Force-mediated kinetics of single P-selectin/ligand complexes observed by atomic force microscopy

Leukocytes roll along the endothelium of postcapillary venules in response to inflammatory signals. Rolling under the hydrodynamic drag forces of blood flow is mediated by the interaction between selectins and their ligands across the leukocyte and endothelial cell surfaces. Here we present force-spectroscopy experiments on single complexes of P-selectin and P-selectin glycoprotein ligand-1 by atomic force microscopy to determine the intrinsic molecular properties of this dynamic adhesion process. By modeling intermolecular and intramolecular forces as well as the adhesion probability in atomic force microscopy experiments we gain information on rupture forces, elasticity, and kinetics of the P-selectin/P-selectin glycoprotein ligand-1 interaction. The complexes are able to withstand forces up to 165 pN and show a chain-like elasticity with a molecular spring constant of 5.3 pN nm−1 and a persistence length of 0.35 nm. The dissociation constant (off-rate) varies over three orders of magnitude from 0.02 s−1 under zero force up to 15 s−1 under external applied forces. Rupture force and lifetime of the complexes are not constant, but directly depend on the applied force per unit time, which is a product of the intrinsic molecular elasticity and the external pulling velocity. The high strength of binding combined with force-dependent rate constants and high molecular elasticity are tailored to support physiological leukocyte rolling.

Corticotropin-releasing factor-binding protein ligand inhibitor blunts excessive weight gain in genetically obese Zucker rats and rats during nicotine withdrawal

Elevation of the neuropeptide corticotropin-releasing factor (CRF) in the brain is associated with a reduction of food intake and body weight gain in normal and obese animals. A protein that binds CRF and the related peptide, urocortin, with high affinity, CRF-binding protein (CRF-BP), may play a role in energy homeostasis by inactivating members of this peptide family in ingestive and metabolic regulatory brain regions. Intracerebroventricular administration in rats of the high-affinity CRF-BP ligand inhibitor, rat/human CRF (6-33), which dissociates CRF or urocortin from CRF-BP and increases endogenous brain levels of “free” CRF or urocortin significantly blunted exaggerated weight gain in Zucker obese subjects and in animals withdrawn from chronic nicotine. Chronic administration of CRF suppressed weight gain nonselectively by 60% in both Zucker obese and lean control rats, whereas CRF-BP ligand inhibitor treatment significantly reduced weight gain in obese subjects, without altering weight gain in lean control subjects. Nicotine abstinent subjects, but not nicotine-naive controls, experienced a 35% appetite suppression and a 25% weight gain reduction following acute and chronic administration, respectively, of CRF-BP ligand inhibitor. In marked contrast to the effects of a CRF-receptor agonist, the CRF-BP ligand inhibitor did not stimulate adrenocorticotropic hormone secretion or elevate heart rate and blood pressure. These results provide support for the hypothesis that the CRF-BP may function within the brain to limit selected actions of CRF and/or urocortin. Furthermore, CRF-BP may represent a novel and functionally selective target for the symptomatic treatment of excessive weight gain associated with obesity of multiple etiology.

Covalent protein-DNA complexes at the 5' strand termini of meiosis-specific double-strand breaks in yeast.

During meiosis in Saccharomyces cerevisiae, the first chemical step in homologous recombination is the occurrence of site-specific DNA double-strand breaks (DSBs). In wild-type cells, these breaks undergo resection of their 5' strand termini to yield molecules with 3' single-stranded tails. We have further characterized the breaks that accumulate in rad50S mutant stains defective in DSB resection. We find that these DSBs are tightly associated with protein via what appears to be a covalent linkage. When genomic DNA is prepared from meiotic rad50S cultures without protease treatment steps, the restriction fragments diagnostic of DSBs selectively partition to the organic-aqueous interphase in phenol extractions and band at lower than normal density in CsCl density gradients. Selective partitioning and decreased buoyant density are abolished if the DNA is treated with proteinase K prior to analysis. Similar results are obtained with sae2-1 mutant strains, which have phenotypes identical to rad50S mutants. The protein is bound specifically to the 5' strand termini of DSBs and is present at both 5' ends in at least a fraction of breaks. The stability of the complex to various protein denaturants and the strand specificity of the attachment are most consistent with a covalent linkage to DSB termini. We propose that the DSB-associated protein is the catalytic subunit of the meiotic recombination initiation nuclease and that it cleaves DNA via a covalent protein-DNA intermediate.

Unbinding forces of single antibody-antigen complexes correlate with their thermal dissociation rates

Point mutants of three unrelated antifluorescein antibodies were constructed to obtain nine different single-chain Fv fragments, whose on-rates, off-rates, and equilibrium binding affinities were determined in solution. Additionally, activation energies for unbinding were estimated from the temperature dependence of the off-rate in solution. Loading rate-dependent unbinding forces were determined for single molecules by atomic force microscopy, which extrapolated at zero force to a value close to the off-rate measured in solution, without any indication for multiple transition states. The measured unbinding forces of all nine mutants correlated well with the off-rate in solution, but not with the temperature dependence of the reaction, indicating that the same transition state must be crossed in spontaneous and forced unbinding and that the unbinding path under load cannot be too different from the one at zero force. The distance of the transition state from the ground state along the unbinding pathway is directly proportional to the barrier height, regardless of the details of the binding site, which most likely reflects the elasticity of the protein in the unbinding process. Atomic force microscopy thus can be a valuable tool for the characterization of solution properties of protein-ligand systems at the single molecule level, predicting relative off-rates, potentially of great value for combinatorial chemistry and biology.

Direct measurement of oligonucleotide binding stoichiometry of gene V protein by mass spectrometry.

The binding stoichiometry of gene V protein from bacteriophage f1 to several oligonucleotides was studied using electrospray ionization-mass spectrometry (ESI-MS). Using mild mass spectrometer interface conditions that preserve noncovalent associations in solution, gene V protein was observed as dimer ions from a 10 mM NH4OAc solution. Addition of oligonucleotides resulted in formation of protein-oligonucleotide complexes with stoichiometry of approximately four nucleotides (nt) per protein monomer. A 16-mer oligonucleotide gave predominantly a 4:1 (protein monomer: oligonucleotide) complex while oligonucleotides shorter than 15 nt showed stoichiometries of 2:1. Stoichiometries and relative binding constants for a mixture of oligonucleotides were readily measured using mass spectrometry. The binding stoichiometry of the protein with the 16-mer oligonucleotide was measured independently using size-exclusion chromatography and the results were consistent with the mass spectrometric data. These results demonstrate, for the first time, the observation and stoichiometric measurement of protein-oligonucleotide complexes using ESI-MS. The sensitivity and high resolution of ESI-MS should make it a useful too] in the study of protein-DNA interactions.

Transcriptional activation of the mouse obese (ob) gene by CCAAT/enhancer binding protein alpha.

Like other adipocyte genes that are transcriptionally activated by CCAAT/enhancer binding protein alpha (C/EBP alpha) during preadipocyte differentiation, expression of the mouse obese (ob) gene is immediately preceded by the expression of C/EBP alpha. While the 5' flanking region of the mouse ob gene contains several consensus C/EBP binding sites, only one of these sites appears to be functional. DNase I cleavage inhibition patterns (footprinting) of the ob gene promoter revealed that recombinant C/EBP alpha, as well as a nuclear factor present in fully differentiated 3T3-L1 adipocytes, but present at a much lower level in preadipocytes, protects the same region between nucleotides -58 and -42 relative to the transcriptional start site. Electrophoretic mobility-shift analysis using nuclear extracts from adipose tissue or 3T3-L1 adipocytes and an oligonucleotide probe corresponding to a consensus C/EBP binding site at nucleotides -55 to -47 generated a specific protein-oligonucleotide complex that was supershifted by antibody against C/EBP alpha. Probes corresponding to two upstream consensus C/EBP binding sites failed to generate protein-oligonucleotide complexes. Cotransfection of a C/EBP alpha expression vector into 3T3-L1 cells with a series of 5' truncated ob gene promoter constructs activated reporter gene expression with all constructs containing the proximal C/EBP binding site (nucleotides -55 to -47). Mutation of this site blocked transactivation by C/EBP alpha. Taken together, these findings implicate C/EBP alpha as a transcriptional activator of the ob gene promoter and identify the functional C/EBP binding site in the promoter.

In vitro integration of human immunodeficiency virus type 1 cDNA into targets containing protein-induced bends.

Integration of human immunodeficiency virus type 1 cDNA into a target DNA can be strongly influenced by the conformation of the target. For example, integration in vitro is sometimes favored in target DNAs containing sequence-directed bends or DNA distortions caused by bound proteins. We have analyzed the effect of DNA bending by studying integration into two well-characterized protein-DNA complexes: Escherichia coli integration host factor (IHF) protein bound to a phage IHF site, and the DNA binding domain of human lymphoid enhancer factor (LEF) bound to a LEF site. Both of these proteins have previously been reported to bend DNA by approximately 140 degrees. Binding of IHF greatly increases the efficiency of in vitro integration at hotspots within the IHF site. We analyzed a series of mutants in which the IHF site was modified at the most prominent hotspot. We found that each variant still displayed enhanced integration upon IHF binding. Evidently the local sequence is not critical for formation of an IHF hotspot. LEF binding did not create preferred sites for integration. The different effects of IHF and LEF binding can be rationalized in terms of the different proposed conformations of the two protein-DNA complexes.

Structure-assisted redesign of a protein-zinc-binding site with femtomolar affinity.

We have inserted a fourth protein ligand into the zinc coordination polyhedron of carbonic anhydrase II (CAII) that increases metal affinity 200-fold (Kd = 20 fM). The three-dimensional structures of threonine-199-->aspartate (T199D) and threonine-199-->glutamate (T199E) CAIIs, determined by x-ray crystallographic methods to resolutions of 2.35 Angstrum and 2.2 Angstrum, respectively, reveal a tetrahedral metal-binding site consisting of H94, H96, H119, and the engineered carboxylate side chain, which displaces zinc-bound hydroxide. Although the stereochemistry of neither engineered carboxylate-zinc interaction is comparable to that found in naturally occurring protein zinc-binding sites, protein-zinc affinity is enhanced in T199E CAII demonstrating that ligand-metal separation is a significant determinant of carboxylate-zinc affinity. In contrast, the three-dimensional structure of threonine-199-->histidine (T199H) CAII, determined to 2.25-Angstrum resolution, indicates that the engineered imidazole side chain rotates away from the metal and does not coordinate to zinc; this results in a weaker zinc-binding site. All three of these substitutions nearly obliterate CO2 hydrase activity, consistent with the role of zinc-bound hydroxide as catalytic nucleophile. The engineering of an additional protein ligand represents a general approach for increasing protein-metal affinity if the side chain can adopt a reasonable conformation and achieve inner-sphere zinc coordination. Moreover, this structure-assisted design approach may be effective in the development of high-sensitivity metal ion biosensors.

Estrogen receptor (ER) modulators each induce distinct conformational changes in ER α and ER β

Estrogen receptor (ER) modulators produce distinct tissue-specific biological effects, but within the confines of the established models of ER action it is difficult to understand why. Previous studies have suggested that there might be a relationship between ER structure and activity. Different ER modulators may induce conformational changes in the receptor that result in a specific biological activity. To investigate the possibility of modulator-specific conformational changes, we have applied affinity selection of peptides to identify binding surfaces that are exposed on the apo-ERs α and β and on each receptor complexed with estradiol or 4-OH tamoxifen. These peptides are sensitive probes of receptor conformation. We show here that ER ligands, known to produce distinct biological effects, induce distinct conformational changes in the receptors, providing a strong correlation between ER conformation and biological activity. Furthermore, the ability of some of the peptides to discriminate between different ER α and ER β ligand complexes suggests that the biological effects of ER agonists and antagonists acting through these receptors are likely to be different.

Conversion of agonist site to metal-ion chelator site in the β2-adrenergic receptor

Previously metal-ion sites have been used as structural and functional probes in seven transmembrane receptors (7TM), but as yet all the engineered sites have been inactivating. Based on presumed agonist interaction points in transmembrane III (TM-III) and -VII of the β2-adrenergic receptor, in this paper we construct an activating metal-ion site between the amine-binding Asp-113 in TM-III—or a His residue introduced at this position—and a Cys residue substituted for Asn-312 in TM-VII. No increase in constitutive activity was observed in the mutant receptors. Signal transduction was activated in the mutant receptors not by normal catecholamine ligands but instead either by free zinc ions or by zinc or copper ions in complex with small hydrophobic metal-ion chelators. Chelation of the metal ions by small hydrophobic chelators such as phenanthroline or bipyridine protected the cells from the toxic effect of, for example Cu2+, and in several cases increased the affinity of the ions for the agonistic site. Wash-out experiments and structure–activity analysis indicated, that the high-affinity chelators and the metal ions bind and activate the mutant receptor as metal ion guided ligand complexes. Because of the well-understood binding geometry of the small metal ions, an important distance constraint has here been imposed between TM-III and -VII in the active, signaling conformation of 7TM receptors. It is suggested that atoxic metal-ion chelator complexes could possibly in the future be used as generic, pharmacologic tools to switch 7TM receptors with engineered metal-ion sites on or off at will.

Adaptor Complex-independent Clathrin Function in Yeast

Clathrin-associated adaptor protein (AP) complexes are major structural components of clathrin-coated vesicles, functioning in clathrin coat assembly and cargo selection. We have carried out a systematic biochemical and genetic characterization of AP complexes in Saccharomyces cerevisiae. Using coimmunoprecipitation, the subunit composition of two complexes, AP-1 and AP-2R, has been defined. These results allow assignment of the 13 potential AP subunits encoded in the yeast genome to three AP complexes. As assessed by in vitro binding assays and coimmunoprecipitation, only AP-1 interacts with clathrin. Individual or combined disruption of AP-1 subunit genes in cells expressing a temperature-sensitive clathrin heavy chain results in accentuated growth and α-factor pheromone maturation defects, providing further evidence that AP-1 is a clathrin adaptor complex. However, in cells expressing wild-type clathrin, the same AP subunit deletions have no effect on growth or α-factor maturation. Furthermore, gel filtration chromatography revealed normal elution patterns of clathrin-coated vesicles in cells lacking AP-1. Similarly, combined deletion of genes encoding the β subunits of the three AP complexes did not produce defects in clathrin-dependent sorting in the endocytic and vacuolar pathways or alterations in gel filtration profiles of clathrin-coated vesicles. We conclude that AP complexes are dispensable for clathrin function in S. cerevisiae under normal conditions. Our results suggest that alternative factors assume key roles in stimulating clathrin coat assembly and cargo selection during clathrin-mediated vesicle formation in yeast.

Molecular determinants of tissue selectivity in estrogen receptor modulators

Interaction of the estrogen receptor/ligand complex with a DNA estrogen response element is known to regulate gene transcription. In turn, specific conformations of the receptor-ligand complex have been postulated to influence unique subsets of estrogen-responsive genes resulting in differential modulation and, ultimately, tissue-selective outcomes. The estrogen receptor ligands raloxifene and tamoxifen have demonstrated such tissue-specific estrogen agonist/antagonist effects. Both agents antagonize the effects of estrogen on mammary tissue while mimicking the actions of estrogen on bone. However, tamoxifen induces significant stimulation of uterine tissue whereas raloxifene does not. We postulate that structural differences between raloxifene and tamoxifen may influence the conformations of their respective receptor/ligand complexes, thereby affecting which estrogen-responsive genes are modulated in various tissues. These structural differences are 4-fold: (A) the presence of phenolic hydroxyls, (B) different substituents on the basic amine, (C) incorporation of the stilbene moiety into a cyclic benzothiophene framework, and (D) the imposition of a carbonyl “hinge” between the basic amine-containing side chain and the olefin. A series of raloxifene analogs that separately exemplify each of these differences have been prepared and evaluated in a series of in vitro and in vivo assays. This strategy has resulted in the development of a pharmacophore model that attributes the differences in effects on the uterus between raloxifene and tamoxifen to a low-energy conformational preference imparting an orthogonal orientation of the basic side chain with respect to the stilbene plane. This three-dimensional array is dictated by a single carbon atom in the hinge region of raloxifene. These data indicate that differences in tissue selective actions among benzothiophene and triarylethylene estrogen receptor modulators can be ascribed to discrete ligand conformations.

Multiple sites of replication initiation in the human β-globin gene locus

The cell cycle-dependent, ordered assembly of protein prereplicative complexes suggests that eukaryotic replication origins determine when genomic replication initiates. By comparison, the factors that determine where replication initiates relative to the sites of prereplicative complex formation are not known. In the human globin gene locus previous work showed that replication initiates at a single site 5′ to the β-globin gene when protein synthesis is inhibited by emetine. The present study has examined the pattern of initiation around the genetically defined β-globin replicator in logarithmically growing HeLa cells, using two PCR-based nascent strand assays. In contrast to the pattern of initiation detected in emetine-treated cells, analysis of the short nascent strands at five positions spanning a 40 kb globin gene region shows that replication initiates at more than one site in non-drug-treated cells. Quantitation of nascent DNA chains confirmed that replication begins at several locations in this domain, including one near the initiation region (IR) identified in emetine-treated cells. However, the abundance of short nascent strands at another initiation site ∼20 kb upstream is ∼4-fold as great as that at the IR. The latter site abuts an early S phase replicating fragment previously defined at low resolution in logarithmically dividing cells.

Vasopressin mRNA localization in nerve cells: Characterization of cis-acting elements and trans-acting factors

mRNA localization is a complex pathway. Besides mRNA sorting per se, this process includes aspects of regulated translation. It requires protein factors that interact with defined sequences (or sequence motifs) of the transcript, and the protein/RNA complexes are finally guided along the cytoskeleton to their ultimate destinations. The mRNA encoding the vasopressin (VP) precursor protein is localized to the nerve cell processes in vivo and in primary cultured nerve cells. Sorting of VP transcripts to dendrites is mediated by the last 395 nucleotides of the mRNA, the dendritic localizer sequence, and it depends on intact microtubules. In vitro interaction studies with cytosolic extracts demonstrated specific binding of a protein, enriched in nerve cell tissues, to the radiolabeled dendritic localizer sequence probe. Biochemical purification revealed that this protein is the multifunctional poly(A)-binding protein (PABP). It is well known for its ability to bind with high affinity to poly(A) tails of mRNAs, prerequisite for mRNA stabilization and stimulation of translational initiation, respectively. With lower affinities, PABP can also associate with non-poly(A) sequences. The physiological consequences of these PABP/RNA interactions are far from clear but may include functions such as translational silencing. Presumably, the translational state of mRNAs subject to dendritic sorting is influenced by external stimuli. PABP thus could be a component required to regulate local synthesis of the VP precursor and possibly of other proteins.

A novel assay for drug-DNA binding mode, affinity, and exclusion number: scanning force microscopy.

Determining the mode-of-binding of a DNA ligand is not always straightforward. Here, we establish a scanning force microscopic assay for mode-of-binding that is (i) direct: lengths of individual DNA-ligand complexes are directly measured; (ii) rapid: there are no requirements for staining or elaborate sample preparation; and (iii) unambiguous: an observed increase in DNA length upon addition of a ligand is definitive evidence for an intercalative mode-of-binding. Mode-of-binding, binding affinity, and site-exclusion number are readily determined from scanning force microscopy measurements of the changes in length of individual drug-DNA complexes as a function of drug concentration. With this assay, we resolve the ambiguity surrounding the mode of binding of 2,5-bis(4-amidinophenyl) furan (APF) to DNA and show that it binds to DNA by nonintercalative modes. APF is a member of an important class of aromatic dicationic drugs that show significant activity in the treatment of Pneumocystis carinii pneumonia, an opportunistic infection that is the leading cause of death in AIDS patients.