Ligand Docking to Biochemical Targets. Crystallography and modelling


J. Hašek, J. Dohnálek, J. Dušková, P. Kolenko, T. Skálová


Institute of Macromolecular Chemistry, Academy of Sciences, 16206 Praha 6


Keywords:   Ligand-protein docking, polymer, drug design, X-ray structure analysis


X-ray structure analysis is an excellent tool for study of molecular recognition (i.e. specific adhesion between two macromolecules of biological origin) and therefore it plays a key role in elucidation of molecular mechanisms of many biochemical processes. A high interest of crystallographers was also devoted to specific interactions of drugs in active sites of enzymes and their research has already been reflected in many practical results of the rational drug design. Also in the case of drugs, we can usually see high affinity of the ligand to the target protein because it is the aim of the human effort to break down the enzymatic function permanently. The molecules of interest are in these cases usually well ordered in crystal and therefore relatively easily resolved by protein crystallography.

The situation is not so easy with ligands possessing lower affinity to the protein molecule (as for example polymers). One has to cope usually with  molecules which are only partly localized on the protein surface with the remaining parts floating freely in the crystallization buffer (for diffraction experiment invisible). In addition, parts of the ligand adhering to protein surface (the only visible fragments in the maps of electron density) have often lower occupancy and also can appear in multiple conformations accompanied by multiple configuration of the surrounding water molecules of buffer. Therefore to localize these low affinity ligands, we need relatively good experimental data and additional work connected with careful localization of water molecule networks forming the hydration shell of the protein. It was believed until recently that protein crystallography cannot describe well the structure of solvent near the protein surface and thus neither localize well the low affinity ligands on the protein surface. These difficulties are probably the reason why crystallographers did not pay attention to these low adhesion molecules in past.

However, many low affinity ligands have already been proved as highly efficient tools practically used in health care. Several tens of studies testing practical usage of these low affinity ligands have been published every year (drug carriers for safe delivery and release of drugs in the target tissue, coating materials protecting the biologically degradable molecules during their transport, control of drug release rate, artificial additives in food, etc.). In spite of a clear  importance of the subject, our knowledge of how the low affinity ligands bind to protein surface remains limited until now.

In spite of the fact that polyethyleneglycol (mostly PEG2000) has been intensively used in crystallization experiments for a long time [1],  only marginal attention has been paid to soaking of various polymers into the protein crystals and to crystallographic studies of adhesion between proteins and polymers (low affinity materials of non-biological origin). Here we show on several examples [2-10] a number of general problems tackled when one tries to study polymers and the complex molecular systems mentioned above. Namely we focus on the problems connected with determination of all conformation states, conformational freedom of the bound ligand influencing the entropy of the system, the flipping problem of His, Gln, Asn, determination of  water sites in the first hydration shell, verification of water molecule networks and localization of the relevant parts of  polymer ligands.

It can be said generally that the experimentally derived view of the molecular structure (the 3D-map of electron density) contains information about all conformational states and motions of the molecular system realized during the time of the measurement (usually several minutes). In the case of a good measurement, parts of the molecular system remaining stable during the measurement are usually well resolved in the map of electron density, any single electron can be resolved and the individual atom positions can be localized with an accuracy up to 1 pm (0.01 Å). However, electron density of atoms in areas with higher mobility of atoms is blurred over larger areas. It is usually described by higher temperature factors and by disorder of some functional groups. A better alternative way, although less compact, is to deposit a definite number of models similarly as it is generally used in the NMR structure analysis [10].

The fact that we cannot expect high specificity of these interactions, because proteins did not pass any genetic selection with respect to these compounds in past, is on the other hand a great advantage. Molecular adhesion of such low affinity materials is not critically dependent on a specific protein, and thus we hope that it will be much easier to generalize the observations received on a number of experimentally determined structures of proteins of different origin and to form rules which govern the properties of these molecular complexes.


The research is supported by grants of the Academy of Sciences of the Czech Republic (project T40050040) and of the Grant Agency of the Czech Republic (project 305/07/1073).


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