1 Introduction
1.1 Physiological and pathophysiological protein-ligand interactions
Physiological and pathophysiological protein-ligand interactions have created many challenges and raised many questions in the field of biochemistry and molecular biology, such as in drug discovery [1-4].
In protein-ligand interactions, the study of the biological and physical manifestations of disease has been of interest as of late since they correlate with the underlying abnormalities and physiological disturbances. Molecular interactions play a most important role in biological processes, including DNA replication, transcription and translation, gene splicing, protein secretion, cell cycle control, signal transduction, drug binding, antigen recognition and enzyme-substrate interactions [3, 5-11].
Therefore, it is crucial to identify and characterise components and structures of protein-ligand complexes for definition and identification of protein functions [8, 9].
Fundamental in protein-ligand interactions is the recognition of a ligand at a unique binding site and/or surface such that it binds in a defined way in order to carry out its function. Often a ligand is a signal-triggering molecule that fits into specific receptor
change in the receptor proteins structure can then activate or inhibit another biological mechanism linked to it. In order to understand the receptor proteins functions and molecular causes of malfunction, the receptor proteins interactions with themselves and/or with other biomolecules need to be characterised and analysed. For explaining such interactions, also called bioaffinities, a major paradigm has been introduced more than 100 years ago by the “lock-and-key” model, formulated for explaining the specificity of enzymatic hydrolysis of glycosides [12] and suggested to be strongly correlated with the threedimensional (3D) structure of a protein.
Molecular recognition is the result of interaction processes such as hydrophobic interactions, hydrogen bonding, electrostatic interactions and van der Waals forces [7].
Molecular structural changes during the formation of a protein-ligand complex can contribute to, or disturb the complex formation. To understand the biological functions of proteins, not only their structures have to be determined, but also the binding reactions have to be characterised. This includes such properties as ligand binding site flexibility, distortion energies, desolvation effects, entropy, molecular electrostatic field complementarity, and kinetics determinations. Therefore, it is of high general interest to identify the binding regions and, above all, to examine the binding kinetics and affinities [7, 8, 13]. Protein-ligand interactions have been studied by many scientists in the last years, and identified for hundreds of proteins and their interaction partners [1, 14, 15].
Protein-ligand interactions are integral to a wide range of biological processes, including hormone, neurotransmitter or drug binding, antigen recognition, and enzyme-substrate interactions. Fundamental to each of these interactions is the recognition by a ligand of a unique binding surface whereby it binds in a defined way in order to carry out its function. Through an understanding of these specific interactions, it may be possible to design or discover analogous ligands with altered binding properties and, therefore, to intervene in the chemical pathway in a specific manner. The ligand of interest may be an organic small molecule, a peptide or a carbohydrate [8].
Protein-ligand interactions can be disturbed in human diseases such that the disease is the result of, e.g. genetic alterations in physiologically important signalling
pathways. Disease-derived protein variants have been frequently used to provide biomedical reference for the study of protein-ligand interactions. Therefore, it is important to delineate the structural characteristics, as well the affinity binding of the proteins, in order to develop a model of the binding interaction. One pathophysiological type of protein-protein interaction is oligomerisation and aggregation. Deposition of aggregates in the brain comprises diseases, including Alzheimer´s, Parkinson´s and Prion Disease [16-19]. Therefore, structural characterisation of these aggregates and the protein sequences which lead to the deposition of these aggregates in brain is very important. Mass spectrometry has been applied with success in the structural identification and characterisation of such proteins and their aggregation [16, 20, 21].
In addition to aggregation/oligomerization, post-translational modifications in proteins may also lead to disease and/or disturbed pathways occurring at physiological conditions [16, 22-24]. For example, oxidation of a protein can lead to aggregation, fragmentation, denaturation, and destruction of secondary and tertiary structure resulting in increased proteolytic susceptibility of the oxidized proteins. Free radicals can lead to oxidation of amino acid side chains, cleavage of peptide bonds, and formation of covalently cross-linked protein derivatives [25]. Oxidative modifications of proteins can result in functional inactivation or activation through the site-selective oxidative modification of specific amino acids. These are not only indicators of toxic and destructive processes in living systems, but can also serve to control enzyme activity [26].
One of the most studied oxidative post-translational modifications is tyrosine nitration which can occur under physiological conditions, including signal transduction [27], and may be substantially enhanced under various pathophysiological conditions associated with oxidative stress. For the molecular correlation of protein nitration with pathogenic mechanisms of human diseases and with animal or cellular models of diseases, it is essential to identify the protein targets of nitration and the specific individual modification sites. Researchers have been interested in the role of the tyrosine nitration for understanding the potential
Parkinson’s disease [27-32]. Tyrosine nitration occurred in both in vitro and in vivo samples under physiological and pathophysiological conditions, and has been successfully characterised and analysed by mass spectrometry [33-38]. For example, nitration of prostacyclin synthase (PCS), upon treatment of bovine aortic microsomes by peroxynitrite (PN), has been identified at Tyr-430 residue using a combination of proteolytic fragmentation, HPLC detection and high resolution mass spectrometry [38]. The specificity of this single post-translational modification, nitration of the Tyr-430 residue, producing inactivation of prostacycline synthase may be explained by heme catalysis. The Tyr-430 residue is located near a heme group and is also accessible to a reactive nitrogen species. Therefore, mutation of the Tyr-430 residue should give further informations to the importance of this amino acid (Figure 1a) [38].
In contrast, physiological tyrosine nitration in human eosinophil granule proteins (eosinophil peroxidase (EPO), eosinophil cationic protein (ECP), eosinophil neurotoxin protein (EDN)), isolated from patients with hypereosinophilia, has been successfullly identified by mass spectrometry [37]. The close relationship between eosinophilia and nitro-tyrosine formation suggested that the EPO itself is an important factor in promoting protein nitration [35, 37]. By using high resolution affinity-mass spectrometry specific single nitration sites at Tyr-349 in EPO and Tyr-33 in both ECP and EDN have been identified [37] (Figure 1b).
a b
Figure 1: (a) Reconstructed 3D structural model of human PCS based on the P450BM-3 X-ray structure [39]. The red structure denotes heme and the arrow indicates the probable localisation of nitrated fragment 427-430 in a tight fold around the heme binding site. (b) Diagram representation of ECP. Nitrated Tyr-33 is represented as a ball-and-stick model [35, 37-39].
Oxidation represents only one type of protein modifications which occurs under physiological and pathophysiological conditions. Once one or more protein-ligand interactions have been identified, it is desirable to investigate the interaction(s) for (i) understanding the mechanism in which the proteins are involved at molecular level;
(ii) understanding the interactions functional significance in vivo and (iii) developing methods to specifically disturb the interaction in vivo [40]. Through an understanding or identification of these specific interactions it may be possible to design or discover analogous ligands/drug with altered binding properties and, therefore, to intervene in the biochemical pathway in a highly specific manner.
Proteins regulate all biological processes in cells, including gene expression, cell growth, morphology, mobility, intercellular communication and apoptosis. Therefore, structural characterisation and the affinity binding of the protein-ligand interaction is of crucial importance for understanding the functions of the proteins as well their mechanism. The proteins that are used to complete specific functions may not always be expressed or activated, or they are expressed in a cell type-dependent manner.
Therefore, highly sensitive and specific approaches are needed in the structural identification and characterisation of proteins. In recent years, it has been
characterisation and identification of protein modifications and complex formation in physiological and pathophysiological conditions [33, 36, 37, 41, 42].