Metals and ions in protein structures – from essential to marginal questions in identification of ions in enzymes and receptors
J. Dohnálek1,2
1Institute of Macromolecular Chemistry, Academy of Sciences of the
Czech Republic, v.v.i., Heyrovského nám. 2, 16206 Praha 6, Czech Republic,
2Institute of Physics, Academy of Sciences of the Czech Republic, v.v.i., Na Slovance 2, 182 21 Praha 8, Czech Republic
Biological macromolecules, such as proteins, nucleic acids, and their complexes, frequently require presence of metal or other ions to perform their native functions. In single crystal studies of biological macromolecules identification of ligands or generally solvent molecules very often represents an uneasy task. The studied molecules undergo a lengthy process of expression, modification (and secretion), purification and sometimes even special pre-crystallization treatment. Proteins and nucleic acids are commonly crystallized in solutions of salts and presence of unwanted metal ions in the used chemicals is not excluded. Even “trace” impurities in chemicals of known identity can represent a sufficient source of potential ligands of studied molecules. Therefore the resulting set of potential “binders” for a given protein or nucleic acid encompasses both the natural cofactors and the complete cohort of chemicals that were encountered on the long journey to the studied state.
If structure resolution allows, the details of an X-ray structure of an enzyme or receptor can tell us more than a pure coincidence of localization of an ion in a certain protein site. When carefully analyzed, such sites can bring better insight into the structure-function relationship and also a better understanding of the relevance of the analyzed state of a given protein. Metal binding to an enzyme can be on one hand a pure consequence of a particular crystallization condition and can be viewed as such. On the other hand it can indicate a real binding site where a metal ion is necessary for proper function or stability (extracellular hydrolases requiring sodium or calcium ions to maintain their stability, copper, zinc or other metal storage and transport proteins, etc.).
For many standard cases typical metal-ligand distances and standard coordination are observed. For example an observation of the typical octahedral coordination of Ca2+ ion mostly by oxygen atoms with a majority of the O-Ca2+ distances in the range of 2.3-2.4 Å (1500 observations in the PDB) does not leave much space for speculation and identity of such an ion for an extracellular enzyme with added calcium in the crystallization condition is far from doubtful. In spite of that even dependence of a given enzyme say on manganese does not necessarily imply that an ion localized in its X-ray structure is the same. Many protein metal binding sites are promiscuous and can bind for example a few different types of metals of the fourth period. For some of them activity towards the same substrate can be measured with different divalent metals occupying the relevant sites. There are serious implications following from this knowledge: 1) Not all structural data in the PDB (or indeed in your local set of solved structures) contain correct information on metal or ion identity, 2) Special attention should be paid to ion identification in all structural studies of metal-binding or ion-binding, and especially metal-dependent proteins.
A majority of protein crystals do not provide diffraction data to atomic or subatomic diffraction limits (1.2 Å or better). To date some 76 thousand X-ray structures deposited in the Protein Data Bank have the high diffraction limit of data 1.2 Å or worse (98% of all X-ray structures) and about 1800 1.2 Å and better (2%). Therefore protein crystallographers must mostly rely on other indicators of the nature of an ion than just the height of an electron density maximum. Moreover, protein X-ray structures very often provide an accurate picture of a mixture of structural states and this can be true even for a metal or ion binding site. Therefore, essential features of an observation (relative heights of unbiased electron density maxima, presence of anomalous signal, the shortest interatomic distances, stability of atomic positions in refinement, nature of coordinating atoms/ions) must be distinguished from marginal signs which can be easily smeared by worse quality of the diffraction data, resolution limits, position in the protein chain (termini, surface) or local disorder (atomic displacement parameters, occurrence of some longer coordination distances, missing vertices of the first coordination sphere).
In our structural studies of enzymes (chitinases, nucleases, glycosyl hydrolases, anhydrolases, oxidases etc.) and natural killer cell receptors we regularly apply the described approach to help us identify a particular protein ligand. Examples include identification of metal ions such as Cu2+, Mn2+, Zn2+, Ni2+, Ca2+, Mg2+, Na+ and other ionic ligands, for instance Cl-, PO43-, SO42- [1-6].
The sum of the available tools for identification of metals/ions in protein structures includes anomalous scattering signal, typical coordination and bonding distances [7], statistical evaluation of typical cases, assessment of the local environment, experimental conditions such as pH, and other. Access to tunable X-ray sources with fluorescence detectors enables absorption edge checks and fluorescence analysis in some cases [4] and availability of a micrometer high energy proton beam allows element identification by microbeam Proton Induced X-ray Emission (microPIXE) [8].
Lighter ions, such as Na+, Mg2+and Cl-, belong to a special category as their presence in protein structures either remains unnoticed or is misinterpreted. In such cases the correct assignment of an ion type and its distinction from a water molecule rely on sufficient evidence from all available information sources.
Grant support from the Czech Science Foundation is gratefully acknowledged (project no. 310/09/1407).
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