Conformational energy range of ligands in protein crystal structures: The difficult quest for accurate understanding

In this review, we address a fundamental question: What is the range of conformational energies seen in ligands in protein‐ligand crystal structures? This value is important biophysically, for better understanding the protein‐ligand binding process; and practically, for providing a parameter to be used in many computational drug design methods such as docking and pharmacophore searches. We synthesize a selection of previously reported conflicting results from computational studies of this issue and conclude that high ligand conformational energies really are present in some crystal structures. The main source of disagreement between different analyses appears to be due to divergent treatments of electrostatics and solvation. At the same time, however, for many ligands, a high conformational energy is in error, due to either crystal structure inaccuracies or incorrect determination of the reference state. Aside from simple chemistry mistakes, we argue that crystal structure error may mainly be because of the heuristic weighting of ligand stereochemical restraints relative to the fit of the structure to the electron density. This problem cannot be fixed with improvements to electron density fitting or with simple ligand geometry checks, though better metrics are needed for evaluating ligand and binding site chemistry in addition to geometry during structure refinement. The ultimate solution for accurately determining ligand conformational energies lies in ultrahigh‐resolution crystal structures that can be refined without restraints.     

Cation-pi (Na+-Trp) interactions in the crystal structure of tetragonal lysozyme.

Experimental evidence of a cation-pi interaction between a sodium cation (Na+) and the indole ring of residue Trp123 in a structure (2.0 A) of hen egg-white lysozyme is presented. The geometry of the metal ion-pi interaction observed in the protein structure (distance between the aromatic plane and the cation approximately 4 A) is consistent with geometries observed among small molecules crystal structures and quantum chemistry ab initio calculations. The present crystal structure of lysozyme provides unique structural information about the geometry of binding of cations to pi systems in proteins. It shows that the metal ion-pi interaction within proteins is not significantly different from similar bindings found in small molecules and that it can be modeled by theoretical methods.     

Crystal structure and functional analysis of the SurE protein identify a novel phosphatase family

Homologs of the Escherichia coli surE gene are present in many eubacteria and archaea. Despite the evolutionary conservation, little information is available on the structure and function of their gene products. We have determined the crystal structure of the SurE protein from Thermotoga maritima. The structure reveals the dimeric arrangement of the subunits and an active site around a bound metal ion. We also demonstrate that the SurE protein exhibits a divalent metal ion-dependent phosphatase activity that is inhibited by vanadate or tungstate. In the vanadate- and tungstate-complexed structures, the inhibitors bind adjacent to the divalent metal ion. Our structural and functional analyses identify the SurE proteins as a novel family of metal ion-dependent phosphatases.     

Can the propensity of protein crystallization be increased by using systematic screening with metals?

Protein crystallization is one of the major bottlenecks in protein structure elucidation with new strategies being constantly developed to improve the chances of crystallization. Generally, well‐ordered epitopes possessing complementary surface and capable of producing stable inter‐protein interactions generate a regular three‐dimensional arrangement of protein molecules which eventually results in a crystal lattice. Metals, when used for crystallization, with their various coordination numbers and geometries, can generate such epitopes mediating protein oligomerization and/or establish crystal contacts. Some examples of metal‐mediated oligomerization and crystallization together with our experience on metal‐mediated crystallization of a putative rRNA methyltransferase from Sinorhizobium meliloti are presented. Analysis of crystal structures from protein data bank (PDB) using a non‐redundant data set with a 90% identity cutoff, reveals that around 67% of proteins contain at least one metal ion, with ～14% containing combination of metal ions. Interestingly, metal containing conditions in most commercially available and popular crystallization kits generally contain only a single metal ion, with combinations of metals only in a very few conditions. Based on the results presented in this review, it appears that the crystallization screens need expansion with systematic screening of metal ions that could be crucial for stabilizing the protein structure or for establishing crystal contact and thereby aiding protein crystallization.     

Structural analysis of ABC-family periplasmic zinc binding protein provides new insights into mechanism of ligand uptake and release

ATP-binding cassette superfamily of periplasmic metal transporters are known to be vital for maintaining ion homeostasis in several pathogenic and non-pathogenic bacteria. We have determined crystal structure of the high-affinity zinc transporter ZnuA from Escherichia coli at 1.8 A resolution. This structure represents the first native (non-recombinant) protein structure of a periplasmic metal binding protein. ZnuA reveals numerous conformational features, which occur either in Zn(2+) or in Mn(2+) transporters, and presents a unique conformational state. A comprehensive comparison of ZnuA with other periplasmic ligand binding protein structures suggests vital mechanistic differences between bound and release states of metal transporters. The key new attributes in ZnuA include a C-domain disulfide bond, an extra alpha-helix proximal to the highly charged metal chelating mobile loop region, alternate conformations of secondary shell stabilizing residues at the metal binding site, and domain movements potentially controlled by salt bridges. Based on in-depth structural analyses of five metal binding transporters, we present here a mechanistic model termed as "partial domain slippage" for binding and release of Zn(2+).     

Accurate reproduction of 161 small-molecule complex crystal structures using the EUDOC program: expanding the use of EUDOC to supramolecular chemistry

EUDOC is a docking program that has successfully predicted small-molecule-bound protein complexes and identified drug leads from chemical databases. To expand the application of the EUDOC program to supramolecular chemistry, we tested its ability to reproduce crystal structures of small-molecule complexes. Of 161 selected crystal structures of small-molecule guest-host complexes, EUDOC reproduced all these crystal structures with guest structure mass-weighted root mean square deviations (mwRMSDs) of <1.0 A relative to the corresponding crystal structures. In addition, the average interaction energy of these 161 guest-host complexes (-50.1 kcal/mol) was found to be nearly half of that of 153 previously tested small-molecule-bound protein complexes (-108.5 kcal/mol), according to the interaction energies calculated by EUDOC. 31 of the 161 complexes could not be reproduced with mwRMSDs of <1.0 A if neighboring hosts in the crystal structure of a guest-host complex were not included as part of the multimeric host system, whereas two of the 161 complexes could not be reproduced with mwRMSDs of <1.0 A if water molecules were excluded from the host system. These results demonstrate the significant influence of crystal packing on small molecule complexation and suggest that EUDOC is able to predict small-molecule complexes and that it is useful for the design of new materials, molecular sensors, and multimeric inhibitors of protein-protein interactions.     

Structural implications for heavy metal-induced reversible assembly and aggregation of a protein: the case of Pyrococcus horikoshii CutA

CutA is a small protein that appears to be involved in the mechanism of divalent metal cation tolerance in microorganisms. Here we report the crystal structure of Pyrococcus horikoshii CutA (PhoCutA), with and without Cu(2+), and its metal-binding properties. Crystallographic analyses revealed that PhoCutA forms a stable trimeric structure with intertwined antiparallel beta-strands. The crystal structure of the Cu(2+)-PhoCutA complex shows that the Cu(2+) is located at a trimer-trimer interface and is recognized by the side chains of one Asp(48) from each trimer. In an in vitro experiment, PhoCutA bound to several heavy metals, most of which led to reversible aggregation of the protein; i.e. the aggregates could be completely solubilized by addition of ethylenediamine tetraacetic acid (EDTA) or dialysis against metal free buffer. Substitution of Asp(48) with Ala led to a decrease in the amount of aggregates, suggesting the significant contribution of Asp(48) to the reversible aggregation. To the best of our knowledge, this is the first report which provides the structural evidence for heavy metal-induced multimerization of a protein.     

Structural basis for copper transfer by the metallochaperone for the Menkes/Wilson disease proteins

The Hah1 metallochaperone protein is implicated in copper delivery to the Menkes and Wilson disease proteins. Hah1 and the N-termini of its target proteins belong to a family of metal binding domains characterized by a conserved MT/HCXXC sequence motif. The crystal structure of Hah1 has been determined in the presence of Cu(I), Hg(II), and Cd(II). The 1.8 A resolution structure of CuHah1 reveals a copper ion coordinated by Cys residues from two adjacent Hah1 molecules. The CuHah1 crystal structure is the first of a copper chaperone bound to copper and provides structural support for direct metal ion exchange between conserved MT/HCXXC motifs in two domains. The structures of HgHah1 and CdHah1, determined to 1.75 A resolution, also reveal metal ion coordination by two MT/HCXXC motifs. An extended hydrogen bonding network, unique to the complex of two Hah1 molecules, stabilizes the metal binding sites and suggests specific roles for several conserved residues. Taken together, the structures provide models for intermediates in metal ion transfer and suggest a detailed molecular mechanism for protein recognition and metal ion exchange between MT/HCXXC containing domains.     

Structural and enzymatic characterization of DR1281: A calcineurin-like phosphoesterase from Deinococcus radiodurans

We have determined the crystal structure of DR1281 from Deinococcus radiodurans. DR1281 is a protein of unknown function with over 170 homologs found in prokaryotes and eukaryotes. To elucidate the molecular function of DR1281, its crystal structure at 2.3 A resolution was determined and a series of biochemical screens for catalytic activity was performed. The crystal structure shows that DR1281 has two domains, a small alpha domain and a putative catalytic domain formed by a four-layered structure of two beta-sheets flanked by five alpha-helices on both sides. The small alpha domain interacts with other molecules in the asymmetric unit and contributes to the formation of oligomers. The structural comparison of the putative catalytic domain with known structures suggested its biochemical function to be a phosphatase, phosphodiesterase, nuclease, or nucleotidase. Structural analyses with its homologues also indicated that there is a dinuclear center at the interface of two domains formed by Asp8, Glu37, Asn38, Asn65, His148, His173, and His175. An absolute requirement of metal ions for activity has been proved by enzymatic assay with various divalent metal ions. A panel of general enzymatic assays of DR1281 revealed metal-dependent catalytic activity toward model substrates for phosphatases (p-nitrophenyl phosphate) and phosphodiesterases (bis-p-nitrophenyl phosphate). Subsequent secondary enzymatic screens with natural substrates demonstrated significant phosphatase activity toward phosphoenolpyruvate and phosphodiesterase activity toward 2',3'-cAMP. Thus, our structural and enzymatic studies have identified the biochemical function of DR1281 as a novel phosphatase/phosphodiesterase and disclosed key conserved residues involved in metal binding and catalytic activity.     

The structural and biochemical characterization of human RNase H2 complex reveals the molecular basis for substrate recognition and Aicardi-Goutières syndrome defects

RNase H2 cleaves RNA sequences that are part of RNA/DNA hybrids or that are incorporated into DNA, thus, preventing genomic instability and the accumulation of aberrant nucleic acid, which in humans induces Aicardi-Goutières syndrome, a severe autoimmune disorder. The 3.1 Å crystal structure of human RNase H2 presented here allowed us to map the positions of all 29 mutations found in Aicardi-Goutières syndrome patients, several of which were not visible in the previously reported mouse RNase H2. We propose the possible effects of these mutations on the protein stability and function. Bacterial and eukaryotic RNases H2 differ in composition and substrate specificity. Bacterial RNases H2 are monomeric proteins and homologs of the eukaryotic RNases H2 catalytic subunit, which in addition possesses two accessory proteins. The eukaryotic RNase H2 heterotrimeric complex recognizes RNA/DNA hybrids and (5′)RNA-DNA(3′)/DNA junction hybrids as substrates with similar efficiency, whereas bacterial RNases H2 are highly specialized in the recognition of the (5′)RNA-DNA(3′) junction and very poorly cleave RNA/DNA hybrids in the presence of Mg²⁺ ions. Using the crystal structure of the Thermotoga maritima RNase H2-substrate complex, we modeled the human RNase H2-substrate complex and verified the model by mutational analysis. Our model indicates that the difference in substrate preference stems from the different position of the crucial tyrosine residue involved in substrate binding and recognition.     

Anti-cholinesterase hybrids as multi-target-directed ligands against Alzheimer’s disease (1998–2018)

Alzheimer’s disease (AD) is a genetically complex, progressive and irreversible neurodegenerative disorder of the brain which involves multiple associated etiological targets. The complex pathogenesis of AD gave rise to multi-target-directed ligands (MTDLs) principle to combat this dreaded disease. Within this approach, the design and synthesis of hybrids prevailed greatly because of their capability to simultaneously target the intertwined pathogenesis components of the disease. The hybrids include pharmacophoric hybridization of two or more established chemical scaffolds endowed with the desired pharmacological properties into a single moiety. In AD, the primary foundation of medication therapy and drug design strategies includes the inhibition of cholinesterase (ChE) enzymes. Hence the development of ChE inhibition based hybrids is the central choice of AD medicinal chemistry research. To illustrate the progress of ChE inhibition based hybrids and novel targets, we reviewed the medicinal chemistry and pharmacological properties of the multi-target molecules published since 1998-December 2018. We hope that this article will allow the readers to easily follow the evolution of this prominent medicinal chemistry approach to develop a more efficient inhibitor.     

Discovery of a potent CDK2 inhibitor with a novel binding mode, using virtual screening and initial, structure-guided lead scoping

Virtual screening against a pCDK2/cyclin A crystal structure led to the identification of a potent and novel CDK2 inhibitor, which exhibited an unusual mode of interaction with the kinase binding motif. With the aid of X-ray crystallography and modelling, a medicinal chemistry strategy was implemented to probe the interactions seen in the crystal structure and to establish SAR. A fragment-based approach was also considered but a different, more conventional, binding mode was observed. Compound selectivity against GSK-3beta was improved using a rational design strategy, with crystallographic verification of the CDK2 binding mode.     

Structure and electron transfer mechanism of pyruvate:ferredoxin oxidoreductase

The first crystal structure of pyruvate:ferredoxin oxidoreductase to be determined has provided significant new information on its structural organization and redox chemistry. Spectroscopic analyses of a radical reaction intermediate have shed more light on its thiamin-based mechanism of catalysis. Different approaches have been used to study the interaction between the enzyme and ferredoxin, its redox partner.     

Proton and metal ion-dependent assembly of a model diiron protein

DF1 is a small, idealized model for carboxylate-bridged diiron proteins. This protein was designed to form a dimeric four-helix bundle with a dimetal ion-binding site near the center of the structure, and its crystal structure has confirmed that it adopts the intended conformation. However, the protein showed limited solubility in aqueous buffer, and access to its active site was blocked by two hydrophobic side chains. The sequence of DF1 has now been modified to provide a very soluble protein (DF2) that binds metal ions in a rapid and reversible manner. Furthermore, the DF2 protein shows significant ferroxidase activity, suggesting that its dimetal center is accessible to oxygen. The affinity of DF2 for various first-row divalent cations deviates from the Irving-Willliams series, suggesting that its structure imparts significant geometric preferences on the metal ion-binding site. Furthermore, in the absence of metal ions, the protein folds into a dimer with concomitant binding of two protons. The uptake of two protons is expected if the structure of the apo-protein is similar to that of the crystal structure of dizinc DF1. Thus, this result suggests that the active site of DF2 is retained in the absence of metal ions.     

Crystal structure studies on rubrerythrin: enzymatic activity in relation to the zinc movement

Rubrerythrin (Rr) is a non-heme iron protein isolated from anaerobic sulfate-reducing bacteria. Rr is a dimeric molecule, each monomer contains a Fe(SCys)(4) center in the C-terminal domain and a binuclear metal center in the N-terminal domain. Rr structures with different protein sources and/or preparation procedures have been studied. Two Rr crystal structures have been solved with significant differences in their binuclear metal centers. The first structure, which was obtained from expressed protein under aerobic conditions, has a diiron-oxo center. The second structure, which was obtained from native protein of Desulfovibrio vulgaris under aerobic conditions, has an Fe-Zn center with the zinc position differing from the corresponding iron position in the former structure by approximately 2 A. The crystal structures of Rr isolated from D. vulgaris (Hildenborough, NCIB 8303), the same as the second structured but prepared under anaerobic conditions, are reported in this paper. The binuclear metal center in these structures is an Fe-Zn center. When the crystal was exposed to air, the zinc atom moved gradually, approximately 2 A, accompanied by the entrance of a water molecule (or hydroxyl group) and changes in the binuclear metal center microenvironment. This finding can explain the differences between the two different structures. The results suggest that the zinc movement may be related to the enzymatic activity of Rr.     

Refined crystal structure of Cd, Zn metallothionein at 2.0 A resolution

The crystal structure of Cd5,Zn2-metallothionein from rat liver has been refined at 2.0 A resolution of a R-value of 0.176 for all observed data. The five Cd positions in the asymmetric unit of the crystal create a pseudo-centrosymmetric constellation about a crystallographic 2-fold axis. Consequently, the distribution of anomalous differences is almost ideally centrosymmetric. Therefore, the previously reported metal positions and the protein model derived therefrom are incorrect. Direct methods were applied to the protein amplitudes to locate the Cd positions. The new positions were used to calculate a new electron density map based on the Cd anomalous scattering and partial structure to model the metal clusters and the protein. Phases calculated from this model predict the positions of three sites in a (NH4)2WS4 derivative. Single isomorphous replacement phases calculated with these tungsten sites confirm the positions of the Cd sites from the new direct methods calculations. The refined metallothionein structure has a root-mean-square deviation of 0.016 A from ideality of bonds and normal stereochemistry of phi, phi and chi torsion angles. The metallothionein crystal structure is in agreement with the structures for the alpha and beta domains in solution derived by nuclear magnetic resonance methods. The overall chain folds and all metal to cysteine bonds are the same in the two structure determinations. The handedness of a short helix in the alpha-domain (residues 41 to 45) is the same in both structures. The crystal structure provides information concerning the metal cluster geometry and cysteine solvent accessibility and side-chain stereochemistry. Short cysteine peptide sequences repeated in the structure adopt restricted conformations which favor the formation of amide to sulfur hydrogen bonds. The crystal packing reveals intimate association of molecules about the diagonal 2-fold axes and trapped ions of crystallization (modeled as phosphate and sodium). Variation in the chemical and structural environments of the metal sites is in accord with data for metal exchange reactions in metallothioneins.     

Combined quantum and molecular mechanics calculations on metalloproteins

The combination of quantum mechanics and molecular mechanics (QM/MM) methods is one of the most promising approaches to study the structure, function and properties of proteins. The number of QM/MM applications on metalloproteins is steadily increasing, especially studies with density functional methods on redox-active metal centres. Recent developments include new parameterised methods to treat covalent bonds between the quantum and classical systems, methods to obtain free energy from QM/MM results, and the combination of quantum chemistry and protein crystallography.     

Site-specific substituted cobalt(II) horse liver alcohol dehydrogenases. Preparation and characterization in solution, crystalline and immobilized state.

The specific substitution, using highly selective techniques, of catalytic and/or noncatalytic zinc ions by cobaltous ions in horse liver alcohol dehydrogenase (EC 1.1.1.1) has been studied with dissolved, crystalline and agarose-immobilised enzyme, in order to examine the effect of protein structure on the specificity of the metal exchange. The different binding sites can be clearly distinguished by the absorption spectra of their cobalt derivatives. In solution an anaerobic column chromatographic method made it possible to exchange half of the zinc in the enzyme by cobalt ions in a much shorter time than previous procedures. By raising the temperature in the exchange step, even the slowly exchanging zinc ions were substituted by cobalt, yielding products similar to cobalt alcohol dehydrogenases described earlier. Treatment of crystal suspensions of the enzyme with chelating agents (preferentially dipicolinic acid) gave an inactive protein with two zinc ions remaining bound. The enzyme could be reactivated by treatment of the crystalline protein with 5 mM zinc or cobaltous ions or by dialysis of dissolved inactive protein against 20 microM zinc or 1 mM cobaltous ions. Higher metal concentrations led to denaturation but the inactive protein could be crystallized from solution and then reactivated completely at higher metal concentrations. The preparation and absorption spectrum show that cobalt is bound specifically at the catalytic sites. Since metal substitution at these sites critically depends on the maintenance of the correct tertiary and quaternary structure, these must be preserved in the crystal lattice and partially altered in solution when the catalytic zinc ions are removed (or when excess of metal ions is applied), thus demonstrating the structure-stabilizing role of the catalytic metal ions. The enzyme immobilised on agarose, with unchanged content of active sites [Schneider-Bernl?hr et al. (1978) Eur. J. Biochem. 41, 475--484], was treated like the crystal suspensions. Although half of the zinc was removed, some activity remained. After reactivation with cobaltous ions, a loss of about 30% active sites was measured. Thus the apparently homogenous bound enzyme was rather heterogeneous in the properties of its catalytic metal binding sites. These results are taken as further proof for the dependence of the metal substitution on the proper tertiary and quaternary structure which is strained by multiple interactions in the covalently immobilised enzyme.     

Evidence from X-ray absorption fine structure spectroscopy for significant differences in the structure of concanavalin A in solution and in the crystal

We have used X-ray absorption fine structure spectroscopy (XAFS) to study and compare the structure of concanavalin A in crystals and in aqueous solution. Significant differences were found between crystal and solution in the configuration of the transition-metal site of the protein. The metal has six ligands in solution but only five in the crystal. The ligand bond lengths are shorter in the crystal than in solution. The vibrational disorder in the crystal and possibly the corresponding bond length show a negative temperature dependence whereas in solution they vary normally with temperature. The anomalous temperature dependence in the crystal suggests that as the temperature decreases the protein molecules are subject to additional stresses, which are transmitted as a tensile stress at the metal site leading to distorted geometry and lengthening and weakening of metal-ligand bonds.     

FINDSITE-metal: integrating evolutionary information and machine learning for structure-based metal-binding site prediction at the proteome level

The rapid accumulation of gene sequences, many of which are hypothetical proteins with unknown function, has stimulated the development of accurate computational tools for protein function prediction with evolution/structure‐based approaches showing considerable promise. In this article, we present FINDSITE‐metal, a new threading‐based method designed specifically to detect metal‐binding sites in modeled protein structures. Comprehensive benchmarks using different quality protein structures show that weakly homologous protein models provide sufficient structural information for quite accurate annotation by FINDSITE‐metal. Combining structure/evolutionary information with machine learning results in highly accurate metal‐binding annotations; for protein models constructed by TASSER, whose average Cα RMSD from the native structure is 8.9 Å, 59.5% (71.9%) of the best of top five predicted metal locations are within 4 Å (8 Å) from a bound metal in the crystal structure. For most of the targets, multiple metal‐binding sites are detected with the best predicted binding site at rank 1 and within the top two ranks in 65.6% and 83.1% of the cases, respectively. Furthermore, for iron, copper, zinc, calcium, and magnesium ions, the binding metal can be predicted with high, typically 70% to 90%, accuracy. FINDSITE‐metal also provides a set of confidence indexes that help assess the reliability of predictions. Finally, we describe the proteome‐wide application of FINDSITE‐metal that quantifies the metal‐binding complement of the human proteome. FINDSITE‐metal is freely available to the academic community at http://cssb.biology.gatech.edu/findsite‐metal/ . Proteins 2011. © 2010 Wiley‐Liss, Inc.