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D F Mierke

Publications and source records attributed to D F Mierke.

At least 19 recordsLinked to original sources
PubMed1998

Parathyroid hormone-receptor interactions identified directly by photocross-linking and molecular modeling studies.

Direct mapping of the interface between parathyroid hormone (PTH) and its receptor (hPTH1-Rc) was carried out by photoaffinity scanning studies. Photoreactive analogs of PTH singularly substituted with a p-benzoylphenylalanine (Bpa) at each of the first six N-terminal positions have been prepared. Among these, the analog [Bpa1,Nle8,18,Arg13,26,27,L-2-Nal23,Tyr34]bPTH-(1-34)N H2 (Bpa1-PTH-(1-34)) displayed in vitro activity with potency similar to that of PTH-(1-34). The radioiodinated analog 125I-Bpa1-PTH-(1-34) cross-linked specifically to the hPTH1-Rc stably expressed in human embryonic kidney cells. A series of chemical and enzymatic digestions of the hPTH1-Rc-125I-Bpa1-PTH-(1-34) conjugate suggested that a methionine residue (either Met414 or Met425) within the contact domain hPTH1-Rc-(409-437), which includes the transmembrane helix 6 and part of the third extracellular loop, as the putative contact point. Site-directed mutagenesis (M414L or M425L) identified Met425 as the putative contact point. Molecular modeling of the hPTH1-Rc together with the NMR-derived high resolution structure of hPTH-(1-34), guided by the cross-linking data, strongly supports Met425, at the extracellular end of transmembrane helix 6, as the residue interacting with the N-terminal residue of the hPTH-(1-34). The photocross-linking and molecular modeling studies provide insight into the topologic arrangement of the receptor-ligand complex.

Animals↗
PubMed1998

Vancomycin: conformational consequences of the sugar substituent.

High-resolution, three-dimensional structures of vancomycin and aglyco-vancomycin in DMSO were determined by nuclear magnetic resonance, metric matrix distance geometry, and molecular dynamics calculations. Conformational flexibility fast on the NMR time scale was examined by ensemble-based calculations which apply the experimentally derived restraints as an ensemble average. Two families of conformations of vancomycin, differing in the positioning of the vancosamine substituent, were observed. In contrast, the aglyco-vancomycin adopts only one conformation in solution. The conformations of vancomycin and the aglyco-vancomycin differ in the alignment of the amide protons which participate in the hydrogen-bonding network with the cell-wall precursor and orientation of the aromatic rings relative to the backbone. Therefore, the high-resolution structural characterization provides insight into a possible role of glycosylation on the activity of this important family of antibiotics.

Anti-Bacterial Agents↗
PubMed1998

Addressing the tertiary structure of human parathyroid hormone-(1-34).

Parathyroid hormone (PTH) regulates mineral metabolism and bone turnover by activating specific receptors located on osteoblastic and renal tubular cells and is fully functional as the N-terminal 1-34 fragment, PTH-(1-34). Previously, a "U-shaped" conformation with N- and C-terminal helices brought in close proximity by a turn has been postulated. The general acceptance of this hypothesis, despite limited experimental evidence, has altered the direction of the design of PTH-analogs. Examining the structure of human PTH-(1-34) under conditions that encompass the different environments the hormone may experience in the approach to and interaction with the G-protein-coupled receptor (including benign aqueous and saline solutions and in the presence of dodecylphosphocholine), we observe no evidence for a U-shape conformation or any tertiary structure. Instead, the N- and C-terminal helical domains, which vary in length and stability depending on the conditions, are separated by a highly flexible region of undefined conformation. These observations are in complete accord with recent conformational studies of PTH-related protein analogs containing lactams (Mierke, D. F., Maretto, S., Schievano, E. , DeLuca, D., Bisello, A., Mammi, S., Rosenblatt, M., Peggion, E., and Chorev, M. (1997) Biochemistry 36, 10372-10383) or a model amphiphilic alpha-helix (Pellegrini, M., Bisello, A., Rosenblatt, M., Chorev, M., and Mierke, D. F. (1997) J. Med. Chem. 40, 3025-3031). Reliable structural data from different environmental conditions are absolutely requisite for the next step in the design of non-peptide PTH analogs.

Circular Dichroism↗
PubMed1998

NMR determination of the major solution conformation of a peptoid pentamer with chiral side chains.

Polymers of N-substituted glycines ("peptoids") containing chiral centers at the alpha position of their side chains can form stable structures in solution. We studied a prototypical peptoid, consisting of five para-substituted (S)-N-(1-phenylethyl)glycine residues, by NMR spectroscopy. Multiple configurational isomers were observed, but because of extensive signal overlap, only the major isomer containing all cis-amide bonds was examined in detail. The NMR data for this molecule, in conjunction with previous CD spectroscopic results, indicate that the major species in methanol is a right-handed helix with cis-amide bonds. The periodicity of the helix is three residues per turn, with a pitch of approximately 6 A. This conformation is similar to that anticipated by computational studies of a chiral peptoid octamer. The helical repeat orients the amide bond chromophores in a manner consistent with the intensity of the CD signal exhibited by this molecule. Many other chiral polypeptoids have similar CD spectra, suggesting that a whole family of peptoids containing chiral side chains is capable of adopting this secondary structure motif. Taken together, our experimental and theoretical studies of the structural properties of chiral peptoids lay the groundwork for the rational design of more complex polypeptoid molecules, with a variety of applications, ranging from nanostructures to nonviral gene delivery systems.

Circular Dichroism↗
PubMed1997

Conformational studies of RS-66271, an analog of parathyroid hormone-related protein with pronounced bone anabolic activity.

Both the parathyroid hormone (PTH) and the functionally similar parathyroid hormone-related protein (PTHrP) have served as templates for the development of novel bone anabolic agents for the treatment of osteoporosis. The PTHrP analog RS-66271 (Vickery, B. H.; Avnur, Z.; Cheng Y.; Chiou, S.-S.; Leaffer, D.; Caulfield, J. P.; Kimmel, D. B.; Ho, T.; Krstenansky, J. L. J. Bone Miner. Res. 1996, 11, 1943-1951), in which the amino acids 22-31 have been substituted by the sequence E22-L-L-E-K-L-L-E-K-L31 (a model amphiphilic peptide), is a potent bone anabolic agent in vivo. Therefore, RS-66271 is a good candidate for structural analysis with the aim of developing a structure-activity relationship. The structural characterization described here was carried out in aqueous solution employing circular dichroism and nuclear magnetic resonance spectroscopy. We find that the incorporated amphiphilic decapeptide is indeed helical. In addition, it induces the adjacent residues, up to residue 16, to adopt the helical conformation. The helical domain, including residues 16-32, incorporates most of the previously identified principal receptor binding domain PTHrP(25-34). We discuss the relevance of the distinct and extensive helicity in light of the reduced in vitro receptor affinity/ activity and the enhanced in vivo bone anabolic efficacy of RS-66271.

Amino Acid Sequence↗
PubMed1997

Conformational studies of mono- and bicyclic parathyroid hormone-related protein-derived agonists.

Parathyroid hormone-related protein (PTHrP) is expressed in a wide variety of cells where it acts as an autocrine and/or paracrine factor involved in regulation of cellular growth, differentiation, and embryonic development. It may also play a physiological endocrine role in calcium transport across the placenta or during lactation. The N-terminal portion, PTHrP-(1-34), retains all the calciotropic parathyroid hormone-like activity and is a lead structure for the design of novel, bone anabolic agents for the treatment of bone disorders such as osteoporosis. To characterize the putative bioactive conformation, we have carried out a detailed structural analysis of a series of three conformationally constrained PTHrP-(1-34)-based mono- and bicyclic lactam-containing biologically active analogs: (III) The conformational properties were studied by circular dichroisim, nuclear magnetic resonance spectroscopy, distance geometry calculations, and molecular dynamic simulations in water/trifluoroethanol (TFE) mixtures. The helical content in water of both monocyclic analogs I and II is approximately 22%; that of the bicyclic analog III is approximately 40%. In 30% TFE, all analogs reached a maximal helical content of 80%, corresponding to 26 or 27 residues out of 34 in a helical conformation. High-resolution structures obtained with 50:50 TFE/water revealed that all three analogs display two helical domains and a hinge region around Gly12-Lys13. The highly potent mono- and bicyclic agonists I and III display a second hinge around Arg19-Arg20 which is shifted to Ser14-Asp17 in the weakly potent monocyclic agonist II. We suggest that the presence and localization of discrete hinges in the sequence together with the high propensity for helicity of the C-terminal sequence and the enhancement of helical nucleation at the N-terminal sequence are essential for generating a PTH/PTHrP receptor-compatible bioactive conformation.

Circular Dichroism↗
PubMed1997

Mono- and bicyclic analogs of parathyroid hormone-related protein. 2. Conformational analysis of antagonists by CD, NMR, and distance geometry calculations.

The conformation of the three cyclic antagonist analogs of parathyroid hormone-related protein (PTHrP)-(7-34) [[Lys13,Asp17]PTHrP-(7-34)NH2,[Lys26,Asp30 ]PTHrP-(7-34)NH2,[Lys13,Asp17,Lys26, Asp30]PTHrP-(7-34)NH2] is investigated by CD, NMR, and extensive computer simulations in aqueous solution and a TFE:water mixture. The structural analysis of these peptides, designed to stabilize different regions of the sequence in alpha-helical conformations, is an important step in addressing the correlation between helical content and binding affinity and bioactivity in this hormone-receptor system. Results from CD and NMR spectroscopy of all three analogues in aqueous solution indicate the presence of alpha-helix only in regions containing a 20-membered lactam ring. Upon addition of TFE, the three analogues display differences in the anticipated increase in helical content. The high-resolution structures produced at 50:50 TFE:water indicate specific differences in the extent and location of the helical regions. These conformations provide insight into the biological profiles of these analogues, reported in the previous manuscript [Bisello et al. (1997) Biochemistry 36, 3293-3299]. Since all three analogues are alpha-helical in the C-terminal region (residues 25-34 have been previously identified as containing the binding domain) and display similar binding affinities, we conclude that this conformational feature is important for the interaction between the peptide and the receptor. The extent of the helix (toward the N-terminus) and the presence of a hinge in the central region of the peptide play roles in the observed efficacy as measured by antagonism of PTH-stimulated adenylyl cyclase activity. The most active analogue consists of helical segments from residues 13-18 and 20-34, separated by a kink centered at Arg19.

Amino Acid Sequence↗
PubMed1997

Threonine6-bradykinin: molecular dynamics simulations in a biphasic membrane mimetic.

The natural peptide [Thr6]-bradykinin, Arg1-Pro2-Pro3-Gly4-Phe5-Thr6-Pro7-Phe8-Arg9, has been conformationally examined by molecular dynamics simulations using a two-phase box consisting of H2O and CCl4 to mimic the micellar environment utilized in the 1H-NMR studies. The different conformations generated from distance geometry calculations were refined with extensive molecular dynamics simulations. The resulting conformations provide additional structural insight into the differing biological activities of native bradykinin and [Thr6]-bradykinin, produced by the one conservative substitution Thr6 for Ser6. In addition, the simulations give some indication of the interaction of the peptide with the biphasic, hydrophilic/hydrophobic environment of the micelle. Such information is vital given the accumulating data indicating that the peptide first interacts with the membrane before the membrane-bound receptor. The structures of membrane-bound [Thr6]-bradykinin developed here provide experimental support for the interaction of residues 7 and 8 with the core of the membrane-bound receptor and the N-terminus and C-terminal arginine interacting with the extracellular portion of the receptor.

Bradykinin↗
PubMed1997

Threonine6-bradykinin: structural characterization in the presence of micelles by nuclear magnetic resonance and distance geometry.

The conformation of the natural peptide [Thr6]-bradykinin, Arg1-Pro2-Pro3-Gly4-Phe5-Thr6-Pro7-Phe8-Arg9, is investigated by NMR spectroscopy and computer simulations in an aqueous solution of sodium dodecyl sulfate micelles. The structural analysis of the peptide is of particular interest since it displays a different biological profile from bradykinin despite the high sequence homology (only one conservative substitution: Ser6/Thr6) and the fact that both peptides bind and activate common receptors. The SDS micelles provide a model system for the membrane-interface environment the peptide experiences when interacting with the membrane-embedded receptor and allow for the conformational examination of the peptide using high-resolution NMR techniques. The NMR spectra show that the micellar system induces a secondary structure in the otherwise inherently flexible peptide (as observed in benign aqueous solution). The distance geometry calculations indicate a beta-turn of type I about residues 7-8 as the preferred conformation. The results of ensemble calculations reveal conformational changes occurring rapidly on the NMR time scale and allow for the identification of three different families of conformations that average to reproduce the NMR observables. The three families differ in the type of conformation adopted at the C-terminus: type I beta-turn, type II beta-turn and a third conformation, intermediate between the two beta-turns. The structural results support the hypothesis of the determining role of the C-terminal conformation for biological activity and can provide an explanation of the different activities observed for bradykinin and [Thr6]-bradykinin.

Bradykinin↗
PubMed1997

Conformational consequences of i, i + 3 cystine linkages: nucleation for alpha-helicity?

Methods to introduce specific secondary structural elements into peptides and proteins are vital for the rational design of peptide and non-peptide drug candidates as well as in the de novo design of proteins. Here the incorporation of a disulfide linkage between cysteine residues spaced three amino acids apart (i, i + 3) as a method to induce helicity is examined. Two dodecamer peptides, A-V-S-E-C-Q-L-C-H-D-K-G-NH2, differing in the chirality of the cysteine at the fifth position (the i position), have been synthesized and conformationally studied both in the linear and cyclized form. This peptide sequence, derived from the N-terminal sequence of parathyroid hormone related protein, does not form helices, even as part of the 1-34 fully active domain of the protein. The four analogs (two cyclic and two linear) were analyzed both in aqueous solution and in the presence of sodium dodecyl sulfate micelles. In aqueous solution the linear peptides display no evidence for secondary structure, while the cyclization induces a turn centered about the cysteine residues. In the presence of micelles the linear form of the peptides adopts bent conformations, containing turns, but results from both NMR and CD provide no evidence of helices. The oxidized L,L-peptide in the micellar solution does not present a well defined conformation, although the presence of one helical turn is evident. The cyclic D,L analog adopts a helical structure (not an alpha-helix) extending from residue 2 to 9, with non-standard phi, psi values caused by the presence of the D-amino acid. These results clearly illustrate that the ability of D-Cys(i), Cys(i + 3) cyclization to initiate helix formation depends greatly on the solvent used. Therefore, any drug-design principle utilizing this modification for helix nucleation must keep the environment in which the peptide is biologically active in mind.

Amino Acid Sequence↗
PubMed1996

Secondary structural elements as a basis for antibody recognition in the immunodominant region of human immunodeficiency viruses 1 and 2.

Synthetic peptide antigens corresponding to the entire third variable region V3, the principal neutralizing determinant of the human immunodeficiency virus (HIV) envelope glycoprotein of HIV-1 subtype B (1), HIV-2 subtype A (5), and HIV-2 subtype B (7) were synthesized by solid-phase peptide synthesis (Table 1). 1 and 5 were also prepared as their GlcNAc-glycosylated forms at the natural N-glycosylation site NXT (positions 6-8; peptides 4 and 6). Additionally, the proposed beta-turn region of 1 (GPGR; positions 15-18) was altered by introducing D-Ala17 (2) and D-Pro16 (3). All compounds have been studied by two-dimensional NMR techniques. Interproton distances and 3JNH/H alpha coupling constants derived from NMR data are used as restraints in distance geometry and ENSEMBLE-Distance and angle-bound driven dynamics calculations. The stimulations led to disordered conformations except for a high propensity of a beta II-turn in the region GPXR (positions 15-18) in 1, 2, and 4. In 3 (G-D-ProGR, positions 15-18), a type beta I'-turn was mainly found instead. For peptide 7, the consensus sequence of HIV-2 subtype B, a type beta II-turn was also found although the primary structure (VSGL; positions 15-18) differs grossly from the HIV-1 peptide 1. With the exception of 2, all beta II-turns were able to form a canonically opened beta-turn by a 180 degree rotation of phi(G17). Surprisingly, compounds 5 and 6 that are highly similar to 7 showed no beta II-type turn within MSGL (positions 15-18). They form a type beta VIII-turn across the tetrapeptide SGLV (positions 16-19) together with a non-canonical turn conformation across LMSG (positions 14-17) leading to an S-conformation. The reaction of the peptides with HIV-positive sera from patients infected with different subtypes of HIV-1 and HIV-2 was tested in enzyme-linked immunosorbent assays (ELISA reactions). No HIV-2 sera reacted peptide 1 and no HIV-1 sera showed reactivity to peptide 5. We propose that certain amino acid exchanges within the V3 domain lead to altered conformations of the V3 loop resulting in antibodies that show altered binding properties to the peptide antigens used in the ELISA reactions.

Amino Acid Sequence↗
PubMed1996

Conformational characterization of a peptide mimetic of the third cytoplasmic loop of the G-protein coupled parathyroid hormone/parathyroid hormone related protein receptor.

The third-cytoplasmic loop of the G-protein coupled receptor responsible for the activity of parathyroid hormone and parathyroid hormone-related protein has been structurally characterized in aqueous solution in the presence of sodium dodecylsulfate and dodecylphosphocholine micelles. The high-resolution conformation of the 29-amino acid peptide containing the sequence of the cytoplasmic loop was obtained by CD and nmr. The structure was refined using a two-step distance geometry based method that first includes the removal of all side chains to quickly locate the globular fold of the peptide. After a simulated annealing protocol, the side chains are added in a random fashion. The resulting conformation was further refined with nuclear Overhauser enhancement restrained molecular dynamics using a two-phase simulation cell consisting of carbon tetrachloride and water as a mimetic of the biphasic, hydrophobic/hydrophilic character of the micelles in which the experimental measurements were carried out. The topological orientation of the cycloplasmic loop within the micelle was determined by addition of 5-doxylstearate and monitoring the decrease of nmr signal intensities from the radical-induced relaxation. The conformation and relative orientation of the peptide provided insight into the mechanism by which the cytoplasmic portion of the receptor activates the heterotrimeric, guanine nucleotide-binding regulatory protein, one of the first steps in signal transduction.

Amino Acid Sequence↗
PubMed1994

Molecular structure of the cyanobacterial tumor-promoting microcystins.

The three-dimensional structure of the two hepatotoxic microcystins LR and LY has been determined by two-dimensional nuclear magnetic resonance (2D NMR) spectroscopy and distance geometry calculations. For the microcystin LY a single family of highly convergent structures was obtained. This family is characterized by a relatively compact boat-like ring structure with the large side chain of the Adda residue protruding from the concave side, in close proximity to the Tyr side chain. Conversely, for the microcystin LR the calculations result in three conformational families characterized by an even more compact ring structure. The Adda and Arg side chains protrude from the ring distal from one another caused by the repulsion between the guanido function of Arg and the hydrophobic Adda. The lower toxicity of the LY microcystin could result from the restricted access of the Adda side chain, an essential residue for activity, which results from the close proximity of the aromatic Tyr residue. A significant enthalpic cost would be expected for disturbance of this hydrophobic collapse and correspondingly lower binding affinity to receptor molecules would be predicted. From the structures of the two related microcystins, and homology with other known toxins, we propose a working hypothesis of the Adda side chain interacting with a hydrophobic pore of the phosphatases while the rest of the microcystin acts as a scaffold to help stabilize the interdigitation of the Adda with additional intermolecular interactions.

Amino Acid Sequence↗
PubMed1994

Structure of cobra cardiotoxin CTX I as derived from nuclear magnetic resonance spectroscopy and distance geometry calculations.

The structure of cardiotoxin CTX I from Naja naja atra has been investigated by NMR spectroscopy. Sequence specific resonance assignments have been obtained for all backbone protons as well as for most side-chain protons. Distance geometry calculations were carried out using a metric matrix DG program. A total of 715 NOE constraints, 27 phi angle constraints and a list of the hydrogen bond donors were used for the metric matrix DG calculations and refinement. The average pairwise r.m.s.d. of the resulting structures was 1.01 A for the backbone heavy atoms, and 1.69 A for all heavy atoms. The protein is rich in beta structure and consists of a large triple-stranded, antiparallel beta sheet as well as a short double-stranded, antiparallel beta sheet. Non-regular hydrogen bonding is found between side-chains of the carboxy-terminal end and the rest of the core region. The structure is discussed in terms of evolutionary aspects as well as recent investigations about the biological function and active site.

Amino Acid Sequence↗
PubMed1994

Comparison of the conformation of active and nonactive backbone cyclic analogs of substance P as a tool to elucidate features of the bioactive conformation: NMR and molecular dynamics in DMSO and water.

The conformations of two backbone-cyclized substance P analogs as derived from 1H NMR and molecular dynamics simulations carried out in DMSO and water are described. The method of floating chiralities is used in the simulations to facilitate the diastereotopic assignment of methylene protons. One of the analogs, cyclo-[-(CH2)3-NH-CO-(CH2)4-Arg-Phe-Phe-N-]-CH2-CO-Leu-Met-NH2, is a highly active, selective agonist for the NK-receptor, while the other, cyclo[-(CH2)2-NH-CO-(CH2)2-Gly-Arg-Phe-Phe-N-]-CH2-CO-Leu-Met-NH2, is inactive. Both analogs contain cyclic ring systems of the same size, varying in only the number of amide linkages. From the conformational analysis, the lack of activity can be attributed to the introduction of too much constraint into the ring system. This has an effect on the topological array of the important residues Arg-Phe-Phe. The results presented here are compared with biologically active analogs previously examined. The differences between conformations of active and inactive compounds are used to develop insight into the conformational requirements for biological activity.

Amino Acid Sequence↗