Zdeněk Kříž

Activation and inhibition of cyclin-dependent kinase-2 by phosphorylation; a molecular dynamics study reveals the functional importance of the glycine-rich loop

Nanoseconds long molecular dynamics (MD) trajectories of differently active complexes of human cyclin‐dependent kinase 2 (inactive CDK2/ATP, semiactive CDK2/Cyclin A/ATP, fully active pT160‐CDK2/Cyclin A/ATP, inhibited pT14‐; pY15‐; and pT14,pY15,pT160‐CDK2/Cyclin A/ATP) were compared. The MD simulations results of CDK2 inhibition by phosphorylation at T14 and/or Y15 sites provide insight into the structural aspects of CDK2 deactivation. The inhibitory sites are localized in the glycine‐rich loop (G‐loop) positioned opposite the activation T‐loop. Phosphorylation of T14 and both inhibitory sites T14 and Y15 together causes ATP misalignment for phosphorylation and G‐loop conformational change. This conformational change leads to the opening of the CDK2 substrate binding box. The phosphorylated Y15 residue negatively affects substrate binding or its correct alignment for ATP terminal phospho‐group transfer to the CDK2 substrate. The MD simulations of the CDK2 activation process provide results in agreement with previous X‐ray data.

Acetylcholinesterases--the structural similarities and differences.

Acetylcholinesterase (AChE) is a widely spread enzyme playing a very important role in nerve signal transmission. As AChE controls key processes, its inhibition leads to the very fast death of an organism, including humans. However, when this feature is to be used for killing of unwanted organisms (i.e. mosquitoes), one is faced with the question - how much do AChEs differ between species and what are the differences? Here, a theoretical point of view was utilized to identify the structural basis for such differences. The various primary and tertiary alignments show that AChEs are very evolutionary conserved enzymes and this fact could lead to difficulties, for example, in the search for inhibitors specific for a particular species.

Efficiency of a second‐generation HIV‐1 protease inhibitor studied by molecular dynamics and absolute binding free energy calculations

A subnanomolar inhibitor of human immunodeficiency virus type 1 (HIV‐1) protease, designated QF34, potently inhibits the wild‐type and drug‐resistant enzyme. To explain its broad activity, the binding of QF34 to the wild‐type HIV‐1 protease is investigated by molecular dynamics simulations and compared to the binding of two inhibitors that are used clinically, saquinavir (SQV) and indinavir (IDV). Analysis of the flexibility of protease residues and inhibitor segments in the complex reveals that segments of QF34 were more mobile during the dynamics studies than the segments of SQV and IDV. The dynamics of hydrogen bonding show that QF34 forms a larger number of stable hydrogen bonds than the two inhibitors that are used clinically. Absolute binding free energies were calculated with molecular mechanics–generalized Born surface area (MM–GBSA) methodology using three protocols. The most consistent results were obtained using the single‐trajectory approach, due to cancellation of errors and inadequate sampling in the separate‐trajectory protocols. For all three inhibitors, energy components in favor of binding include van der Waals and electrostatic terms, whereas polar solvation and entropy terms oppose binding. Decomposition of binding energies reveals that more protease residues contribute significantly to the binding of QF34 than to the binding of SQV and IDV. Moreover, contributions from protease main chains and side chains are balanced in the case of QF34 (52:48 ratio, respectively), whereas side chain contributions prevail in both SQV and IDV (main‐chain:side‐chain ratios of 41:59 and 45:55, respectively). The presented results help explain the ability of QF34 to inhibit multiple resistant mutants and should be considered in the design of broad‐specificity second‐generation HIV‐1 protease inhibitors. Proteins 2004.

Influence of the Acetylcholinesterase Active Site Protonation on Omega Loop and Active Site Dynamics

Abstract Existence of alternative entrances in acetylcholinesterase (AChE) could explain the contrast between the very high AChE catalytic efficiency and the narrow and long access path to the active site revealed by X-ray crystallography. Alternative entrances could facilitate diffusion of the reaction products or at least water and ions from the active site. Previous molecular dynamics simulations identified side door and back door as the most probable alternative entrances. The simulations of non-inhibited AChE suggested that the back door opening events occur only rarely (0.8% of the time in the 10ns trajectory). Here we present a molecular dynamics simulation of non-inhibited AChE, where the back door opening appears much more often (14% of the time in the 12ns trajectory) and where the side door opening was observed quite frequently (78% of trajectory time). We also present molecular dynamics, where the back door does not open at all, or where large conformational changes of the AChE omega loop occur together with alternative passage opening events. All these differences in AChE dynamical behavior are caused by different protonation states of two glutamate residues located on bottom of the active site gorge (Glu202 and G450 in Mus Musculus AChE). Our results confirm the results of previous molecular dynamics simulations, expand the view and suggest the probable reasons for the overall conformational behavior of AChE omega loop.

Different mechanisms of CDK5 and CDK2 activation as revealed by CDK5/p25 and CDK2/cyclin A dynamics.

A detailed analysis is presented of the dynamics of human CDK5 in complexes with the protein activator p25 and the purine-like inhibitor roscovitine. These and other findings related to the activation of CDK5 are critically reviewed from a molecular perspective. In addition, the results obtained on the behavior of CDK5 are compared with data on CDK2 to assess the differences and similarities between the two kinases in terms of (i) roscovitine binding, (ii) regulatory subunit association, (iii) conformational changes in the T-loop following CDK/regulatory subunit complex formation, and (iv) specificity in CDK/regulatory subunit recognition. An energy decomposition analysis, used for these purposes, revealed why the binding of p25 alone is sufficient to stabilize the extended active T-loop conformation of CDK5, whereas the equivalent conformational change in CDK2 requires both the binding of cyclin A and phosphorylation of the Thr160 residue. The interaction energy of the CDK5 T-loop with p25 is about 26 kcal·mol-1 greater than that of the CDK2 T-loop with cyclin A. The binding pattern between CDK5 and p25 was compared with that of CDK2/cyclin A to find specific regions involved in CDK/regulatory subunit recognition. The analyses performed revealed that the αNT-helix of cyclin A interacts with the α6-α7 loop and the α7 helix of CDK2, but these regions do not interact in the CDK5/p25 complex. Further differences between the CDK5/p25 and CDK2/cyclin A systems studied are discussed with respect to their specific functionality.

The mechanism of inhibition of the cyclin-dependent kinase-2 as revealed by the molecular dynamics study on the complex CDK2 with the peptide substrate HHASPRK

Molecular dynamics (MD) simulations were used to explain structural details of cyclin‐dependent kinase‐2 (CDK2) inhibition by phosphorylation at T14 and/or Y15 located in the glycine‐rich loop (G‐loop). Ten‐nanosecond‐long simulations of fully active CDK2 in a complex with a short peptide (HHASPRK) substrate and of CDK2 inhibited by phosphorylation of T14 and/or Y15 were produced. The inhibitory phosphorylations at T14 and/or Y15 show namely an ATP misalignment and a G‐loop shift (∼5 Å) causing the opening of the substrate binding box. The biological functions of the G‐loop and GxGxxG motif evolutionary conservation in protein kinases are discussed. The position of the ATP γ‐phosphate relative to the phosphorylation site (S/T) of the peptide substrate in the active CDK2 is described and compared with inhibited forms of CDK2. The MD results clearly provide an explanation previously not known as to why a basic residue (R/K) is preferred at the P2 position in phosphorylated S/T peptide substrates.

Photochemistry of Valerophenone in Solid Solutions

Abstract Photoreactivity of valerophenone was investigated in frozen solid solvents: benzene, cyclohexane, t -butanol, hexadecane, and water. Different product and mass distributions were followed during the course of the photoreaction. It was evidenced that a portion of ketone molecules is almost unreactive in the solid state due to physical restraints of the solid solvent cavity. Free rotation along the C–C bonds becomes difficult inside the cavity and it is probable that larger conformational changes are totally restricted. It was shown that a fraction of molecules having the favorable conformation for hydrogen abstraction reacts with the same photochemical efficiency no matter what solvent was used. The elimination/cyclization ratio of the Norrish Type II reaction was studied as a function of temperature. Variation of the ratios, characteristic for each solvent, diminished with decreasing temperature what has been rationalized in terms of the effective reaction cavity. The semi-empirical PM3 method and molecular mechanics MM3 force field calculations were performed to evaluate stabilities of the ground state valerophenone conformations.

TRITON: a graphical tool for ligand-binding protein engineering

Summary: The new version of the TRITON program provides user-friendly graphical tools for modeling protein mutants using the external program MODELLER and for docking ligands into the mutants using the external program AutoDock. TRITON can now be used to design ligand-binding proteins, to study protein–ligand binding mechanisms or simply to dock any ligand to a protein. Availability: Executable files of TRITON are available free of charge for academic users at http://ncbr.chemi.muni.cz/triton/ Contact: triton@chemi.muni.cz Supplementary information: Supplementary data are available at Bioinformatics online.

Dynamics and Binding Modes of Free cdk2 and its Two Complexes with Inhibitors Studied by Computer Simulations

Abstract This article presents a molecular dynamics (MD) study of the cdk2 enzyme and its two complexes with the inhibitors isopentenyladenine and roscovitine using the Cornell et al. force field from the AMBER software package. The results show that inserting an inhibitor into the enzyme active site does not considerably change enzyme structure but it seemingly changes the distribution of internal motions. The inhibitor causes differences in the domain motions in free cdk2 and in its complexes. It was found out that repulsion of roscovitine N9 substituent causes conformational change on Lys 33 side chain. Isopentenyladenine forms with Lys 33 side chain terminal amino group a hydrogen bond. It implies that the cavity, where N9 substituent of roscovitine is buried, can adopt larger substituent due to Lys 33 side chain flexibility. The composition of electrostatic and van der Waals interactions between the inhibitor and the enzyme were also calculated along both cdk2/inhibitor MD trajectories together with MM-PB/GBSA analysis. These results show that isopentenyladenine-like inhibitors could be more effective after modifications leading to an increase in their van der Waals contact with the enzyme. We suggest that a way leading to better inhibitors occupying isopentenyladenine binding mode could be: to keep N9 and N7 purine positions free, to keep 3,3-dimethylallylamino group at C6 position, and to add, e.g., benzylamino group at C2 position. The results support the idea that the isopentenyladenine binding mode can be used for cdk2 inhibitors design and that all possibilities to improve this binding mode were not uncovered yet.

Conformational features of linear and cyclic enkephalins. A computational study

A theoretical conformational study using the CICADA program package (J. Mol. Struct. (Theochem), 1995, 337, 17-24) was performed for two linear enkephalins, Leu-enkephalin and Met-enkephalin, and two cyclic analogues, DLFE and DPDPE. The conformational flexibilities of whole molecules and selected torsions were calculated. The low energy conformers obtained were compared with structures obtained by spectroscopic methods. The mutual space positions of key elements for receptor recognition were analyzed. Conformations were clustered using RMS deviation computed for selected atoms. The different conformational behavior of aromatic rings in cyclic analogues of enkephalins was observed. While aromatic rings of cyclic analogues exhibit different conformational behavior, the linear enkephalins show similar behavior in these key parts. Hydrogen bonds predicted by spectroscopic measurements were confirmed by our calculations. Also very specific conformational features, like concerted conformational movement, were analyzed.