Two-dimensional arrays of proteins adsorbed at lipid membranes in living organisms have attracted exceptional interest in biomedical studies. According to modern conceptions, the real biochemistry processes in a cell proceed not in three-dimensional (3D), but in two-dimensional (2D) (or even one-dimensional (1D)) space. Various micro- and nanostructures in the cell are organized in such a way that proteins and other biomolecules are either permanently bound to their surfaces, or adhere on surfaces, reacting with different molecular species. As a result of such interactions, the physico-chemical parameters of protein macromolecules, as well as the general mechanism of action, can significantly alter.
High precision X-ray techniques have been recognized to be very promising for investigations of two-dimensional bioorganic nanostructures. Nowadays, X-ray reflectometry, grazing incidence diffraction, grazing incidence small-angle scattering, the X-ray standing wave method, etc. are extensively used to study adsorbed protein layers at fluid interfaces [1
]. However, these techniques do not provide atomically resolved structural information about protein macromolecules.
X-ray absorption spectroscopy (XAS) expands opportunities to characterize the local structure of the absorbing sites in biological materials. Analysis of XAS spectra for metalloproteins and metal-protein complexes allows one to obtain detailed information about the 3D geometry of a metal site, the number and type of ligands, metal-ligand distances, and the oxidation state of metal ions [4
]. Metalloproteins play a key role in many vital biological processes, such as enzymatic catalysis, gas transport, electron transfer, and redox signal transmission. Transition metals (Zn, Cu, Ni, Co, Fe, and Mn) in proteins coordinate with a wide range of ligand systems and are involved in the regulation of the reactivity of active centers in metalloproteins by modulating its electronic structure, redox potential, steric factor, dielectric properties, etc. Even subtle changes in the structural parameters of the metal-ligand system can induce severe alterations of the biological functions of metalloproteins.
In recent years, much attention has been paid to metal complexes that have occurred at the interface between biological molecules, with special regard for the role that metal ions play in formation of supramolecular protein ensembles. Numerous investigations have demonstrated that metal ions act as linkage agents for protein macromolecules facilitating the assembling of protein structures with very specific properties, such as high adhesion, rigidity, abrasion resistance, and self-healing [9
For a long time, XAS has been successfully used to study protein macromolecules both in protein crystals and in protein solutions. X-ray absorption near edge structure (XANES) measurements in the fluorescent mode under total external reflection conditions at liquid interfaces, implemented at European Synchrotron Radiation Facility (ESRF) on the ID10 beamline, for the first time and presented in this work, significantly extend the general capabilities of X-ray structural methods. These experiments combine several benefits important for biological studies, i.e., two-dimensional arrays of proteins can be investigated directly at liquid interface and structural changes in reacting protein molecules can be monitored in real-time under physiological conditions.
XANES spectra at liquid interfaces were first obtained by Wang et al [11
]. These studies were focused on the interaction between the lipid monolayer, deposited on the water surface in a Langmuir trough, and metal ions (iron) present in the water subphase. In the present work we applied XANES measurement for the characterization of the local environment around metal ions in protein molecules adsorbed at liquid surface in a Langmuir trough. Our object was the parkin protein. Parkin is traditionally mentioned as E3 ubiquitin ligase, but its official name, according to enzyme nomenclature, is RING-type (really interesting new gene) ubiquitin transferase (EC 184.108.40.206). Thus, it belongs to a ssecond class of enzymes, transferases, which catalyze transfer of specific functional groups from the donor molecule to another acceptor molecule. Parkin catalyzes the transfer of ubiquitin residue (ubiquitinyl) from an E2 enzyme conjugated with ubiquitin to an acceptor protein. E1, E2, and E3 are the conventional names of enzymes catalyzing different stages of protein ubiquitination, i.e., the process of marking proteins for proteasome degradation. Parkin is an E3 enzyme and provides the substrate specificity of the ubiquitin-proteasome system [12
] and plays a central role in proteasome-mediated degradation of misfolded proteins in dopaminergic neurons.
Parkin belongs to a group of Zn-finger proteins and contains eight Zn binding clusters in four RING domains. Alterations in the structure of parkin are caused by mutations in the gene encoding the protein or posttranslational modifications, and have been shown to be a predominant cause of the accumulation of protein aggregates (mostly the presynaptic protein alpha-synuclein) in the neuron cytoplasm that has a significant implication in the pathogenesis of Parkinson disease. Mutations of cysteine (Cys) residues that disrupt zinc coordination in parkin RING domains have been demonstrated to correlate to the loss of the ligase activity of parkin [13
]. These findings highlighted the particular relevance of Zn binding clusters for parkin functioning and have attracted great interest in investigating possible mechanisms responsible for destabilization of the zinc ligand environment in the parkin protein.
Zinc belongs to the group of most abundant transition metal in protein macromolecules. Over 10% of human proteins contain zinc [29
]. Zinc is known to play a key role in stabilizing the protein structure and to exert the stabilization effect at all levels of protein structure organization, i.e., secondary, tertiary, and quaternary. In most proteins (~90%) zinc ions are coordinated to cysteine, histidine, aspartate, and glutamate residues [29
]. Depending on their functional role, zinc binding sites in protein can be divided into the following five main classes: catalytic, structural, cluster, transport, and intermolecular [30
]. The most common are structural sites consisting of Cys and His residues in a tetrahedral geometry [29
]. Cysteine is the preferential amino acid in such sites.
In the present studies, we investigated the parkin protein that belongs to the Zn-finger family and contains eight tetracoordinated Zn binding sites. On the basis of the obtained XANES data, we identified two types of local zinc environment in our parkin preparations depending on X-ray radiation load. According to our experimental results, under mild conditions, the zinc sites are four-coordinated sites; but it appeared that local zinc environment does not correspond to that determined in parkin crystallographically. Essentially, we found that zinc site structure in our parkin preparations was very similar to that identified in hemoglobin, treated with a solution of ZnCl2 salt. Under high X-ray radiation load, considerable changes in the zinc site structure were detected; zinc coordination number increased to six, while the ligand environment became almost identical to that defined in Zn-containing enzyme alkaline phosphatase.
The biological reasons for occurrence of similar metal binding sites in metalloproteins have been actively discussed in the past years. Structurally conservative binding sites in Zn- and Ca-containing proteins were investigated in [33
]. Torrance et al. checked the Protein Data Bank and used the protein structure comparison methods to search the cases of structural templates with the same number of liganding residues in similar geometry. It has been demonstrated that the structure of Zn binding sites can differ greatly in a group of related proteins (with high level of sequence identity) and be very similar in unrelated proteins that have completely different conformations. These sites have independently evolved in a large number of proteins, indicating that unrelated proteins use the same set of residues to bind metals.
Recently, Rosato et al. proposed the concept of minimal functional sites in order to describe structure-function relations in metalloproteins [34
]. Minimal functional sites were defined as 3D structures with conserved geometry that include the nearby region around the metal site and did not depend on the macromolecular structure of the protein. Structural bioinformatics analysis demonstrated, that equivalent minimal functional sites can occur in significantly divergent proteins. Generally, Rosato et al. defined minimal functional sites as essential structural units, that are “grafted onto the protein fold” and play an important role in maintaining protein functions. The close similarity between Zn binding sites in the studied parkin preparations and two unrelated proteins (hemoglobin and alkaline phosphatase), observed in our experiments, is consistent with the concepts mentioned above.
Remarkable is the fact that specific Zn binding templates with distinct topologies in our parkin preparations occurred as a result of random changes in protein conformation (due to misfolding or X-ray radiation damage). These findings highlight the biological relevance of metal binding templates and are in the mainstream of the general idea that tends to specify metal sites as substantive elements in protein macromolecules.
In conclusion, the presented results clearly demonstrated the great potential of XANES measurements at liquid surface for structural studies of biological materials. Due to a drastic decrease in background scattering intensity under total external reflection conditions (accordingly a considerable increase in the signal-to-noise ratio) low spectroscopic signal from metal atoms bound in trace amounts by protein molecules can be detected in such experiments. Concentration of protein molecules at the liquid interface due to self-assembling is another important experimental advantage, enabling XANES spectra to be collected for a single-molecule thick protein layer. Additionally, the possibility to examine non-crystalline protein systems under nearly physiological conditions is critical in biomedical researches. All the mentioned benefits are extremely challenging for studying biophysical properties of proteins and understanding their functions in complex cellular processes.