Dynamic simulations of 13 TATA variants refine kinetic hypotheses of sequence/activity relationships.

The fundamental relationship between DNA sequence/deformability and biological function has attracted numerous experimental and theoretical studies. A classic prototype system used for such studies in eukaryotes is the complex between the TATA element transcriptional regulator and the TATA-box binding protein (TBP). The recent crystallographic study by Burley and co-workers demonstrated the remarkable structural similarity contrasted to different transcriptional activity of 11 TBP/DNA complexes in which the DNAs differed by single base-pairs. By simulating these TATA variants and two other single base-pair variants that were not crystallizable, we uncover sequence-dependent structural, energetic, and flexibility properties that tailor TATA elements to TBP interactions, complementing many previous studies by refining kinetic hypotheses on sequence/activity correlations. The factors that combine to produce favorable elements for TBP activity include overall flexibility; minor groove widening, as well as roll, rise, and shift increases at the ends of the TATA element; untwisting within the TATA element accompanied by large roll at the TATA element ends; and relatively low maximal water densities around the DNA. These features accompany the severe deformation induced by the minor-groove binding protein, which kinks the TATA element at the ends and displaces local water molecules to form stabilizing hydrophobic contacts. Interestingly, the preferred bending direction itself is not a significant predictor of activity disposition, although certain variants (such as wild-type AdMLP, 5'-TATA4G-3', and inactive A29, 5'-TA6G-3') exhibit large preferred bends in directions consistent with their activity or inactivity (major groove and minor groove bends, respectively). These structural, flexibility, and hydration preferences, identified here and connected to a new crystallographic study of a larger group of DNA variants than reported to date, highlight the profound influence of single base-pair DNA variations on DNA motion. Our refined kinetic hypothesis suggests the functional implications of these motions in a kinetic model of TATA/TBP recognition, inviting further theoretical and experimental research.

A-Tract bending: insights into experimental structures by computational models.

While solution structures of adenine tract (A-tract) oligomers have indicated a unique bend direction equivalent to negative global roll (commonly termed "minor-groove bending"), crystallographic data have not unambiguously characterized the bend direction; nevertheless, many features are shared by all A-tract crystal and solution structures (e.g. propeller twisting, narrow minor grooves, and localized water spines). To examine the origin of bending and to relate findings to the crystallographic and solution data, we analyze molecular dynamics trajectories of two solvated A-tract dodecamers: 1D89, d(CGCGA(6)CG), and 1D98, d(CGCA(6)GCG), using a new general global bending framework for analyzing bent DNA and DNA/protein complexes. It is significant that the crystallographically-based initial structures are converted from dissimilar to similar bend directions equivalent to negative global roll, with the average helical-axis bend ranging from 10.5 degrees to 14.1 degrees. The largest bend occurs as positive roll of 12 degrees on the 5' side of the A-tracts (supporting a junction model) and is reinforced by gradual curvature at each A-tract base-pair (bp) step (supporting a wedge model). The precise magnitude of the bend is subtly sequence dependent (consistent with a curved general sequence model). The conversion to negative global roll only requires small local changes at each bp, accumulated over flexible moieties both outside and inside the A-tract. In contrast, the control sequence 1BNA, d(CGCGA(2)TTCGCG), bends marginally (only 6.9 degrees ) with no preferred direction. The molecular features that stabilize the bend direction in the A-tract dodecamers include propeller twisting of AT base-pairs, puckering differences between A and T deoxyriboses, a narrow minor groove, and a stable water spine (that extends slightly beyond the A-tract, with lifetimes approaching 0.2 ns). The sugar conformations, in particular, are proposed as important factors that support bent DNA. It is significant that all these curvature-stabilizing features are also observed in the crystallographic structures, but yield overall different bending paths, largely due to the effects of sequences outside the A-tract. These results merge structural details reported for A-tract structures by experiment and theory and lead to structural and dynamic insights into sequence-dependent DNA flexibility, as highlighted by the effect of an A-tract variant of a TATA-box element on bending and flexibility required for TBP binding.

Comparative modeling and molecular dynamics studies of the delta, kappa and mu opioid receptors.

Molecular models of the trans-membrane domains of delta, kappa and mu opioid receptors, members of the G-protein coupled receptor (GPCR) superfamily, were developed using techniques of homology modeling and molecular dynamics simulations. Structural elements were predicted from sequence alignments of opioid and related receptors based on (i) the consensus, periodicities and biophysical interpretations of alignment-derived properties, and (ii) tertiary structure homology to rhodopsin. Initial model structures of the three receptors were refined computationally with energy minimization and the result of the first 210 ps of a 2 ns molecular dynamics trajectory at 300K. Average structures from the trajectory obtained for each receptor subtype after release of the initial backbone constraints show small backbone deviations, indicating stability. During the molecular dynamics phase, subtype-differentiated residues of the receptors developed divergent structures within the models, including changes in regions common to the three subtypes and presumed to belong to ligand binding regions. The divergent features developed by the model structures appear to be consistent with the observed ligand binding selectivities of the opioid receptors. The results thus implicate identifiable receptor microenvironments as primary determinants of some of the observed subtype specificities in opiate ligand binding and in functional effects of mutagenesis. Networks of interacting residues observed in the models are common to the opiate receptors and other GPCRs, indicating core interfaces that are potentially responsible for structural integrity and signal transduction. Analysis of extended molecular dynamics trajectories reveals concerted motions of distant parts of ligand-binding regions, suggesting motion-sensitive components of ligand binding. The comparative modeling results from this study help clarify experimental observations of subtype differences and suggest both structural and dynamic rationales for differences in receptor properties.

DNA conformational changes associated with the cooperative binding of cI-repressor of bacteriophage lambda to OR.

The cI repressor protein (cI) maintains bacteriophage lambda in the lysogenic state in infected Escherichia coli cells by binding cooperatively to three tandemly repeated sequences comprising the right operator (OR). Cooperative interactions occur between alternate pairs of cI dimers bound to adjacent sites. Although crystallographic studies have revealed the structure of the DNA in the 92 amino acid residue amino-terminal fragment-OL1 complex, the structure of the DNA within the OR-cI complex with intact, cooperatively bound cI has not been described. In this study, the structure of the DNA within OR was quantitatively examined using sequence and structure-dependent nuclease cleavage patterns as a function of cI binding. The cooperative binding of cI to OR1 and OR2 induces a conformational change in the DNA of OR3 that is detectable by both DNase I and 5-phenyl-1,10-phenanthroline. Hydroxyl radical footprinting indicates the presence of an "A-tract" between OR1 and OR2 at the site of a run of four adenine-thymine base-pairs, implying a stable bend between the sites of approximately 18 degrees. 5-Phenyl-1,10-phenanthroline footprinting reports conformational changes within the central base-pairs of all three sites that is dependent upon the sequence-specific binding of cI. The observed conformational changes are more extensive within OR2 and OR3 compared with OR1, consistent with an "induced-fit" model of sequence-specific recognition. A number of changes in nuclease reactivity within the individual binding sites were quantitatively correlated with cI binding at the other sites within OR. These results demonstrate that changes in the DNA structure are propagated among the sites in response to the binding of cI and imply a role for DNA sequence-dependent conformational changes in the mechanisms of both the intrinsic and cooperative binding reactions of cI to OR.

Role of bile salt hydrophobicity in hepatic microtubule-dependent bile salt secretion.

Under basal conditions, bile salt secretion by the liver is not affected by microtubule disruption. However, when a bile salt load is imposed on the liver, a microtubule-dependent secretion mechanism is recruited (J. Lipid Res. 1988. 29: 144-156). We tested the hypothesis that recruitment of this microtubule-dependent mechanism is influenced by the relative hydrophobicity of the bile salts being secreted. Intact male rats were depleted of bile salts by overnight biliary diversion, pretreated with colchicine (a microtubule inhibitor) or its inactive isomer, lumicolchicine (control), and reinfused intravenously with bile salts of increasing hydrophobicity (taurodehydrocholate < tauroursodeoxycholate < taurocholate) at 200 nmol/min.100 g. After 45 min, when steady-state bile salt secretion was achieved, tracer [3H]taurocholate was administered intravenously. The colchicine-insensitive component of bulk bile salt secretion was constant at approximately 130 nmol/min.100 g, and the colchicine-sensitive component increased from approximately 0 to 35 and 60 nmol/min.100 g, respectively, with reinfusion of the more hydrophobic bile salts. Retained bile salts accumulated in the liver and serum and were detectable in urine. Peak biliary secretion of [3H]taurocholate in control animals increased linearly from 15.3 to 18.0% administered dose/min with increasing hydrophobicity of the secreted bile salts (P < 0.002). In colchicine-pretreated animals, peak secretion rates decreased linearly from 13.8 to 9.2%/min (P < 0.001), with maximal inhibition in taurocholate-reinfused animals (P < 0.01). Utilization of a microtubule-dependent secretion mechanism increases with increasing bile salt hydrophobicity. This mechanism permits more efficient hepatic secretion of bile salts, but increases the susceptibility of bile salt secretion to microtubule disruption. We postulate that microtubule-dependent insertion of bile salt transporters into the canalicular membrane underlies the enhanced bile salt secretion observed when a bile salt load is imposed upon the liver.

Microtubule-dependent transport of bile salts through hepatocytes: cholic vs. taurocholic acid.

Studies with taurine-conjugated bile salts have demonstrated two pathways for hepatocellular delivery of bile salts to bile: a cytosolic, microtubule-independent pathway and a membrane-based, microtubule-dependent pathway. However, a significant portion of circulating bile salts may be unconjugated. To determine whether free bile salts utilize similar pathways, we examined the effect of colchicine on the biliary excretion of intravenously administered cholic acid and taurocholate in intact rats. Basal rats were pretreated with low-dose colchicine or its inactive isomer, lumicolchicine, 1 hr before placement of intravenous and biliary cannulas and 2.75 hr before intravenous injection of [14C]cholic acid and [3H]taurocholate. Superfused rats were prepared as above but with intravenous infusion of taurocholate at 200 nmol/min.100 gm beginning 0.75 hr before [14C]cholic acid/[3H]taurocholate injection. Depleted/reinfused rats were subjected to biliary diversion for 20 hr before colchicine or lumicolchicine pretreatment, infusion of taurocholate and [14C]cholic acid/[3H]taurocholate injection. In each group, biliary excretion of [14C]taurocholate and [3H]taurocholate was inhibited equally by colchicine; for peak excretion rates the respective inhibition values were 33% and 35% in basal rats, 63% and 65% in superfused rats, and 74% and 76% in depleted/reinfused rats. Biliary excretion of [14C]taurocholate occurred consistently later than excretion of [3H]taurocholate, and maximal rates of excretion were reduced. In contrast, plasma uptake rates of [14C]cholic acid and [3H]taurocholate were essentially the same in depleted/reinfused rats. Deconvolution analysis of [14C]taurocholate vs. [3H]taurocholate biliary excretion curves revealed no significant differences among experimental groups. We conclude that conversion of [14C]cholic acid to [14C]taurocholate slightly retards its biliary excretion and diminishes its peak excretion rate compared with exogenous [3H]taurocholate.(ABSTRACT TRUNCATED AT 250 WORDS)

'Footprinting' proteins on DNA with peroxonitrous acid.

The peroxonitrite anion (ONOO-) is a stable species in alkaline solution that quickly generates a strong oxidant at neutral pH. A convenient procedure for the preparation of ONOOK has been developed based on the procedure of Keith & Powell [(1969) J. Chem. Soc. A, 90], which when added to a sample of duplex DNA buffered at neutral pH rapidly generates a strong oxidant capable of nonspecifically cleaving the DNA present. We show that this solution containing ONOOK can be used to hydroxyl radical footprint the binding the cl-repressor (cl) of phage lambda with the right operator, OR. In addition, we show that the individual-site binding isotherms determined by quantitative DNase I, Fe-EDTA and ONOOK footprinting are identical within experimental error. The identical isotherms obtained with the three different reagents with greatly differing sampling times indicates that the sampling time of the footprinting probe need not be short relative to the kinetic dissociation constants that govern protein-DNA interactions.