Quantitative and sequence-specific analysis of DNA-ligand interaction by means of fluorescent intercalator probes

A novel method of analysis of double-stranded DNA-ligand interaction is presented. The interaction is monitored by the fluorescence of a DNA bis-intercalator oxazole homodimer YoYo-3. The fluorescence intensity or its decay time reflects the modification of the DNA double helix. The DNA sequence is scanned by hybridization with short oligomers having consecutively overlapping complementary sequences to analyse the sequence specificity of binding. In our experiments we used as ligands the minor groove binders netropsin, SN6999 (both with AT-preference), the GC-specific ligand chromomycin A3 as well as the derivative SN6113 (non-specific interaction), which displace the bis-intercalator YoYo-3 or influence the duplex structure in such away that the fluorescence intensity and lifetime decrease in comparison to a ligand-free screening. The changes of fluorescence emission clearly define the binding motif and indicate minor groove interactions with a reduced DNA binding site. Titration of the ligand quantitatively characterizes its binding by determining the dependence of the binding constant on the oligonucleotide sequence.     

Recognition of alpha 2-macroglobulin by the low density lipoprotein receptor-related protein requires the cooperation of two ligand binding cluster regions

The low density lipoprotein receptor-related protein (LRP) is a scavenger receptor that binds several ligands including the activated form of the pan-proteinase inhibitor alpha(2)-macroglobulin (alpha(2)M*) and amyloid precursor protein, two ligands genetically linked to Alzheimer's disease. To delineate the contribution of LRP to this disease, it will be necessary to identify the sites on this receptor which are responsible for recognizing these and other ligands to assist in the development of specific inhibitors. Structurally, LRP contains four clusters of cysteine-rich repeats, yet studies thus far suggest that only two of these clusters (clusters II and IV) bind ligands. Identifying binding sites within LRP for certain ligands, such as alpha(2)M*, has proven to be difficult. To accomplish this, we mapped the binding site on LRP for two inhibitors of alpha(2)M* uptake, monoclonal antibody 8G1 and an amino-terminal fragment of receptor-associated protein (RAP D1D2). Surprisingly, the inhibitors recognized different clusters of ligand binding repeats: 8G1 bound to repeats within cluster I, whereas the RAP fragment bound to repeats within cluster II. A recombinant LRP mini-receptor containing the repeats from cluster I along with three ligand binding repeats from cluster II was effective in mediating the internalization of (125)I-labeled alpha(2)M*. Together, these studies indicate that ligand binding repeats from both cluster I and II cooperate to generate a high affinity binding site for alpha(2)M*, and they suggest a strategy for developing specific inhibitors to block alpha(2)M* binding to LRP by identifying molecules capable of binding repeats in cluster I.     

Identification of a cDNA encoding an active asparaginyl endopeptidase of Schistosoma mansoni and its expression in Pichia pastoris

Novel affinity ligands, consisting of ATP-resembling part coupled with specificity determining peptide fragment, were proposed for purification of protein kinases. Following this approach affinity sorbents based on two closely similar ligands AdoC-Aoc-Arg4-Lys and AdoC-Aoc-Arg4-NH(CH2)6NH2, where AdoC stands for adenosine-5'-carboxylic acid and Aoc for amino-octanoic acid, were synthesized and tested for purification of recombinant protein kinase A catalytic subunit directly from crude cell extract. Elution of the enzyme with MgATP as well as L-arginine yielded homogeneous protein kinase A preparation in a single purification step. Also protein kinase A from pig heart homogenate was selectively isolated using MgATP as eluting agent. Protein kinase with acidic specificity determinant (CK2) as well as other proteins possessing nucleotide binding site (L-type pyruvate kinase) or sites for wide variety of different ligands (bovine serum albumin) did not bind to the column, pointing to high selectivity of the bi-functional binding mode of the affinity ligand.     

Structural requirements for ligand binding by a probable plant vacuolar sorting receptor

How sorting receptors recognize amino acid determinants on polypeptide ligands and respond to pH changes for ligand binding or release is unknown. The plant vacuolar sorting receptor BP-80 binds polypeptide ligands with a central Asn-Pro-Ile-Arg (NPIR) motif. tBP-80, a soluble form of the receptor lacking transmembrane and cytoplasmic sequences, binds the peptide SSSFADSNPIRPVTDRAASTYC as a monomer with a specificity indistinguishable from that of BP-80. tBP-80 contains an N-terminal region homologous to ReMembR-H2 (RMR) protein lumenal domains, a unique central region, and three C-terminal epidermal growth factor (EGF) repeats. By protease digestion of purified secreted tBP-80, and from ligand binding studies with a secreted protein lacking the EGF repeats, we defined three protease-resistant structural domains: an N-terminal/RMR homology domain connected to a central domain, which together determine the NPIR-specific ligand binding site, and a C-terminal EGF repeat domain that alters the conformation of the other two domains to enhance ligand binding. A fragment representing the central domain plus the C-terminal domain could bind ligand but was not specific for NPIR. These results indicate that two tBP-80 binding sites recognize two separate ligand determinants: a non-NPIR site defined by the central domain-EGF repeat domain structure and an NPIR-specific site contributed by the interaction of the N-terminal/RMR homology domain and the central domain.     

Spectroscopic and kinetic features of allocolchicine binding to tubulin

Allocolchicine is a structural isomer of colchicine in which colchicine's tropone C ring is replaced with an aromatic ester. In spite of the structural differences between the two ligands, the association parameters for both molecules binding to tubulin are quite similar. The association constant for allocolchicine binding to tubulin was determined by fluorescence titration to be 6.1 x 10(5) M-1 at 37 degrees C, which is about a factor of 5 less than that of the colchicine-tubulin association. In particular, analysis of the kinetics of the association of allocolchicine with tubulin yielded nearly equivalent activation parameters for the two ligands. The activation energy of the allocolchicine binding reaction was found to be 18.4 +/- 1.5 kcal/mol, which is only slightly less than the activation energy for colchicine binding to tubulin. This finding argues against conformational flexibility of the C ring as the structural feature of colchicine responsible for the slow kinetics of colchicinoid-tubulin binding reactions. Tubulin binding promote a dramatic enhancement of allocolchicine fluorescence. Unlike colchicine, the emission energy and intensity of the tubulin-bound allocolchicine fluorescence can be mimicked by solvent, and a general hydrophobic environment for the ligand binding site is indicated. The excitation spectrum of the protein-bound species, however, is shown to possess two bands which center at higher and lower energy than the energy maximum of the spectrum of the ligand in apolar solvents, indicating that properties of the colchicine binding site in addition to a low dielectric constant contribute to the fluorescence of the bound species.(ABSTRACT TRUNCATED AT 250 WORDS)     

A metal ion-binding site in the kringle region of bovine prothrombin fragment 1.

45Ca(II) binding studies (equilibrium dialysis) on the kringle domain of bovine prothrombin fragment 1 were conducted using a mixture of peptides (residues 43-156 and 46-156) resulting from limited alpha-chymotryptic hydrolysis of fragment 1. Analysis of the Scatchard plot of these data indicates a single, low affinity Ca(II)-binding site to be present. Similar results were obtained from studies on the decarboxylated fragment 1 derivative, 10-gamma-MGlu-fragment 1. Acetylation of bovine fragment 1 in the absence of Ca(II) or Mg(II) ions results in the loss of the metal ion-promoted quenching of the intrinsic Trp fluorescence of the protein and the Ca(II)-mediated binding to phosphatidylserine/phosphatidylcholine (PS/PC) vesicles. The acetylation of the NH2 alpha-group of Ala-1 has been shown (Welsch, D. J., and Nelsestuen, G. L. (1988) Biochemistry 27, 4946-4952) to abolish the PS/PC binding property of fragment 1. The present study demonstrates that acetylation of a second site possibly Ser-79 or Thr-81 using the conditions described in the preceding paper results in loss of both the fluorescence transition and the Ca(II)-mediated PS/PC binding of the resulting protein derivative. Removal of the O-acetyl group at the Ser-79/Thr-81 site is accomplished by aminolysis with 0.2 M hydroxylamine, pH 10, 50 degrees C; the fluorescence transition is partially restored. PS/PC binding is partially restored if the NH2 alpha-group of Ala-1 is trinitrophenylated but is not restored if the NH2 alpha-group of Ala-1 is acetylated. We conclude that the Ser-79/Thr-81 site may represent a portion of the metal ion-binding site within the kringle domain of fragment 1. Occupancy of this site by a Ca(II) ion appears to be important in the binding of the protein to PS/PC vesicles.     

Listeria monocytogenes ActA protein interacts with phosphatidylinositol 4,5-bisphosphate in vitro

The N-terminal region of the Listeria monocytogenes ActA protein, in conjunction with host cell factors, is sufficient for actin polymerization at the bacterial surface. Previous data suggested that ActA could protect barbed ends from capping proteins. We tested this hypothesis by actin polymerization experiments in the presence of the ActA N-terminal fragment and capping protein. ActA does not protect barbed ends from capping protein. In contrast, this polypeptide prevents PIP(2) from inhibiting the capping activity of capping protein. Gel filtration and tryptophan fluorescence experiments showed that the purified ActA N-terminal fragment binds to PIP(2) and PIP, defining phosphoinositides as novels ligands for this functional domain of ActA. Phosphoinositide binding to the N-terminal region of ActA may induce conformational changes in ActA and/or facilitate binding of other cell components, important for ActA-induced actin polymerization.     

Promiscuous binding of ligands by beta-lactoglobulin involves hydrophobic interactions and plasticity

Bovine beta-lactoglobulin (betaLG) binds a variety of hydrophobic ligands, though precisely how is not clear. To understand the structural basis of this promiscuous binding, we studied the interaction of betaLG with palmitic acid (PA) using heteronuclear NMR spectroscopy. The titration was monitored using tryptophan fluorescence and a HSQC spectrum confirmed a 1:1 stoichiometry for the PA-betaLG complex. Upon the binding of PA, signal disappearances and large changes in chemical shifts were observed for the residues located at the entrance and bottom of the cavity, respectively. This observation indicates that the lower region makes a rigid connection with PA whereas the entrance is more flexible. The result is in contrast to the binding of PA to intestinal fatty acid-binding protein, another member of the calycin superfamily, in which structural consolidation occurs upon ligand binding. On the other hand, the ability of betaLG to accommodate various hydrophobic ligands resembles that of GroEL, in which a large hydrophobic cavity and flexible binding site confer the ability to bind various hydrophobic substrates. Considering these observations, it is suggested that, in addition to the presence of the hydrophobic cavity, the plasticity of the entrance region makes possible the binding of hydrophobic ligands of various shapes. Thus, in contrast to the specific binding seen for many enzymes, betaLG provides an example of binding with low specificity but high affinity, which may play an important role in protein-ligand and protein-protein networks.     

Escherichia coli glutaminyl-tRNA synthetase is electrostatically optimized for binding of its cognate substrates

Natural evolution has resulted in protein molecules displaying a wide range of binding properties that include extremes of affinity and specificity. A detailed understanding of the principles underlying protein structure-function relationships, particularly with respect to binding properties, would greatly enhance molecular engineering and ligand design studies. Here, we have analyzed the interactions of an aminoacyl-tRNA synthetase for which strong evolutionary pressure has enforced high specificity for substrate binding and catalysis. Electrostatic interactions have been identified as one efficient mechanism for enhancing binding specificity; as such, the effects of charged and polar groups were the focus of this study. The binding of glutaminyl-tRNA synthetase from Escherichia coli to several ligands, including the natural substrates, was analyzed. The electrostatic complementarity of the enzyme to its ligands was assessed using measures derived from affinity optimization theory. The results were independent of the details of the calculational parameters, including the value used for the protein dielectric constant. Glutamine and ATP, two of the natural ligands, were found to be extremely complementary to their binding sites, particularly in regions seen to make electrostatic interactions in the structure. These data suggest that the optimization of electrostatic interactions has played an important role in guiding the evolution of this enzyme. The results also show that the enzyme is able to effectively select for high affinity and specificity for the same chemical moieties both in the context of smaller substrates, and in that of a larger reactive intermediate. The regions of greatest non-complementarity between the enzyme and ligands are the portions of the ligand that make few polar contacts with the binding site, as well as the sites of chemical reaction, where overly strong electrostatic binding interactions with the substrate could hinder catalysis. The results also suggest that the negative charge on the phosphorus center of glutaminyl-adenylate plays an important role in the tight binding of this intermediate, and thus that adenylate analogs that preserve the negative charge in this region may bind substantially tighter than analogs where this group is replaced with a neutral group, such as the sulfamoyl family, which can make similar hydrogen bonds but is uncharged.     

Assessing the binding and endocytosis activity of cellular receptors using GFP-ligand fusions

We have developed a simple scheme for characterizing ligand-receptor binding and post-binding activity on living cells. Our approach makes use of green fluorescent protein (GFP) as an auto-fluorescent tag to label protein ligands. We have constructed GFP-tagged ligands that can be expressed in bacteria as soluble fusion proteins. A cell-binding assay using fluorescence-activated cell sorting (FACS) demonstrates that GFP-tagged proteins retain their wild-type receptor-binding specificity. Using this assay, we measure ligand binding on unfixed cells and demonstrate receptor specificity using specific competitors. To determine the ability of receptor targets to internalize, we developed a second FACS-based assay to detect the rate and percentage of internalized ligand in living cells. Noninternalizing control ligands and fluorescence microscopy of treated cells confirm that our assay is reliable for determining receptor internalization activity.     

Binding of regulatory ligands to rabbit muscle phosphofructokinase. A model for nucleotide binding as a function of temperature and pH.

The binding of nucleoside triphosphates to rabbit muscle phosphofructokinase has been determined in 0.05 M phosphate buffers by changes in intrinsic protein fluorescence and by direct binding measurements. These experiments have been performed over a wide range of pH, temperature, and effector concentration. Quenching of protein fluorescence is shown to measure binding of nucleotides to a site which is not the active site but rather a site responsible for inhibition of the kinetic activity. This site is relatively specific for either ATP or MgATP with free ATP binding about 10-fold more tightly than MgATP. A model to describe binding to this site as a function of pH and temperature is proposed. This model assumes that the apparent affinity for ATP is determined by protonation of two ionizable groups (per subunit) and that ATP binds exclusively to protonated enzyme forms. Several ligands which affect the apparent affinity for nucleotide binding at the inhibitory site act by shifting the apparent pK of the ionizable groups. NH4+ and citrate do not influence nucleotide binding to the inhibitory site. At pH 6.9 in 0.05 M phosphate, low concentrations of MgATP or MgGTP enhance the protein fluorescence due to binding at the active site. The fluorescence studies and direct binding studies show that there is one active site and one inhibitory site per subunit. As described elsewhere (Pettigrew, D. W., and Frieden, C. (1978) J. Biol. Chem. 253, 3623-3627), there is a third nucleotide binding site on each subunit which is specific for cAMP, AMP, and ADP.     

Heme binding by the N-terminal fragment 1-44 of human growth hormone

Fragment 1-44 of human growth hormone (hGH), prepared in vitro by limited proteolysis of the hormone with pepsin at low pH, encompasses in full the N-terminal helix of this four-helix bundle protein [Spolaore, B., Polverino de Laureto, P., Zambonin, M., and Fontana, A. (2004) Biochemistry 40, 9460-9468]. Here, we report the new and interesting observation that fragment 1-44 can bind heme. The binding property is specific for the N-terminal helix of hGH, since heme binding does not occur with fragment 45-191 or the entire protein. The spectral characteristics of Fe-protoporphyrin IX are those of a low-spin, hexacoordinated iron ligated by two imidazole rings of His residues or His and Met residues. Far-UV circular dichroism (CD) measurements revealed that fragment 1-44 acquires a helical secondary structure upon heme binding. Heme appears to be bound to the fragment in a stereospecific way, since an induced dichroic signal is observed in the Soret region of the CD spectrum. The heme-fragment complex occurs in a 1:1 molar ratio, as determined by spectrophotometric titration, as well as by electrospray-ionization mass spectrometric analysis of the complex. The fragment alone is much more susceptible to tryptic digestion than the heme complex, implying a more folded and rigid structure of this last species. It is proposed that the molecular features of fragment 1-44 determining its heme-binding property reside in the amphipathic character of the helix adopted by the fragment, as well as in the presence in its polypeptide chain of His18, His21, and Met14. These residues can act as specific ligands for the heme-iron, as observed with cytochromes.     

A microbead-based system for identifying and characterizing RNA-protein interactions by flow cytometry

We present a high throughput, versatile approach to identify RNA-protein interactions and to determine nucleotides important for specific protein binding. In this approach, oligonucleotides are coupled to microbeads and hybridized to RNA-protein complexes. The presence or absence of RNA and/or protein fluorescence indicates the formation of an oligo-RNA-protein complex on each bead. The observed fluorescence is specific for both the hybridization and the RNA-protein interaction. We find that the method can discriminate noncomplementary and mismatch sequences. The observed fluorescence reflects the affinity and specificity of the RNA-protein interaction. In addition, the fluorescence patterns footprint the protein recognition site to determine nucleotides important for protein binding. The system was developed with the human protein U1A binding to RNAs derived from U1 snRNA but can also detect RNA-protein interactions in total RNA backgrounds. We propose that this strategy, in combination with emerging coded bead systems, can identify RNAs and RNA sequences important for interacting with RNA-binding proteins on genomic scales.     

Biochemical characterisation of a hydrophobic ligand binding protein from the tapeworm Hymenolepis diminuta

The cestode Hymenolepis diminuta contains an abundant, cytoplasmic, hydrophobic ligand, binding protein (H-HLBP). Studies with polarity sensitive probes suggest a single hydrophobic binding site, the results also indicate that the single tryptophan in the molecule (Trp41) is involved in ligand binding. Of the possible physiological ligands tested, only haematin and retinoids (retinol and retinoic acid) show appreciable binding in addition to fatty acids. H-HLBP also binds a range of anthelmintics, again with K(D) values in the nM range. The interaction of anthelmintics with hydrophobic binding proteins may be important in determining drug specificity and site of action and could have a role in the development of drug resistance.     

Computational protocol for predicting the binding affinities of zinc containing metalloprotein-ligand complexes

Zinc is one of the most important metal ions found in proteins performing specific functions associated with life processes. Coordination geometry of the zinc ion in the active site of the metalloprotein-ligand complexes poses a challenge in determining ligand binding affinities accurately in structure-based drug design. We report here an all atom force field based computational protocol for estimating rapidly the binding affinities of zinc containing metalloprotein-ligand complexes, considering electrostatics, van der Waals, hydrophobicity, and loss in conformational entropy of protein side chains upon ligand binding along with a nonbonded approach to model the interactions of the zinc ion with all the other atoms of the complex. We examined the sensitivity of the binding affinity predictions to the choice of Lennard-Jones parameters, partial atomic charges, and dielectric treatments adopted for system preparation and scoring. The highest correlation obtained was R2 = 0.77 (r = 0.88) for the predicted binding affinity against the experiment on a heterogenous dataset of 90 zinc containing metalloprotein-ligand complexes consisting of five unique protein targets. Model validation and parameter analysis studies underscore the robustness and predictive ability of the scoring function. The high correlation obtained suggests the potential applicability of the methodology in designing novel ligands for zinc-metalloproteins. The scoring function has been web enabled for free access at www.scfbio-iitd.res.in/software/drugdesign/bapplz.jsp as BAPPL-Z server (Binding Affinity Prediction of Protein-Ligand complexes containing Zinc metal ions).     

Basement-membrane heparan sulfate proteoglycan binds to laminin by its heparan sulfate chains and to nidogen by sites in the protein core.

A large, low-density form of heparan sulfate proteoglycan was isolated from the Engelbreth-Holm-Swarm (EHS) tumor and demonstrated to bind in immobilized-ligand assays to laminin fragment E3, collagen type IV, fibronectin and nidogen. The first three ligands mainly recognize the heparan sulfate chains, as shown by inhibition with heparin and heparan sulfate and by the failure to bind to the proteoglycan protein core. Nidogen, obtained from the EHS tumor or in recombinant form, binds exclusively to the protein core in a heparin-insensitive manner. Studies with other laminin fragments indicate that the fragment E3 possesses a unique binding site of laminin for the proteoglycan. A major binding site of nidogen was localized to its central globular domain G2 by using overlapping fragments. This allows for the formation of ternary complexes between laminin, nidogen and proteoglycan, suggesting a key role for nidogen in basement-membrane assembly. Evidence is provided for a second proteoglycan-binding site in the C-terminal globule G3 of nidogen, but this interaction prevents the formation of such ternary complexes. Therefore, the G3-mediated nidogen binding to laminin and proteoglycan are mutually exclusive.     

Interaction of DNA with the Klenow fragment of DNA polymerase I studied by time-resolved fluorescence spectroscopy

The interaction of a fluorescent duplex DNA oligomer with the Klenow fragment of DNA polymerase I from Escherichia coli has been studied in solution by using time-resolved fluorescence spectroscopy. An aminonaphthalenesulfonate (dansyl) fluorescent probe was linked by a propyl chain to a C5-modified uridine base located at a specific site in the primer strand of the DNA oligomer. The fluorescent oligomer bound tightly to the Klenow fragment (KD = 7.9 nM), and the probe's position within the DNA-protein complex was varied by stepwise elongation of the primer strand upon addition of the appropriate deoxynucleoside triphosphates. The decay of the total fluorescence intensity and the polarization anisotropy were measured with a picosecond laser and a time-correlated single photon counting system. The fluorescence lifetimes, the correlation time for internal rotation, and the angular range of internal rotation varied according to the probe's position within the DNA-protein complex. These results showed that five or six bases of the primer strand upstream of the 3' terminus were in contact with the protein and that within this contact region there were differences in the degree of solvent accessibility and the closeness of contact. Further, a minor binding mode of the DNA-protein complex was identified, on the basis of heterogeneity of the probe environment observed when the probe was positioned seven bases upstream from the primer 3' terminus, which resulted in a distinctive "dip and rise" in the anisotropy decay. Experiments with an epoxy-terminated DNA oligomer and a site-directed mutant protein established that the labeled DNA was binding at the polymerase active site (major form) and at the spatially distinct 3'----5' exonuclease active site (minor form). The abundance of each of these distinct binding modes of the DNA-protein complex was estimated under solution conditions by analyzing the anisotropy decay of the dansyl probe. About 12% of the labeled DNA was bound at the 3'----5' exonuclease site. This method should be useful for investigating the editing mechanism of this important enzyme.     

Active site-independent recognition of substrates and product by bovine prothrombinase: a fluorescence resonance energy transfer study

The conversion of prothrombin to thrombin is catalyzed by prothrombinase, an enzyme complex composed of the serine proteinase factor Xa and a cofactor protein, factor Va, assembled on membranes. Kinetic studies indicate that interactions with extended macromolecular recognition sites (exosites) rather than the active site of prothrombinase are the principal determinants of binding affinity for substrate or product. We now provide a model-independent evaluation of such ideas by physical studies of the interaction of substrate derivatives and product with prothrombinase. The enzyme complex was assembled using Xa modified with a fluorescent peptidyl chloromethyl ketone to irreversibly occlude the active site. Binding was inferred by prethrombin 2-dependent perturbations in the fluorescence of Oregon Green(488) at the active site of prothrombinase. Active site-independent binding was also unequivocally established by fluorescence resonance energy transfer between 2,6-dansyl tethered to the active site of Xa and eosin tethered to the active sites of either thrombin or meizothrombin des fragment 1. Comparable interprobe distances obtained from these measurements suggest that substrate and product interact equivalently with the enzyme. Competition established the ability of a range of substrate or product derivatives to bind in a mutually exclusive fashion to prothrombinase. Equilibrium dissociation constants obtained for the active site-independent binding of prothrombin, prethrombin 2, meizothrombin des fragment 1 and thrombin to prothrombinase were comparable with their affinities inferred from kinetic studies using active enzyme. Our findings directly establish that binding affinity is principally determined by the exosite-mediated interaction of either the substrate, both possible intermediates, or product with prothrombinase. A single type of exosite binding interaction evidently drives affinity and binding specificity through the stepwise reactions necessary for the two cleavage reactions of prothrombin activation and product release.     

Thermodynamic compensation upon binding to exosite 1 and the active site of thrombin

Several lines of experimental evidence including amide exchange and NMR suggest that ligands binding to thrombin cause reduced backbone dynamics. Binding of the covalent inhibitor dPhe-Pro-Arg chloromethyl ketone to the active site serine, as well as noncovalent binding of a fragment of the regulatory protein, thrombomodulin, to exosite 1 on the back side of the thrombin molecule both cause reduced dynamics. However, the reduced dynamics do not appear to be accompanied by significant conformational changes. In addition, binding of ligands to the active site does not change the affinity of thrombomodulin fragments binding to exosite 1; however, the thermodynamic coupling between exosite 1 and the active site has not been fully explored. We present isothermal titration calorimetry experiments that probe changes in enthalpy and entropy upon formation of binary ligand complexes. The approach relies on stringent thrombin preparation methods and on the use of dansyl-l-arginine-(3-methyl-1,5-pantanediyl)amide and a DNA aptamer as ligands with ideal thermodynamic signatures for binding to the active site and to exosite 1. Using this approach, the binding thermodynamic signatures of each ligand alone as well as the binding signatures of each ligand when the other binding site was occupied were measured. Different exosite 1 ligands with widely varied thermodynamic signatures cause a similar reduction in ΔH and a concomitantly lower entropy cost upon DAPA binding at the active site. The results suggest a general phenomenon of enthalpy-entropy compensation consistent with reduction of dynamics/increased folding of thrombin upon ligand binding to either the active site or exosite 1.     

Human glutathione-dependent formaldehyde dehydrogenase. Structural changes associated with ternary complex formation

Human glutathione-dependent formaldehyde dehydrogenase plays an important role in the metabolism of glutathione adducts such as S-(hydroxymethyl)glutathione and S-nitrosoglutathione. The role of specific active site residues in binding these physiologically important substrates and the structural changes during the catalytic cycle of glutathione-dependent formaldehyde dehydrogenase was examined by determining the crystal structure of a ternary complex with S-(hydroxymethyl)glutathione and the reduced coenzyme to 2.6 A resolution. The formation of the ternary complex caused the movement of the catalytic domain toward the coenzyme-binding domain. This represents the first observation of domain closure in glutathione-dependent formaldehyde dehydrogenase in response to substrate binding. A water molecule adjacent to the 2'-ribose hydroxyl of NADH suggests that the alcohol proton is relayed to solvent directly from the coenzyme, rather than through the action of the terminal histidine residue as observed in the proton relay system for class I alcohol dehydrogenases. S-(Hydroxymethyl)glutathione is directly coordinated to the active site zinc and forms interactions with the highly conserved residues Arg114, Asp55, Glu57, and Thr46. The active site zinc has a tetrahedral coordination environment with Cys44, His66, and Cys173 as the three protein ligands in addition to S-(hydroxymethyl)glutathione. This is in contrast to zinc coordination in the binary coenzyme complex where all of the ligands were contributed by the enzyme and included Glu67 as the fourth protein ligand. This change in zinc coordination is accomplished by an approximately 2.3 A movement of the catalytic zinc.