Unusual Binding Research Articles

Unusual coordination mode of tetradentate Schiff base cobalt(iii) complexes

Contrary to the stereotype, Jacobsen's catalyst, chiral (salcy)Co(III)OAc adopts an unusual binding mode. The tetradentate {ONNO} ligand does not form a square plane but wraps cobalt in a cis-β fashion while acetate is chelating.

Stereoselectivity for DNA Threading Intercalation of Short Binuclear Ruthenium Complexes

Threading intercalation is an unusual DNA binding mode with significantly slower association and dissociation rates compared with classical intercalation. The latter has been shown to correlate well with cytotoxicity, and therefore, threading intercalating compounds are of great interest in the search for new DNA binding drugs. Thus, there is a need for better understanding of the mechanisms behind this type of binding. In this work, we have investigated the threading intercalation ability of the four stereoisomers of the AT-specific binuclear ruthenium complex [μ-dppzip(phen)(4)Ru(2)](4+) using different spectroscopic techniques. This complex contains an unsymmetrical bridging ligand consisting of a dipyridophenazine and an imidazophenanthroline ring system, in which the photophysical properties of the Ru-dipyridophenazine complex moiety make it possible to distinguish the intercalating part from the nonintercalating part. We have found that Δ geometry around the ruthenium on the intercalating dipyridophenazine moiety and Λ geometry on the nonintercalating imidazophenanthroline moiety is the optimal configuration for threading intercalation of this complex and that the chirality on the ruthenium of the nonintercalating half dominates the stereospecificity in the threaded state. This is the cause of the reversed enantioselectivity compared with the parent threading intercalating complex [μ-bidppz(phen)(4)Ru(2)](4+), in which the enantioselectivity is controlled by the chirality on the intercalating half. The differences in the interactions with DNA between the two complexes are most likely due to the fact that [μ-dppzip(phen)(4)Ru(2)](4+) has a slightly shorter bridging ligand than the parent complex.

Mutagenesis of Beryllium-Specific TCRs Suggests an Unusual Binding Topology for Antigen Recognition

Unconventional Ags, such as metals, stimulate T cells in a very specific manner. To delineate the binding landscape for metal-specific T cell recognition, alanine screens were performed on a set of Be-specific TCRs derived from the lung of a chronic beryllium disease patient. These TCRs are HLA-DP2-restricted and express nearly identical TCR Vβ5.1 chains coupled with different TCR α-chains. Site-specific mutagenesis of all amino acids comprising the CDRs of the TCRA and TCRB genes showed a dominant role for Vβ5.1 residues in Be recognition, with little contribution from the TCR α-chain. Solvent-exposed residues along the α-helices of the HLA-DP2 α- and β-chains were also mutated to alanine. Two β-chain residues, located near the proposed Be binding site of HLA-DP2, played a dominant role in T cell recognition with no contribution from the HLA-DP2 α-chain. These findings suggest that Be-specific T cells recognize Ag using an unconventional binding topology, with the majority of interactions contributed by TCR Vβ5.1 residues and the HLA-DP2 β1-chain. Thus, unusual docking topologies are not exclusively used by autoreactive T cells, but also for the recognition of unconventional metal Ags, such as Be.

Capreomycin — A polypeptide antitubercular antibiotic with unusual binding properties toward copper(II)

Capreomycin is an important therapeutic agent having intriguing and diverse molecular features. Its polypeptidic structure rich in nitrogen donors makes the drug a promising chelating agent for a number of transition metal ions, especially for copper(II). The results of the model investigational studies suggest that capreomycin anchors Cu 2+ ion with an amino function of the α,β-diaminopropionic acid residue at pH around 5. At physiological pH copper(II) ion is coordinated by two deprotonated amide nitrogen atoms of the α,β-diaminopropionic acid, the serine residue as well as the amino function deriving from the β-lysine. Above that pH value we observe a rearrangement within the coordination sphere leading to movement of Cu 2+ to the center of the peptide ring with concurrent coordination of four nitrogen donors. Spin–lattice relaxation enhancements and potentiometric measurements clearly indicate that deprotonated amide nitrogen atom from the β-ureidodehydroalanine moiety is the fourth donor atom.

Structural analysis of human dihydrofolate reductase as a binary complex with the potent and selective inhibitor 2,4-diamino-6-{2'-O-(3-carboxypropyl)oxydibenz[b,f]-azepin-5-yl}methylpteridine reveals an unusual binding mode.

In order to understand the structure-activity profile observed for a series of substituted dibenz[b,f]azepine antifolates, the crystal structure of the binary complex of human dihydrofolate reductase (hDHFR) with the potent and selective inhibitor 2,4-diamino-6-{2'-O-(3-carboxypropyl)oxydibenz[b,f]-azepin-5-yl}methylpteridine (PT684) was determined to 1.8 Å resolution. These data revealed that the carboxylate side chain of PT684 occupies two alternate positions, neither of which interacts with the conserved Arg70 in the active-site pocket, which in turn hydrogen bonds to water. These observations are in contrast to those reported for the ternary complex of mouse DHFR (mDHFR) with NADPH [Cody et al. (2008), Acta Cryst. D64, 977-984], in which the 3-carboxypropyl side chain of PT684 was hydrolyzed to its hydroxyl derivative, PT684a. The crystallization conditions differed for the human and mouse DHFR crystals (100 mM K2HPO4 pH 6.9, 30% ammonium sulfate for hDHFR; 15 mM Tris pH 8.3, 75 mM sodium cacodylate, PEG 4K for mDHFR). Additionally, the side chains of Phe31 and Gln35 in the hDHFR complex have a single conformation, whereas in the mDHFR complex they occupied two alternative conformations. These data show that the hDHFR complex has a decreased active-site volume compared with the mDHFR complex, as reflected in a relative shift of helix C (residues 59-64) of 1.2 Å, and a shift of 1.5 Å compared with the ternary complex of Pneumocystis carinii DHFR (pcDHFR) with the parent dibenz[b,f]azepine PT653. These data suggest that the greater inhibitory potency of PT684 against pcDHFR is consistent with the larger active-site volume of pcDHFR and the predicted interactions of the carboxylate side chain with Arg75.

Crystallographic Trapping of Heme Loss Intermediates during the Nitrite-Induced Degradation of Human Hemoglobin

Heme is an important cofactor in a large number of essential proteins and is often involved in small molecule binding and activation. Loss of heme from proteins thus negatively affects the function of these proteins but is also an important component of iron recycling. The characterization of intermediates that form during the loss of heme from proteins has been problematic, in a large part, because of the instability of such intermediates. We have characterized, by X-ray crystallography, three compounds that form during the nitrite-induced degradation of human α(2)β(2) hemoglobin (Hb). The first is an unprecedented complex that exhibits a large β heme displacement of 4.8 Å toward the protein exterior; the heme displacement is stabilized by the binding of the distal His residue to the heme Fe, which in turn allows for the unusual binding of an exogenous ligand on the proximal face of the heme. We have also structurally characterized complexes that display regiospecific nitration of the heme at the 2-vinyl position; we show that heme nitration is not a prerequisite for heme loss. Our results provide structural insight into a possible pathway for nitrite-induced loss of heme from human Hb.

An extended dsRBD with a novel zinc-binding motif mediates nuclear retention of fission yeast Dicer

Dicer proteins function in RNA interference (RNAi) pathways by generating small RNAs (sRNAs). Here, we report the solution structure of the C-terminal domain of Schizosaccharomyces pombe Dicer (Dcr1). The structure reveals an unusual double-stranded RNA binding domain (dsRBD) fold embedding a novel zinc-binding motif that is conserved among dicers in yeast. Although the C-terminal domain of Dcr1 still binds nucleic acids, this property is dispensable for proper functioning of Dcr1. In contrast, disruption of zinc coordination renders Dcr1 mainly cytoplasmic and leads to remarkable changes in gene expression and loss of heterochromatin assembly. In summary, our results reveal novel insights into the mechanism of nuclear retention of Dcr1 and raise the possibility that this new class of dsRBDs might generally function in nucleocytoplasmic trafficking and not substrate binding. The C-terminal domain of Dcr1 constitutes a novel regulatory module that might represent a potential target for therapeutic intervention with fungal diseases.

Slow Threading Intercalation of Monomeric Ru(II) Complexes with 10,13-Diarylsubstituted dppz Ligands

Threading intercalation is an unusual DNA binding mode that displays extremely slow dissociation kinetics, which is an important feature for cytotoxicity, making threading intercalating compounds interesting as model compounds in the search for new DNA binding drugs. This type of binding has for ruthenium complexes previously only been observed for complexes containing 11-substituted dipyridophenazine ligands. In this work we have synthesized and investigated the DNA binding properties of two new 10,13-diarylsubstituted dipyridophenazine ruthenium complexes, using spectroscopic techniques, and found that this substitution pattern provides a new strategy for development of drugs with slow dissociation kinetics. However, the nature of the aryl substituents largely affects the binding properties of the complexes as it was found that a dithienyl substituted complex exhibit slow dissociation kinetics characteristic for threading intercalation while its diphenyl substituted analogue seems to bind DNA by partial intercalation of one phenyl substituent resulting in faster dissociation.

Solution Structure of a Repeated Unit of the ABA-1 Nematode Polyprotein Allergen of Ascaris Reveals a Novel Fold and Two Discrete Lipid-Binding Sites

BackgroundNematode polyprotein allergens (NPAs) are an unusual class of lipid-binding proteins found only in nematodes. They are synthesized as large, tandemly repetitive polyproteins that are post-translationally cleaved into multiple copies of small lipid binding proteins with virtually identical fatty acid and retinol (Vitamin A)-binding characteristics. They are probably central to transport and distribution of small hydrophobic compounds between the tissues of nematodes, and may play key roles in nutrient scavenging, immunomodulation, and IgE antibody-based responses in infection. In some species the repeating units are diverse in amino acid sequence, but, in ascarid and filarial nematodes, many of the units are identical or near-identical. ABA-1A is the most common repeating unit of the NPA of Ascaris suum, and is closely similar to that of Ascaris lumbricoides, the large intestinal roundworm of humans. Immune responses to NPAs have been associated with naturally-acquired resistance to infection in humans, and the immune repertoire to them is under strict genetic control.Methodology/Principal FindingsThe solution structure of ABA-1A was determined by protein nuclear magnetic resonance spectroscopy. The protein adopts a novel seven-helical fold comprising a long central helix that participates in two hollow four-helical bundles on either side. Discrete hydrophobic ligand-binding pockets are found in the N-terminal and C-terminal bundles, and the amino acid sidechains affected by ligand (fatty acid) binding were identified. Recombinant ABA-1A contains tightly-bound ligand(s) of bacterial culture origin in one of its binding sites.Conclusions/SignificanceThis is the first mature, post-translationally processed, unit of a naturally-occurring tandemly-repetitive polyprotein to be structurally characterized from any source, and it belongs to a new structural class. NPAs have no counterparts in vertebrates, so represent potential targets for drug or immunological intervention. The nature of the (as yet) unidentified bacterial ligand(s) may be pertinent to this, as will our characterization of the unusual binding sites.

Beryllium-specific T cells adopt an unusual binding topology for antigen recognition (100.25)

Abstract Unconventional antigens, such as metals, stimulate T cells in a very specific manner. To delineate the binding landscape for metal-specific T cell recognition, alanine screens were performed on a set of beryllium (Be)-specific T cell receptors (TCR) derived from the lung of a chronic beryllium disease patient. These TCRs were HLA-DP2-restricted and expressed nearly identical Vβ5.1+ chains coupled with different Vα chains. Site-specific mutagenesis of all amino acids comprising the complementarity determining regions of TCR Vα and Vβ chains showed a dominant role for Vβ5.1 residues in Be recognition, with little contribution from the Vα domain, suggesting an abnormal binding topology. To confirm these findings, solvent-exposed residues along the α-helices of the α- and β-chains of HLA-DP2 (i.e., potential TCR contact sites) were mutated to alanine. Two β-chain residues, located near the proposed Be binding site of HLA-DP2, played a dominant role in T cell recognition, while the α-chain mutations had no effect on T cell activation. These findings strongly suggest that Be-specific T cells recognize antigen using an unconventional binding topology, with the majority of interactions contributed by residues in the TCR Vβ5.1 domain and HLA-DP2 β1-chain. We propose that unusual docking topologies are not used exclusively by T cells recognizing self-antigens, but also for the recognition of metal antigens, such as beryllium. Supported by NIH grants: HL062410 and HL092997.

Inside Cover: Sulfonylthiadiazoles with an Unusual Binding Mode as Partial Dual Peroxisome Proliferator‐Activated Receptor (PPAR) γ/δ Agonists with High Potency and in vivo Efficacy (ChemMedChem 4/2011)

Inside Cover: Sulfonylthiadiazoles with an Unusual Binding Mode as Partial Dual Peroxisome Proliferator‐Activated Receptor (PPAR) γ/δ Agonists with High Potency and in vivo Efficacy (ChemMedChem 4/2011)

Structural Basis for the Interaction Between Carbonic Anhydrase and 1,2,3,4-tetrahydroisoquinolin-2-ylsulfonamides

Isoquinolinesulfonamides inhibit human carbonic anhydrases (hCAs) and display selectivity toward therapeutically relevant isozymes. The crystal structure of hCA II in complex with 6,7-dimethoxy-1-methyl-1,2,3,4-tetrahydroisoquinolin-2-ylsulfonamide revealed unusual inhibitor binding. Structural analyses allowed for discerning the fine details of the inhibitor binding mode to the active site, thus providing clues for the future design of even more selective inhibitors for druggable isoforms such as the cancer associated hCA IX and neuronal hCA VII.

Sulfonylthiadiazoles with an Unusual Binding Mode as Partial Dual Peroxisome Proliferator‐Activated Receptor (PPAR) γ/δ Agonists with High Potency and in vivo Efficacy

Compounds that simultaneously activate the peroxisome proliferator-activated receptor (PPAR) subtypes PPARγ and PPARδ have the potential to effectively target dyslipidemia and type II diabetes in a single pharmaceutically active molecule. The frequently observed side effects of selective PPARγ agonists, such as edema and weight gain, are expected to be overcome by using partial instead of full agonists for this nuclear receptor family. Herein we report the discovery, synthesis, and optimization of a novel series of sulfonylthiadiazoles that are active as partial agonists. The initial compound 6 was discovered by high-throughput screening as a moderate partial PPARδ agonist; its optimization was based on the X-ray crystal structure in complex with PPARδ. In contrast to other PPARδ agonists, this ligand does not interact directly with residues from the activation helix AF-2, which might be linked to its partial agonistic effect. Interestingly, the thiadiazole moiety fills a novel subpocket, which becomes accessible after moderate conformational rearrangement. The optimization was focused on introducing conformational constraints and replacing intramolecular hydrogen bonding interactions. Highly potent molecules with activity as dual partial PPARγ/δ agonists in the low nanomolar range were then identified. One of the most active members, compound 20 a, displayed EC₅₀ values of 1.6 and 336 nM for PPARδ and γ, respectively. The X-ray crystal structure of its complex with PPARδ confirms our design hypothesis. Compound 20 a clearly displayed in vivo activity in two chronic mice studies. Lipids were modified in a beneficial way in normolipidemic mice, and the development of overt diabetes could be prevented in pre-diabetic db/db mice. However, body weight gain was similar to that observed with the PPARγ agonist rosiglitazone. Hence, active compounds from this series can be considered as valuable tools to elucidate the complex roles of dual PPARγ/δ agonists for potential treatment of metabolic syndrome.

Mutagenesis Study on the Conformation of Distal Histidine in Dehaloperoxidase-Hemoglobin

Dehaloperoxidase-hemoglobin (DHP A), found in terebellid polychaete Amphitrite ornata, acts as a bi-functional protein. It has the globin fold and acts as an oxygen carrier, but it can also act as the peroxidase, which can oxidatively dehalogenate halophenols found in benthic ecosystems to the corresponding quinones. The distal histidine His55 is the only residue that determines the substrate and heme ligand binding function. His55 also plays a key role in peroxidase catalysis. His55 has two conformations, open conformation and closed conformation, which is related to inhibitor and substrate binding respectively. In myoglobin, Phe46 and Arg45 have been suggested the critical residues effect the C alpha - C beta rotation of distal His64. Moreover, in myoglobin the residue adjacent to His 64 (the distal histidine) is a glycine (Gly65). Although this is a conserved feature in many globins, DHP A has a threonine in this position. To understand the role of the amino acid in this position the mutants T56G, T56A, T56S and T56V were prepared. Kinetic assay results show that all of these mutants have higher activity than wild-type DHP A. Molecular dynamics simulations, Resonance Raman spectroscopy, X-ray crystallography and other methods will be presented to understand how a change in the Thr56 position affects the both the conformation of His55 and peroxidase activity. The inhibitor in DHP A binds in an unusual internal binding site. We will determine the inhibitor binding constants and measure the enzyme inhibition in kinetic assays to understand the role played by the distal histidine conformation inhibition as well.

Unusual Heme Binding in the Bacterial Iron Response Regulator Protein: Spectral Characterization of Heme Binding to the Heme Regulatory Motif

We characterized heme binding in the bacterial iron response regulator (Irr) protein, which is a simple heme-regulated protein having a single "heme-regulatory motif", HRM, and plays a key role in the iron homeostasis of a nitrogen-fixing bacterium. The heme titration to wild-type and mutant Irr clearly showed that Irr has two heme binding sites: one of the heme binding sites is in the HRM, where (29)Cys is the axial ligand, and the other one, the secondary heme binding site, is located outside of the HRM. The Raman line for the Fe-S stretching mode observed at 333 cm(-1) unambiguously confirmed heme binding to Cys. The lower frequency of the Fe-S stretching mode corresponds to the weaker Fe-S bond, and the broad Raman line of the Fe-S bond suggests multiple configurations of heme binding. These structural characteristics are definitely different from those of typical hemoproteins. The unusual heme binding in Irr was also evident in the EPR spectra. The characteristic g-values of the 5-coordinate Cys-ligated heme and 6-coordinate His/His-ligated heme were observed, while the multiple configurations of heme binding were also confirmed. Such multiple heme configurations are not encountered for typical hemoproteins where the heme functions as the active center. Therefore, we conclude that heme binding to HRM in the heme-regulated protein, Irr, is quite different from that in conventional hemoproteins but characteristic of heme-regulated proteins using heme as the signaling molecule.

Congested Cyclometalated Platinum(II) Ditopic Frameworks and Their Phosphorescent Responses to S-Containing Amino Acids

New phosphorescent platinum(II) molecular hosts featuring a tridentate N,C,N-coordinating ligand, a conformationally rigid organic linker, and a binding group have been prepared. The complexes have been fully characterized by various spectroscopic techniques, and the X-ray crystal structure of one derivative has been determined. Their photophysical properties have been studied, and intense green metal-perturbed 3IL emission is observed in solution at room temperature. The luminescent responses of these Pt(II) hosts to amino acids have been investigated: emission quenching and UV−vis absorption changes in polar/aqueous media are detected for terminal thiols only, and unusual preferential binding is apparent for cysteine over homocysteine. The nature of the host−guest interactions has been examined by quantitative and comparative binding studies, mass spectrometry, and DFT calculations, which indicate that these observations may be ascribed to the presence of rigidly positioned ditopic binding sites.

Double Click Reaction for the Acquisition of a Highly Potent and Selective mPTPB Inhibitor

Tuberculosis (TB), which is caused by Mycobacterium tuberculosis (Mtb), is a major worldwide threat to public health. Mycobacterium protein tyrosine phosphatase B (mPTPB) is a virulent phosphatase secreted by Mtb, which is essential for the survival and persistence of the bacterium in the host. Consequently, small-molecule inhibitors of mPTPB are expected to serve as anti-TB agents with a novel mode of action. Herein, we report the discovery of highly potent and selective mPTPB inhibitors using a novel, double Click chemistry strategy. The most potent mPTPB inhibitor from this approach possesses a K(i) value of 160 nM and a >25-fold selectivity for mPTPB over 19 other protein tyrosine phosphatases (PTBs). Molecular docking study of the enzyme-inhibitor complex provides a rationale for the high potency and selectivity of the lead compound and reveals an unusual binding mode, which may guide further optimization effort.

Structural studies of Cu(II) binding to the novel peptidyl derivative of quinoxaline: N-(3-(2,3-di(pyridin-2-yl)quinoxalin-6-yl)alanyl)glycine

The coordination properties of the novel conjugate towards copper ions were investigated. The performed studies exhibited the unusual binding properties of the ligand molecule having two potential strong coordination sites, namely dipeptidic chain and pyridyl nitrogens. On the basis of potentiometric and spectroscopic studies the binding at the low pH values to the aromatic entity is suggested, while the rise of pH (including physiological one) yielded the dimeric head-to-tail complex formation. This stable species possesses three nitrogen donors involved in Cu(II) chelation: N(pirydyl), NH 2 and N −(amide).

Enhanced Anion Binding from Unusual Coordination Modes of Bis(thiourea) Ligands in Platinum Group Metal Complexes

Treatment of a range of bis(thiourea) ligands with inert organometallic transition-metal ions gives a number of novel complexes that exhibit unusual ligand binding modes and significantly enhanced anion binding ability. The ruthenium(II) complex [Ru(η(6)-p-cymene)(κS,S',N-L(3)-H)](+) (2b) possesses juxtaposed four- and seven-membered chelate rings and binds anions as both 1:1 and 2:1 host guest complexes. The pyridyl bis(thiourea) complex [Ru(η(6)-p-cymeme)(κS,S',N(py)-L(4))](2+) (4) binds anions in both 1:1 and 1:2 species, whereas the free ligand is ineffective because of intramolecular NH⋅⋅⋅N hydrogen bonding. Novel palladium(II) complexes with nine- and ten-membered chelate rings are also reported.

Unusual Specificity of a Receptor for Nd3+ Among Other Lanthanide Ions for Selective Colorimetric Recognition

A rare example of a reversible recognition of Nd(3+) by a newly synthesized molecular receptor (L(1)), having a diazo group as the reporter functionality, is reported. Studies revealed that this receptor eventually forms a [Nd(3+)](2)L(1) type complex, through the formation of the intermediate complex [Nd(3+)] x L(1) following a two-step equilibrium process. Respective equilibrium constants for two successive processes were evaluated based on data obtained from the systematic fluorescence titration. Formation of [Nd(3+)](2)L(1) caused a detectable change in color, and associated affinity constants were also evaluated from spectrophotometric titration data. A rather unusual binding mode of L(1) for Nd(3+) ion is established by various spectroscopic studies. The remarkable specificity of L(1) for Nd(3+), constitutes a rare example of a highly selective receptor for this ion in the presence of excess of all other lanthanide ions.