis, PrkC

Paweł Gruszczyński1,2, Michał Obuchowski2, Rajmund Kaźmierkiewicz2

1Department of Chemistry, University of Gdańsk, J. Sobieskiego 18/19, 80-952 Gdańsk, Poland; 2Intercollegiate Faculty of Biotechnology, University of Gdańsk and Medical University of Gdańsk, Kładki 24, 80-822 Gdańsk, Polande-mail: pawelg@chem.univ.gda.pl

The PrkC is a serine/threonine kinase, from Hanks super family [1]. The activity of the enzyme increases consider-ably after phosphorylation of four threonine residues, lo-cated on the activation loop (T162, T163, T165, T167) and one serine in the 214 position, located in the C-terminal lobe of PrkC [2].The purpose of this work was to investigate the structural changes imposed by the phosphorylation and ATP bind-ing of the PrkC intracellular domain (PrkCc) using mo-lecular dynamics (MD) simulations. Four models were built, two unphosphorylated: PrkCc, PrkCc-ATP and two phosphorylated: pPrkCc, pPrkCc-ATP and subjected to 5ns long MD simulations.Sequence alignment of the PrkC shows surprisingly high similarities in the catalytic domain, and in the activation loop, with the PknB kinase from Mycobacterium tubercu-losis [3]. Analysis of the ATP binding pocket (see Fig. 1) within phosphorylated and unphosphorylated complex-es revealed that phosphorylation did not affect nucleotide contact residues, which are highly conserved. The overall flexibility of the activation loop, calculated by the RMSd, as well as visualized, suggests that the ATP binds after phosphorylation of the activation loop, and does not bind to the unphosphorylated kinase, since we find the PrkCc-ATP complex to be unstable.

Residues closer than 4.0 Å from ATP (orange) are present-ed as sticks (black) and the PrkCc interface as a cartoon representation.The structural parts, characteristic for the activity of the enzyme, were studied: DFG-motif, HRD-motif and αC helix. It was shown that the presence of ATP is respon-sible for most changes of their spatial arrangement. DFG-in conformation was seen only when the nucleotide was placed in the binding pocket. At the same time the pres-ence of strong salt-bridge stabilized the position of the αC helix. The overall position of the HRD triad remains un-changed for most of the models (besides PrkCc-ATP) and indicates the active state of the enzyme.Studies on the conformation of the phosphorylated resi-dues provided some ideas about their role. The phos-phorylation loop, by interaction with the positively charged arginine cluster, found in the αC helix, stabilizes the position of the catalytically important residue E59. The conformational diversity of the activation segment increases in the presence of ATP or after phosphoryla-tion. We speculate that the phosphorylated threonine residues do not induce critical conformational changes of the kinase, such as opening/closing conformations of the protein. Their role probably correlates with substrate recognition or dimerization, likewise the phosphorylated serine residue (pS214).Based on our study, we find the phosphorylated catalytic domain of PrkC, complexed with ATP (pPrkCc-ATP), as fully active in the closed conformation. The mechanism of the activation of PrkC is still unknown, but we hope this research brings us a little closer to its understanding.

References:

1. Hanks SK, Quinn AM, Hunter T (1988) The protein kinase family: conserved features and deduced phylogeny of the cata-lytic domains. Science 241: 42–52.2. Madec E, Stensballe A, Kjellstrom S, Cladiere L, Obuchowski M, Jensen ON, Seror SJ (2003) Mass spectrometry and site-direct-ed mutagenesis identify several autophosphorylated residues required for the activity of PrkC, a Ser/Thr kinase from Bacillus subtilis. J Mol Biol 330: 459–472.3. Madec E, Laszkiewicz A, Iwanicki A, Obuchowski M, Seror S (2002) Characterization of a membrane-linked Ser/Thr protein kinase in Bacillus subtilis, implicated in developmental processes. Mol Microbiol 46: 571–586.

Figure 1. ATP binding pocket.

LA2

Co-crystal structures of human protein kinase CK2α with tetrabromo-benzimidazole/ benzotriazole-derived inhibitors

Nils Bischoff1, Maria Bretner2, Karsten Niefind1

1Institute of Biochemistry, Department of Chemistry, University of Cologne, Zuelpicher Str. 47, D-50674 Cologne, Germany; 2Institute of Biochemistry and Biophysics, A. Pawinskiego 5A, 02-106 Warszawa, Polande-mail: Karsten.Niefind@uni-koeln.de

Co-crystallization studies of human CK2α ― the catalytic subunit of human protein kinase CK2 ― with small mol-ecule ligands [1, 2] have revealed the enzyme’s tendency to bind some of these ligands in a “remote cavity” located in a region normally used to form an interface to the non-catalytic subunit CK2β. In the future this remarkable be-haviour of human CK2α may open a way to disturb the CK2α/CK2β interaction by small molecules and even to design drugs of a new type [3].In an attempt to further explore this feature of human CK2α we performed co-crystallization experiments with a number of potential ligands, among them three deriva-tives of the well-known CK2-inhibitors tetrabromo-ben-zotriazole (TBBt) [4] and tetrabromo-benzimidazole (TBBi) [5]. These three derivatives were substituted by hydroxypropyl and show higher inhibitory activity than their parent compounds by testing human CK2α [6].In the complex structures, however, the TBBt and TBBi derivatives do not occupy the “remote cavity”. Rather they bind to the canonical ATP-cleft. In this context one of the inhibitors shows a conspicuous and unexpected fea-ture (Fig. 1): it is the first known substance that replaces some water molecules described as highly conserved before [7]. Thus, this molecule exploits the enzyme’s hy-drogen bonding potential quite efficiently and is a good candidate for further drug design efforts.

References:

1. Raaf J, Brunstein E, Issinger O-G, Niefind K (2008) The CK2α/CK2β interface of human protein kinase CK2 harbors a binding pocket for small molecules. Chem Biol 15: 111–117.

2. Raaf J, Issinger O-G, Niefind K (2009) First inactive conforma-tion of CK2α, the catalytic subunit of protein kinase CK2. J Mol Biol 386: 1212–1221.3. Wells JA, McClendon CL (2007) Reaching for high-hanging fruit in drug discovery at protein-protein interfaces. Nature 450: 1001–1009.4. Battistutta R, De Moliner E, Sarno S, Zanotti G, Pinna LA (2001) Structural features underlying selective inhibition of pro-tein kinase CK2 by ATP site-directed tetrabromo-2-benzotria-zole. Protein Sci 10: 2200–2206.5. Zień P, Bretner M, Zastapiło K, Szyszka R, Shugar D (2003) Se-lectivity of 4,5,6,7-tetrabromobenzimidazole as an ATP-competi-tive inhibitor of protein kinase CK2 from various sources. Bio-chem Biophys Res Commun 306: 129–133.6. Bretner M, Najda-Bernatowicz A, Lebska M, Muszynska G, Kilanowicz A, Sapota A (2008) New inhibitors of protein kinase CK2, analogues of benzimidazole and benzotriazole. Mol Cell Biochem 316: 87–89.7. Battistutta R, Mazzorana M, Cendron L, Bortolato A, Sarno S, Kazimierczuk Z, Zanotti G, Moro S, Pinna LA (2007) The ATP-binding site of protein kinase CK2 holds a positive electrostatic area and conserved water molecules. ChemBioChem 8: 1804–1809.

Figure 1. The active site of CK2. The positions of three conserved water molecules (PDB ID: 1lp4) are occupied by MB 003, a derivative of TBBt, if it is in complex with CK2α. MB 003 is covered with 2Fo–Fc electron density (con-tour level 1.0 σ).

6th International Conference: Inhibitors of Protein KinasesVol. 56 2