The 1.6 angstroms resolution crystal structure of nuclear transport factor 2 (NTF2)

Nuclear transport factor 2 (NTF2) facilitates protein transport into the nucleus and interacts with both the small Ras-like GTPase Ran and nucleoporin p62. We have determined the structure of bacterially expressed rat NTF2 at 1.6 angstroms resolution using X-ray crystallography. The NTF2 polypeptide chain forms an alpha + beta barrel that opens at one end to form a distinctive hydrophobic cavity and its fold is homologous to that of scytalone dehydratase. The NTF2 hydrophobic cavity is a candidate for a potential binding site for other proteins involved in nuclear import such as Ran and nucleoporin p62. In addition, the hydrophobic cavity contains a putative catalytic Asp-His pair, which raises the possibility of an unanticipated enzymatic activity of the molecule that may have implications for the molecular mechanism of nuclear protein import.     

Interaction between the small GTPase Ran/Gsp1p and Ntf2p is required for nuclear transport

Bidirectional movement of proteins and RNAs across the nuclear envelope requires Ran, a Ras-like GTPase. A genetic screen of the yeast Saccharomyces cerevisiae was performed to isolate conditional alleles of GSP1, a gene that encodes a homolog of Ran. Two temperature-sensitive alleles, gsp1-1 and gsp1-2, were isolated. The mutations in these two alleles map to regions that are structurally conserved between different members of the Ras family. Each mutant strain exhibits various nuclear transport defects. Both biochemical and genetic experiments indicate a decreased interaction between Ntf2p, a factor which is required for protein import, and the mutant GSP1 gene products. Overexpression of NTF2 can suppress the temperature sensitive phenotype of gsp1-1 and gsp1-2 and partially rescue nuclear transport defects. However, overexpression of a mutant allele of NTF2 with decreased binding to Gsp1p cannot rescue the temperature sensitivity of gsp1-1 and gsp1-2. Taken together, these data demonstrate that the interaction between Gsp1p and Ntf2p is critical for nuclear transport.     

Nuclear protein import is decreased by engineered mutants of nuclear transport factor 2 (NTF2) that do not bind GDP-Ran

Nuclear transport factor 2 (NTF2) is associated with the translocation stage of nuclear protein import and binds both to nuclear pore proteins (nucleoporins) containing phenylalanine-rich repeats and to the Ras family GTPase Ran. In this study we probed the role of the NTF2-Ran interaction in nuclear protein import using site-directed mutants of NTF2 that interfere with its interaction with GDP-Ran. The design of these mutants was based on the X-ray crystal structure of NTF2 and was concentrated on conserved residues in and around the molecule's hydrophobic cavity. The mutant NTF2 cDNAs were expressed in Escherichia coli. Purified mutant proteins retained the interaction with FxFG-repeat nucleoporins, but several mutants in the negatively charged residues that surround the NTF2 cavity or in residues in the cavity itself were unable to bind GDP-Ran in vitro. The crystal structure of the E42K mutant protein showed significant structural changes only in this side-chain, indicating that it participated directly in the interaction with GDP-Ran. In permeabilised cell nuclear protein import assays, only wild-type NTF2 and mutants that bound GDP-Ran were functional. Furthermore, when the NTF2 E42K and D92N/D94N NTF2 mutants that failed to bind GDP-Ran in vitro were substituted for the chromosomal yeast NTF2, the yeast cells became non-viable, whereas yeast substituted with wild-type human NTF2 remained viable. We conclude that interaction between NTF2 and GDP-Ran is important for efficient nuclear protein import.     

Interactions between a nuclear transporter and a subset of nuclear pore complex proteins depend on Ran GTPase

Proteins to be transported into the nucleus are recognized by members of the importin-karyopherin nuclear transport receptor family. After docking at the nuclear pore complex (NPC), the cargo-receptor complex moves through the aqueous pore channel. Once cargo is released, the importin then moves back through the channel for new rounds of transport. Thus, importin and exportin, another member of this family involved in export, are thought to continuously shuttle between the nuclear interior and the cytoplasm. In order to understand how nuclear transporters traverse the NPC, we constructed functional protein fusions between several members of the yeast importin family, including Pse1p, Sxm1p, Xpo1p, and Kap95p, and the green fluorescent protein (GFP). Complexes containing nuclear transporters were isolated by using highly specific anti-GFP antibodies. Pse1-GFP was studied in the most detail. Pse1-GFP is in a complex with importin-alpha and -beta (Srp1p and Kap95p in yeast cells) that is sensitive to the nucleotide-bound state of the Ran GTPase. In addition, Pse1p associates with the nucleoporins Nsp1p, Nup159p, and Nup116p, while Sxm1p, Xpo1p, and Kap95p show different patterns of interaction with nucleoporins. Association of Pse1p with nucleoporins also depends on the nucleotide-bound state of Ran; when Ran is in the GTP-bound state, the nucleoporin association is lost. A mutant form of Pse1p that does not bind Ran also fails to interact with nucleoporins. These data indicate that transport receptors such as Pse1p interact in a Ran-dependent manner with certain nucleoporins. These nucleoporins may represent major docking sites for Pse1p as it moves in or out of the nucleus via the NPC.     

Structural basis for the recognition of carbohydrates by human galectin-7

Knowledge about carbohydrate recognition domains of galectins, formerly known as S-type animal lectins, is important in understanding their role(s) in cell-cell interactions. Here we report the crystal structure of human galectin-7 (hGal-7), in free form and in the presence of galactose, galactosamine, lactose, and N-acetyl-lactosamine at high resolution. This is the first structure of a galectin determined in both free and carbohydrate-bound forms. The structure shows a fold similar to that of the prototype galectins -1 and -2, but has greater similarity to a related galectin molecule, Gal-10. Even though the carbohydrate-binding residues are conserved, there are significant changes in this pocket due to shortening of a loop structure. The monomeric hGal-7 molecule exists as a dimer in the crystals, but adopts a packing arrangement considerably different from that of Gal-1 and Gal-2, which has implications for carbohydrate recognition.     

A molecular redox switch on p21(ras). Structural basis for the nitric oxide-p21(ras) interaction

We have identified the site of molecular interaction between nitric oxide (NO) and p21(ras) responsible for initiation of signal transduction. We found that p21(ras) was singly S-nitrosylated and localized this modification to a fragment of p21(ras) containing Cys118. A mutant form of p21(ras), in which Cys118 was changed to a serine residue and termed p21(ras)C118S, was not S-nitrosylated. NO-related species stimulated guanine nucleotide exchange on wild-type p21(ras), resulting in an active form, but not on p21(ras)C118S. Furthermore, in contrast to parental Jurkat T cells, NO-related species did not stimulate mitogen-activated protein kinase activity in cells transfected with p21(ras)C118S. These data indicate that Cys118 is a critical site of redox regulation of p21(ras), and S-nitrosylation of this residue triggers guanine nucleotide exchange and downstream signaling.     

The signal recognition particle receptor of Escherichia coli (FtsY) has a nucleotide exchange factor built into the GTPase domain

Targeting of many secretory and membrane proteins to the inner membrane in Escherichia coli is achieved by the signal recognition particle (SRP) and its receptor (FtsY). In E. coli SRP consists of only one polypeptide (Ffh), and a 4.5S RNA. Ffh and FtsY each contain a conserved GTPase domain (G domain) with an alpha-helical domain on its N terminus (N domain). The nucleotide binding kinetics of the NG domain of the SRP receptor FtsY have been investigated, using different fluorescence techniques. Methods to describe the reaction kinetically are presented. The kinetics of interaction of FtsY with guanine nucleotides are quantitatively different from those of other GTPases. The intrinsic guanine nucleotide dissociation rates of FtsY are about 10(5) times higher than in Ras, but similar to those seen in GTPases in the presence of an exchange factor. Therefore, the data presented here show that the NG domain of FtsY resembles a GTPase-nucleotide exchange factor complex not only in its structure but also kinetically. The I-box, an insertion present in all SRP-type GTPases, is likely to act as an intrinsic exchange factor. From this we conclude that the details of the GTPase cycle of FtsY and presumably other SRP-type GTPases are fundamentally different from those of other GTPases.     

Structural determinants required for the interaction between Rho GTPase and the GTPase-activating domain of p190

The Rho family small GTP-binding proteins are subjected to regulation by Rho GTPase-activating proteins (GAPs) in the course of transmitting diverse intracellular signals. To understand the mechanism of GAP-catalyzed GTP hydrolysis of Rho GTPases, we have studied the interaction between RhoA and p190, the RasGAP binding phosphoprotein which has been implicated as a Rho-specific GAP, by delineating the structural determinants of RhoA and p190 GAP domain (p190GD) that are involved in their functional coupling. Besides the conserved residues Tyr34, Thr37, and Phe39 in the switch I region of RhoA which are required for p190GD interaction, chimeras made between RhoA and Cdc42, a close relative of RhoA with which p190GD interacts 50-fold less efficiently, revealed that residues outside the switch I and neighboring regions of RhoA, residues 85-122 in particular, contain the major p190GD-specifying determinant(s). Mutation of the unique Asp90 of RhoA in this region mostly abolished p190GD stimulation, whereas the corresponding reverse mutation of Cdc42 (S88D) was able to respond to p190GD-catalysis similarly as RhoA. Further kinetic analysis of these mutants provided evidence that Asp90 of RhoA contributes primarily to the specific binding interaction with p190GD. On the other hand, two charged residues of p190GD, Arg1283 and Lys1321, which are located in the putative G-protein binding helix pocket of GAP domain, were found to be involved in different aspects of interaction with RhoA. The R1283L mutant of p190GD lost GAP activity but retained the ability to bind to RhoA, while K1321A failed to stimulate and to bind to RhoA. These results indicate that residue Asp90 constitutes the second GAP-interactive site in RhoA which is mostly responsible for conferring p190GD-specificity, and suggest that the role of p190GD in the GTPase reaction of RhoA is in part to supply active site residue Arg1283 for efficient catalysis.     

Structural basis for the pathophysiology of lipoprotein(a) in the athero-thrombotic process

Lipoprotein Lp(a) is a major and independent genetic risk factor for atherosclerosis and cardiovascular disease. The essential difference between Lp(a) and low density lipoproteins (LDL) is apolipoprotein apo(a), a glycoprotein structurally similar to plasminogen, the precursor of plasmin, the fibrinolytic enzyme. This structural homology endows Lp(a) with the capacity to bind to fibrin and to membrane proteins of endothelial cells and monocytes, and thereby to inhibit plasminogen binding and plasmin generation. The inhibition of plasmin generation and the accumulation of Lp(a) on the surface of fibrin and cell membranes favor fibrin and cholesterol deposition at sites of vascular injury. Moreover, insufficient activation of TGF-beta due to low plasmin activity may result in migration and proliferation of smooth muscle cells into the vascular intima. These mechanisms may constitute the basis of the athero-thrombogenic mode of action of Lp(a). It is currently accepted that this effect of Lp(a) is linked to its concentration in plasma. An inverse relationship between Lp(a) concentration and apo(a) isoform size, which is under genetic control, has been documented. Recently, it has been shown that inhibition of plasminogen binding to fibrin by apo(a) is also inversely associated with isoform size. Specific point mutations may also affect the lysine-binding function of apo(a). These results support the existence of functional heterogeneity in apolipoprotein(a) isoforms and suggest that the predictive value of Lp(a) as a risk factor for vascular occlusive disease would depend on the relative concentration of the isoform with the highest affinity for fibrin.     

Structural basis for inhibition of the Hsp90 molecular chaperone by the antitumor antibiotics radicicol and geldanamycin

OBJECTIVES: Prostate cancer recurrence, evidenced by rising prostate-specific antigen (PSA) levels after radical prostatectomy, is an increasingly prevalent clinical problem in need of new treatment options. Preclinical studies have suggested that for tumors in general, settings of minimal cancer volume may be uniquely suitable for recombinant vaccine therapy targeting tumor-associated antigens. A clinical study was undertaken to evaluate the safety and biologic effects of vaccinia-PSA (PROSTVAC) administered to subjects with postprostatectomy recurrence of prostate cancer and to assess the feasibility of interrupted androgen deprivation as a tool for modulating expression of the vaccine target antigen, as well as detecting vaccine bioactivity in vivo. METHODS: A limited Phase I clinical trial was conducted to evaluate the safety and biologic effects of vaccinia-PSA administered in 6 patients with androgen-modulated recurrence of prostate cancer after radical prostatectomy. End points included toxicity, serum PSA rise related to serum testosterone restoration, and immunologic effects measured by Western blot analysis for anti-PSA antibody induction. RESULTS: Toxicity was minimal, and dose-limiting toxicity was not observed. Noteworthy variability in time required for testosterone restoration (after interruption of androgen deprivation therapy) was observed. One subject showed continued undetectable serum PSA (less than 0.2 ng/mL) for over 8 months after testosterone restoration, an interval longer than those reported in previous androgen deprivation interruption studies. Primary anti-PSA IgG antibody activity was induced after vaccinia-PSA immunization in 1 subject, although such antibodies were detectable in several subjects at baseline. CONCLUSIONS: Interrupted androgen deprivation may be a useful tool for modulating prostate cancer bioactivity in clinical trials developing novel biologic therapies. Immune responses against PSA may be present among some patients with prostate cancer at baseline and may be induced in others through vaccinia-PSA immunization.     

Structural characteristics of 2'-O-(2-methoxyethyl)-modified nucleic acids from molecular dynamics simulations

The structure and physical properties of 2'-sugar substituted O -(2-methoxyethyl) (MOE) nucleic acids have been studied using molecular dynamics simulations. Nanosecond simulations on the duplex MOE[CCAACGTTGG]-r[CCAACGUUGG] in aqueous solution have been carried out using the particle mesh Ewald method. Parameters for the simulation have been developed from ab initio calculations on dimethoxyethyl fragments in a manner consistent with the AMBER 4.1 force field database. The simulated duplex is compared with the crystal structure of the self-complementary duplex d[GCGTATMOEACGC]2, which contains a single modification in each strand. Structural details from each sequence have been analyzed to rationalize the stability imparted by substitution with 2'- O -(2-methoxyethyl) side chains. Both duplexes have an A-form structure, as indicated by several parameters, most notably a C3' endo sugar pucker in all residues. The simulated structure maintains a stable A-form geometry throughout the duration of the simulation with an average RMS deviation of 2.0 A from the starting A-form structure. The presence of the 2' substitution appears to lock the sugars in the C3' endo conformation, causing the duplex to adopt a stable A-form geometry. The side chains themselves have a fairly rigid geometry with trans , trans , gauche +/- and trans rotations about the C2'-O2', O2'-CA', CA'-CB' and CB'-OC' bonds respectively.     

Structural basis for the high affinity of amino-aromatic SH2 phosphopeptide ligands

When vertebrate somatic cells are selectively irradiated in the nucleus during late prophase (<30 min before nuclear envelope breakdown) they progress normally through mitosis even if they contain broken chromosomes. However, if early prophase nuclei are similarly irradiated, chromosome condensation is reversed and the cells return to interphase. Thus, the G2 checkpoint that prevents entry into mitosis in response to nuclear damage ceases to function in late prophase. If one nucleus in a cell containing two early prophase nuclei is selectively irradiated, both return to interphase, and prophase cells that have been induced to returned to interphase retain a normal cytoplasmic microtubule complex. Thus, damage to an early prophase nucleus is converted into a signal that not only reverses the nuclear events of prophase, but this signal also enters the cytoplasm where it inhibits e.g., centrosome maturation and the formation of asters. Immunofluorescent analyses reveal that the irradiation-induced reversion of prophase is correlated with the dephosphorylation of histone H1, histone H3, and the MPM2 epitopes. Together, these data reveal that a checkpoint control exists in early but not late prophase in vertebrate cells that, when triggered, reverses the cell cycle by apparently downregulating existing cyclin-dependent kinase (CDK1) activity.     

The molecular basis for cross-reacting material-positive hemophilia A due to missense mutations within the A2-domain of factor VIII

Factor VIII (FVIII) is the protein defective in the bleeding disorder hemophilia A. Approximately 5% of hemophilia A patients have normal amounts of a dysfunctional FVIII protein and are termed cross-reacting material (CRM)-positive. The majority of genetic alterations that result in CRM-positive hemophilia A are missense mutations within the A2-domain. To determine the mechanistic basis of the genetic defects within the A2-domain for FVIII function we constructed six mutations within the FVIII cDNA that were previously found in five CRM-positive hemophilia A patients (R527W, S558F, I566T, V634A, and V634M) and one CRM-reduced hemophilia A patient (DeltaF652/3). The specific activity for each mutant secreted into the conditioned medium from transiently transfected COS-1 cells correlated with published data for the patients plasma-derived FVIII, confirming the basis of the genetic defect. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of immunoprecipitated FVIII protein radiolabeled in COS-1 cells showed that all CRM-positive mutant proteins were synthesized and secreted into the medium at rates similar to wild-type FVIII. The majority of the DeltaF652/3 mutant was defective in secretion and was degraded within the cell. All mutant FVIII proteins were susceptible to thrombin cleavage, and the A2-domain fragment from the I566T mutant had a reduced mobility because of use of an introduced potential N-linked glycosylation site that was confirmed by N-glycanase digestion. To evaluate interaction of FVIII with factor IXa, we performed an inhibition assay using a synthetic peptide corresponding to FVIII residues 558 to 565, previously shown to be a factor IXa interaction site. The concentration of peptide required for 50% inhibition of FVIII activity (IC50) was reduced for the I566T (800 mumol/L) and the S558F (960 mumol/L) mutants compared with wild-type FVIII (> 2,000 mumol/L). N-glycanase digestion increased I566T mutant FVIII activity and increased its IC50 for the peptide (1,400 mumol/L). In comparison to S558F, a more conservative mutant (S558A) had a sixfold increased specific activity that also correlated with an increased IC50 for the peptide. These results provided support that the defects in the I566T and S558F FVIII molecules are caused by steric hindrance for interaction with factor IXa.     

Structural requirements for ligand interactions at the benzodiazepine recognition site of the GABA(A) receptor

We have measured the rebinding of carbon monoxide (CO) to some distal mutants of myoglobin (Mb) in the time range from 10(-8) to 10(-1) s by flash photolysis, in which the photodissociated CO rebinds to the heme iron without escaping to the solvent water from the protein matrix. We have found that the double mutants [His64-->Val/Val68-->Thr (H64V/V68T) and His64-->Val/Val68-->Ser (H64V/V68S)] have an extremely large geminate yield (70-80%) in water at 5 degreesC, in contrast to the 7% of the geminate yield of wild-type Mb. The CO geminate yields for these two mutants are the largest in those of Mb mutants reported so far, showing that the two mutants have a unique heme environment that favors CO geminate rebinding. Comparing the crystal structures and 1H-NMR and vibrational spectral data of H64V/V68T and H64V/V68S with those of other mutants, we discuss factors that may control the nanosecond geminate CO rebinding and CO migration in the protein matrix.     

Utilization of chimeras between human (HM-175) and simian (AGM-27) strains of hepatitis A virus to study the molecular basis of virulence

Chimeras between human (HM-175) and simian (AGM-27) strains of hepatitis A virus (HAV) were constructed to evaluate the effect of the 2C gene of AGM-27 on HAV replication in cell culture and virulence in tamarins (Saguinus mystax) and chimpanzees (Pan troglodytes). Kinetic studies and radioimmunofocus assays demonstrated that replacement of the 2C gene of HAV/7, a cell culture-adapted strain of HM-175, with that of AGM-27 drastically reduced the ability of the virus to replicate in cultured cells. Intragenic chimeras containing AGM-27 sequences in either the 5' or 3' half of the 2C gene replicated in cell culture at an intermediate level. Whereas HAV/7 is attenuated for tamarins, a chimera containing the simian virus 2C gene in the HAV/7 background was virulent in tamarins, demonstrating that the simian virus 2C gene alone can confer the phenotype of virulence to an otherwise attenuated virus. Clusters of AGM-27-specific residues near both ends of the 2C protein were required for virulence since a chimera containing AGM-27 sequences in the carboxy-terminal half of 2C was partially attenuated for tamarins while one containing AGM-27 sequences only in the amino-terminal half of 2C was even more attenuated. Chimeras containing either the entire or only the 3' half of the simian virus 2C gene in the HAV/7 background were attenuated for chimpanzees.     

A molecular mechanics and dynamics study of the minor adduct between DNA and the carcinogen 2-(acetylamino)fluorene (dG-N2-AAF)

Experimental studies involving the carcinogenic aromatic amine 2-(acetylamino)fluorene (AAF) have afforded two acetylated DNA adducts, the major one bound to C8 of guanine and a minor adduct bound to N2 of guanine. The minor adduct may be important in carcinogenesis because it persists, while the major adduct is rapidly repaired. Primer extension studies of the minor adduct have indicated that it blocks DNA synthesis, with some bypass and misincorporation of adenine opposite the lesion [Shibutani, S., and Grollman, A.P. (1993) Chem. Res. Toxicol. 6, 819-824]. No experimental structural information is available for this adduct. Extensive minimized potential energy searches involving thousands of trials and molecular dynamics simulations were used to study the conformation of this adduct in three sequences: I, d(C1-G2-C3-[AAF]G4-C5-G6-C7).d(G8-C9-G10-C11-G12-C13-G14+ ++); II, the sequence of Shibutani and Grollman, d(C1-T2-A3-[AAF]G4-T5-C6-A7).d(T8-G9-A10-C11-T12-A13-G14); and III, which is the same as II but with a mismatched adenine in position 11, opposite the lesion. AAF was located in the minor groove in the low-energy structures of all sequences. In the lowest energy form of the C3-[AAF]G4-C5 sequence I, the fluorenyl rings point in the 3' direction along the modified strand and the acetyl in the 5' direction. These orientations are reversed in the second lowest energy structure of this sequence, and the energy of this structure is 1.4 kcal/mol higher. Watson Crick hydrogen bonding is intact in both structures. In the two lowest energy structures of the A3-[AAF]G4-T5 sequence II, the AAF is also located in the minor groove with Watson-Crick hydrogen bonding intact. However, in the lowest energy form, the fluorenyl rings point in the 5' direction and the acetyl in the 3' direction. The energy of the structure with opposite orientation is 5.1 kcal/mol higher. In sequence III with adenine mismatched to the modified guanine, the lowest energy form also had the fluorenyl rings oriented 5' in the minor groove with intact Watson-Crick base pairing. However, the mispaired adenine adopts a syn orientation with Hoogsteen pairing to the modified guanine. These results suggest that the orientation of the AAF in the minor groove may be DNA sequence dependent. Mobile aspects of favored structures derived from molecular dynamics simulations with explicit solvent and salt support the essentially undistorting nature of this lesion, which is in harmony with its persistence in mammalian systems.     

Structural requirements for peptidic antagonists of the corticotropin-releasing factor receptor (CRFR): development of CRFR2beta-selective antisauvagine-30

Different truncated and conformationally constrained analogs of corticotropin-releasing factor (CRF) were synthesized on the basis of the amino acid sequences of human/rat CRF (h/rCRF), ovine CRF (oCRF), rat urocortin (rUcn), or sauvagine (Svg) and tested for their ability to displace [125I-Tyr0]oCRF or [125I-Tyr0]Svg from membrane homogenates of human embryonic kidney (HEK) 293 cells stably transfected with cDNA coding for rat CRF receptor, type 1 (rCRFR1), or mouse CRF receptor, type 2beta (mCRFR2beta). Furthermore, the potency of CRF antagonists to inhibit oCRF- or Svg-stimulated cAMP production of transfected HEK 293 cells expressing either rCRFR1 (HEK-rCRFR1 cells) or mCRFR2beta (HEK-mCRFR2beta cells) was determined. In comparison with astressin, which exhibited a similar affinity to rCRFR1 (Kd = 5.7 +/- 1.6 nM) and mCRFR2beta (Kd = 4.0 +/- 2.3 nM), [DPhe11,His12]Svg(11-40), [DLeu11]Svg(11-40), [DPhe11]Svg(11-40), and Svg(11-40) bound, respectively, with a 110-, 80-, 68-, and 54-fold higher affinity to mCRFR2beta than to rCRFR1. The truncated analogs of rUcn displayed modest preference (2- to 7-fold) for binding to mCRFR2beta. In agreement with the results of these binding experiments, [DPhe11, His12]Svg(11-40), named antisauvagine-30, was the most potent and selective ligand to suppress agonist-induced adenylate cyclase activity in HEK cells expressing mCRFR2beta.     

Characterization of the bioactive form and molecular determinants of recognition of cyclic enkephalin peptides at the delta-opioid receptor

An extensive and systematic search strategy to determine the conformational profile of 12 cyclic disulfide-bridged opioid peptides with varying affinities at the delta receptor has been carried out to identify the structure that is recognized by the delta receptor for each analogue. The methods and procedures used here for the conformational search have already been validated for [D-Pen2,D-Pen5] enkephalin (DPDPE), one member of this family. Use of these methods led to a low-energy solution conformation of DPDPE in excellent agreement with all the geometric properties deduced from its solution nmr spectra. Each of the analogue was subjected to the same procedure, involving a combination of molecular dynamics simulations at high and low temperature. The study was repeated in two environmental conditions, an apolar environment, simulated by using a distance-dependent dielectric constant, and a polar environment by embedding the peptides in a high constant dielectric (epsilon = 80). An automated comparison of the different conformers based on their backbone rms and average distance between the key aromatic moieties was followed by graphic analysis using maximum structural overlap. The cross-comparison of the conformations for each analogue revealed a unique conformer that may be recognized by the delta receptor for each high-affinity analogue that permitted maintaining the critical elements required for recognition in a simple spatial orientation, while maximizing similarity in other regions.