Solution structure of the PDZ2 domain from human phosphatase hPTP1E and its interactions with C-terminal peptides from the Fas receptor

The solution structure of the second PDZ domain (PDZ2) from human phosphatase hPTP1E has been determined using 2D and 3D heteronuclear NMR experiments. The binding of peptides derived from the C-terminus of the Fas receptor to PDZ2 was studied via changes in backbone peptide and protein resonances. The structure is based on a total of 1387 nonredundant experimental NMR restraints including 1261 interproton distance restraints, 45 backbone hydrogen bonds, and 81 torsion angle restraints. Analysis of 30 lowest-energy structures resulted in rmsd values of 0.41 +/- 0.09 A for backbone atoms (N, Calpha, C') and 1.08 +/- 0.10 A for all heavy atoms, excluding the disordered N- and C-termini. The hPTP1E PDZ2 structure is similar to known PDZ domain structures but contains two unique structural features. In the peptide binding domain, the first glycine of the GLGF motif is replaced by a serine. This serine appears to replace a bound water observed in PDZ crystal structures that hydrogen bonds to the bound peptide's C-terminus. The hPTP1E PDZ2 structure also contains an unusually large loop following strand beta2 and proximal to the peptide binding site. This well-ordered loop folds back against the PDZ domain and contains several residues that undergo large amide chemical shift changes upon peptide binding. Direct observation of peptide resonances demonstrates that as many as six Fas peptide residues interact with the PDZ2 domain.     

Interaction of troponin I and troponin C: 19F NMR studies of the binding of the inhibitory troponin I peptide to turkey skeletal troponin C.

We have used 19F nuclear magnetic resonance spectroscopy to study the interaction of the inhibitory region of troponin (TnI) with apo- and calcium(II)-saturated turkey skeletal troponin C (TnC), using the synthetic TnI analogue N alpha-acetyl[19FPhe106]TnI(104-115)amide. Dissociation constants of Kd = (3.7 +/- 3.1) x 10(-5) M for the apo interaction and Kd = (4.8 +/- 1.8) x 10(-5) M for the calcium(II)-saturated interaction were obtained using a 1:1 binding model of peptide to protein. The 19F NMR chemical shifts for the F-phenylalanine of the bound peptide are different from the apo- and calcium-saturated protein, indicating a different environment for the bound peptide. The possibility of 2:1 binding of the peptide to Ca(II)-saturated TnC was tested by calculating the fit of the experimental titration data to a series of theoretical binding curves in which the dissociation constants for the two hypothetical binding sites were varied. We obtained the best fit for 0.056 mM less than or equal to Kd1 less than or equal to 0.071 mM and 0.5 mM less than or equal to Kd2 less than or equal to 2.0 mM. These results allow the possibility of a second peptide binding site on calcium(II)-saturated TnC with an affinity 10- to 20-fold weaker than that of the first site.     

Energetics of Shaker K channels block by inactivation peptides

A synthetic peptide of the NH2-terminal inactivation domain of the ShB channel blocks Shaker channels which have an NH2-terminal deletion and mimics many of the characteristics of the intramolecular inactivation reaction. To investigate the role of electrostatic interactions in both peptide block and the inactivation process we measured the kinetics of block of macroscopic currents recorded from the intact ShB channel, and from ShB delta 6-46 channels in the presence of peptides, at different ionic strengths. The rate of inactivation and the association rate constants (k(on)) for the ShB peptides decreased with increasing ionic strength. k(on) for a more positively charged peptide was more steeply dependent on ionic strength consistent with a simple electrostatic mechanism of enhanced diffusion. This suggests that a rate limiting step in the inactivation process is the diffusion of the NH2-terminal domain towards the pore. The dissociation rates (k(off)) were insensitive to ionic strength. The temperature dependence of k(on) for the ShB peptide was very high, (Q10 = 5.0 +/- 0.58), whereas k(off) was relatively temperature insensitive (Q10 approximately 1.1). The results suggest that at higher temperatures the proportion of time either the peptide or channel spends in the correct conformation for binding is increased. There were two components to the time course of recovery from block by the ShB peptide, indicating two distinct blocked states, one of which has similar kinetics and dependence on external K+ concentration as the inactivated state of ShB. The other is voltage- dependent and at -120 mV is very unstable. Increasing the net charge on the peptide did not increase sensitivity to knock-off by external K+. We propose that the free peptide, having fewer constraints than the tethered NH2-terminal domain binds to a similar site on the channel in at least two different conformations.     

Mobility of TOAC spin-labelled peptides binding to the Src SH3 domain studied by paramagnetic NMR

Paramagnetic relaxation enhancement provides a tool for studying the dynamics as well as the structure of macromolecular complexes. The application of side-chain coupled spin-labels is limited by the mobility of the free radical. The cyclic, rigid amino acid spin-label TOAC (2,2,6,6-Tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid), which can be incorporated straightforwardly by peptide synthesis, provides an attractive alternative. In this study, TOAC was incorporated into a peptide derived from focal adhesion kinase (FAK), and the interaction of the peptide with the Src homology 3 (SH3) domain of Src kinase was studied, using paramagnetic NMR. Placing TOAC within the binding motif of the peptide has a considerable effect on the peptide-protein binding, lowering the affinity substantially. When the TOAC is positioned just outside the binding motif, the binding constant remains nearly unaffected. Although the SH3 domain binds weakly and transiently to proline-rich peptides from FAK, the interaction is not very dynamic and the relative position of the spin-label to the protein is well-defined. It is concluded that TOAC can be used to generate reliable paramagnetic NMR restraints.     

Structure characterization of the central repetitive domain of high molecular weight gluten proteins. I. Model studies using cyclic and linear peptides.

The high molecular weight (HMW) proteins from wheat contain a repetitive domain that forms 60-80% of their sequence. The consensus peptides PGQGQQ and GYYPTSPQQ form more than 90% of the domain; both are predicted to adopt beta-turn structure. This paper describes the structural characterization of these consensus peptides and forms the basis for the structural characterization of the repetitive HMW domain, described in the companion paper. The cyclic peptides cyclo-[PGQGQQPGQGQQ] (peptide 1), cyclo-[GYYPTSPQQGA] (peptide 2), and cyclo-[PGQGQQGYYPTSPQQ] (peptide 3) were prepared using a novel synthesis route. In addition, the linear peptides (PGQGQQ)n (n = 1, 3, 5) were prepared. CD, FTIR, and NMR data demonstrated a type II beta-turn structure at QPGQ in the cyclic peptide 1 that was also observed in the linear peptides 9PGQGQQ)n. A type I beta-turn was observed at YPTS and SPQQ in peptides 2 and 3, with additional beta-turns of either type I or II at GAGY (peptide 2) and QQGY (peptide 3). The proline in YPTS showed considerable cis/trans isomerization, with up to 50% of the population in the cis-conformation; the other prolines were more than 90% in the trans conformation. The conversion from trans to cis destroys the type I beta-turn at YPTS, but leads to an increase in turn character at SPQQ and GAGY (peptide 2) or QQGY (peptide 3).     

Characterization of high-affinity [3H]TBZOH binding to the human platelet vesicular monoamine transporter.

The present study indicates that human platelets can be used as an accessible peripheral model not only for the plasma membrane serotonin transporter, but also for the vesicular monoamine transporter. The vesicular monoamine transporter (VMAT2) is responsible for the accumulation of monoamines in the synaptic vesicles. VMAT2 differs from the plasma membrane transporters in its capability to recognize serotonin, histamine, norepinephrine and dopamine with almost the same affinity. Dihydrotetrabenazine (TBZOH) is a very potent inhibitor of VMAT2 that binds with high affinity to this transporter. [3H]TBZOH has been used as a ligand to label VMAT2 in human, bovine and rodent brain. In this study we characterized the pharmacodynamic and pharmacokinetic parameters of [3H]TBZOH binding in human platelets as compared to rat brain. The density (Bmax) and affinity (Kd) of [3H]TBZOH specific binding was assessed by Scatchard analysis. Association and dissociation rate constants (k(on), K(off)) were assessed by kinetic binding studies. In this study high-affinity and saturable binding sites for [3H]TBZOH were demonstrated in human platelets. Both the affinity of [3H]TBZOH to its binding site in platelets (Kd = 3.2+/-0.5 nM) and the kinetic rate constants (K(on) = 2.8 x 10(7) M(-1) min(-1); K(off) = 0.099 min(-1)) were similar to that in rat brain (Kd(striatum) = 1.5 nM; Kd(cerebral cortex) = 1.35 nM; K(on) = 2 x 10(7) M(-1) min(-1); K(off) = 0.069 min(-1)). Only the VMAT2 blockers tetrabenazine and reserpine inhibited [3H]TBZOH specific binding.     

An allosteric mechanism controls antigen presentation by the H-2K(b) complex.

The mechanism of assembly/dissociation of a recombinant water-soluble class I major histocompatibility complex (MHC) H-2Kb molecule was studied by a real-time fluorescence resonance energy transfer method. Like the H-2Kd ternary complex [Gakamsky et al. (1996) Biochemistry 35, 14841-14848], the interactions among the heavy chain, beta2-microglobulin (beta2m), and antigenic peptides were found to be controlled by an allosteric mechanism. Association of the heavy chain with beta2m increased peptide binding rate constants by more than 2 orders of magnitude and enhanced affinity of the heavy-chain molecule for peptides. Interaction of peptides with the heavy-chain binding site, in turn, increased markedly the affinity of the heavy chain for beta2m. Binding of peptide variants of the ovalbumin sequence (257-264) to the heavy chain/beta2m heterodimer was found to be a biphasic reaction. The fast phase was a second-order process with nearly the same rate constants as those of binding of peptides derived from the influenza virus nucleoprotein 147-155 to the H-2Kd heavy chain/beta2m heterodimer [(3.0 +/- 1.0) x 10(-6) M-1 s-1 at 37 degrees C]. The slow phase was a result of both the ternary complex assembly from the "free" heavy chain, beta2m, and peptide as well as an intramolecular conformational transition within the heavy chain/beta2m heterodimer to a peptide binding conformation. Biexponential kinetics of peptide or beta2m dissociation from the ternary complex were observed. They suggest that it can exist in two conformations. The rate constants of beta2m dissociation from the H-2Kb ternary complex were, in the limits of experimental accuracy, independent of the structure of the bound peptide, though their affinities differed by an order of magnitude. Dissociation of peptides from the Kb heavy chain was always faster than from the ternary complexes, yet the heavy chain/peptide complexes were considerably more stable compared with their Kd/nucleoprotein peptide counterparts.     

Stopped-flow studies of the binding of 2-n-heptyl-4-hydroxyquinoline-N-oxide to fumarate reductase of Escherichia coli.

We have studied the kinetics of binding of the menaquinol analog 2-n-heptyl-4-hydroxyquinoline-N-oxide (HOQNO) by fumarate reductase (FrdABCD) using the stopped-flow method. The results show that the fluorescence of HOQNO is quenched when HOQNO binds to FrdABCD. The observed quenching of HOQNO fluorescence has two phases and it can be best fitted to a double exponential equation. A two-step equilibrium model is applied to describe the binding process in which HOQNO associates with FrdABCD by a fast bimolecular step to form a loosely bound complex; this is subsequently converted into a tightly bound complex by a slow unimolecular step. The rates of the forward and the reverse reactions for the first equilibrium (k1 and k2) are determined to be k1 = (1.1 +/- 0.1) x 10(7) M-1.s-1, and k2 = 6.0 +/- 0.6 s-1, respectively. The dissociation constants of the first equilibrium (Kd1 = k2/k1) is calculated to be about 550 nM. The overall dissociation constant for the two-step equilibrium, Kd overall = Kd1/[1+ (1/Kd2)], is estimated to be < or = 7 nM. Comparison of the kinetic parameters of HOQNO binding by FrdABCD and by dimethyl sulfoxide reductase provides important information on menaquinol binding by these two enzymes.     

Kinetic analysis of the interactions between troponin C and the C-terminal troponin I regulatory region and validation of a new peptide delivery/capture system used for surface plasmon resonance

Using surface plasmon resonance (SPR)-based biosensor analysis and fluorescence spectroscopy, the apparent kinetic constants, k(on) and k(off), and equilibrium dissociation constant, K(d), have been determined for the binding interaction between rabbit skeletal troponin C (TnC) and rabbit skeletal troponin I (TnI) regulatory region peptides: TnI(96-115), TnI(96-131) and TnI(96-139). To carry out SPR analysis, a new peptide delivery/capture system was utilized in which the TnI peptides were conjugated to the E-coil strand of a de novo designed heterodimeric coiled-coil domain. The TnI peptide conjugates were then captured via dimerization to the opposite strand (K-coil), which was immobilized on the biosensor surface. TnC was then injected over the biosensor surface for quantitative binding analysis. For fluorescence spectroscopy analysis, the environmentally sensitive fluoroprobe 5-((((2-iodoacetyl)amino)ethyl)amino) naphthalene-1-sulfonic acid (1,5-IAEDANS) was covalently linked to Cys98 of TnC and free TnI peptides were added. SPR analysis yielded equilibrium dissociation constants for TnC (plus Ca(2+)) binding to the C-terminal TnI regulatory peptides TnI(96-131) and TnI(96-139) of 89nM and 58nM, respectively. The apparent association and dissociation rate constants for each interaction were k(on)=2.3x10(5)M(-1)s(-1), 2.0x10(5)M(-1)s(-1) and k(off)=2.0x10(-2)s(-1), 1.2x10(-2)s(-1) for TnI(96-131) and TnI(96-139) peptides, respectively. These results were consistent with those obtained by fluorescence spectroscopy analysis: K(d) being equal to 130nM and 56nM for TnC-TnI(96-131) and TnC-TnI(96-139), respectively. Interestingly, although the inhibitory region peptide (TnI(96-115)) was observed to bind with an affinity similar to that of TnI(96-131) by fluorescence analysis (K(d)=380nM), its binding was not detected by SPR. Subsequent investigations examining salt effects suggested that the binding mechanism for the inhibitory region peptide is best characterized by an electrostatically driven fast on-rate ( approximately 1x10(8) to 1x10(9)M(-1)s(-1)) and a fast off-rate ( approximately 1x10(2)s(-1)). Taken together, the determination of these kinetic rate constants permits a clearer view of the interactions between the TnC and TnI proteins of the troponin complex.     

Cyclic peptide inhibitors of HIV-1 integrase derived from the LEDGF/p75 protein

Restricting linear peptides to their bioactive conformation is an attractive way of improving their stability and activity. We used a cyclic peptide library with conformational diversity for selecting an active and stable peptide that mimics the structure and activity of the HIV-1 integrase (IN) binding loop from its cellular cofactor LEDGF/p75 (residues 361-370). All peptides in the library had the same primary sequence, and differed only in their conformation. Library screening revealed that the ring size and linker structure had a huge effect on the conformation, binding and activity of the peptides. One of the cyclic peptides, c(MZ 4-1), was a potent and stable inhibitor of IN activity in vitro and in cells even after 8 days. The NMR structure of c(MZ 4-1) showed that it obtains a bioactive conformation that is similar to the parent site in LEDGF/p75.     

Thermodynamics of calmodulin trapping by Ca2+/calmodulin-dependent protein kinase II: subpicomolar Kd determined using competition titration calorimetry

Calmodulin (CaM) trapping by Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a phenomenon whereby the affinity of CaM for CaMKII increases >1000-fold following CaMKII autophosphorylation. The molecular basis of this effect is not entirely understood. Binding of CaM to the phosphorylated and the unphosphorylated states of CaMKII is well mimicked by the interaction of CaM with two different length peptides taken from the CaM-binding region of CaMKII, peptides we refer to as the long and intermediate peptides. To better understand the conformational change accompanying CaM trapping, we have used isothermal titration calorimetry (ITC) to compare the binding thermodynamics of CaM to these peptides as well as to a shorter CaMKII-based peptide. Calorimetric analysis revealed that the enthalpy, rather than the entropy, distinguished binding of these three peptides. Furthermore, the heat capacity change was found to be similar for the long and intermediate peptides but smaller in magnitude for the short peptide. Direct titration of CaM with peptide provided the Kd value for the short peptide (Kd = 5.9 +/- 2.4 microM), but a novel, two-phased competitive binding strategy was necessary to ascertain the affinities of the intermediate (Kd = 0.17 +/- 0.06 nM) and long (Kd = 0.07 +/- 0.04 pM) peptides. To our knowledge, the Kd for the long peptide is the most potent measured to date using ITC. Together, the findings reported here support a model whereby the final conformational change accompanying CaM trapping buries little additional surface area but does involve formation of new hydrogen bonds and van der Waals contacts that contribute to formation of the high-affinity, CaM-trapped state.     

Functional properties of the recombinant kringle-2 domain of tissue plasminogen activator produced in Escherichia coli

The kringle-2 domain (residues 176-262) of tissue-type plasminogen activator (t-PA) was cloned and expressed in Escherichia coli. The recombinant peptide, which concentrated in cytoplasmic inclusion bodies, was isolated, solubilized, chemically refolded, and purified by affinity chromatography on lysine-Sepharose to apparent homogeneity. [35S]Cysteine-methionine-labeled polypeptide was used to study the interactions of kringle-2 with lysine, fibrin, and plasminogen activator inhibitor-1. The kringle-2 domain bound to lysine-Sepharose and to preformed fibrin with a Kd = 104 +/- 6.2 microM (0.86 +/- 0.012 binding site) and a Kd = 4.2 +/- 1.05 microM (0.80 +/- 0.081 binding site), respectively. Competition experiments and direct binding studies showed that the kringle-2 domain is required for the formation of the ternary t-PA-plasminogen-intact fibrin complex and that the association between the t-PA kringle-2 domain and fibrin does not require plasmin degradation of fibrin and exposure of new COOH-terminal lysine residues. We also observed that kringle-2 forms a complex with highly purified guanidine-activated plasminogen activator inhibitor-1, dissociable by 0.2 M epsilon-aminocaproic acid. The kringle-2 polypeptide significantly inhibited tissue plasminogen activator/plasminogen activator inhibitor-1 interaction. The kringle-2 domain bound to plasminogen activator inhibitor-1 in a specific and saturable manner with a Kd = 0.51 +/- 0.055 microM (0.35 +/- 0.026 binding site). Therefore, the t-PA kringle-2 domain is important for the interaction of t-PA not only with fibrin, but also with plasminogen activator inhibitor-1 and thus represents a key structure in the regulation of fibrinolysis.     

Primary structure requirements for the binding of human high molecular weight kininogen to plasma prekallikrein and factor XI

We recently identified residues 185-224 of the light chain of human high molecular weight kininogen (HMWK) as the binding site for plasma prekallikrein (Tait, J.F., and Fujikawa, K. (1986) J. Biol. Chem. 261, 15396-15401). In the present study, we have further defined the primary structure requirements for binding of HMWK to factor XI and prekallikrein. In a competitive fluorescence polarization binding assay, a 31-residue synthetic peptide (residues 194-224 of the HMWK light chain) bound to prekallikrein with a Kd of 20 +/- 6 nM, indistinguishable from the previously determined value of 18 +/- 5 nM for the light chain. We also prepared three shorter synthetic peptides corresponding to different portions of the 31-residue peptide (residues 205-224, 212-224, and 194-211), but these peptides bound to prekallikrein more than 100-fold more weakly. Factor XI also bound to the same region of the HMWK light chain, but at least 58 residues (185-242) were required for optimal binding (Kd = 69 +/- 4 nM for the light chain; Kd = 130 +/- 50 nM for residues 185-242). The four synthetic peptides inhibited kaolin-activated clotting of blood plasma with potencies paralleling their affinities for prekallikrein and factor XI. Peptide 194-224 can also be used for rapid affinity purification of prekallikrein and factor XI from plasma.     

Selective distinction at equilibrium between the two alpha-neurotoxin binding sites of Torpedo acetylcholine receptor by microtitration

The binding of the monoiodinated alpha-neurotoxin I from Naja mossambica mossambica to the membrane-bound acetylcholine receptor from Torpedo marmorata was investigated using a new picomolar-sensitive microtitration assay. From equilibrium binding studies a non-linear Scatchard plot demonstrated two populations of binding sites characterized by the two dissociation constants Kd1 = 7 +/- 4 pM and Kd2 = 51 +/- 16 pM and having equal binding capacities. These two populations differed in their rate of dissociation (k-1.1 = 25 x 10(-6) s-1 and k-1.2 = 623 x 10(-6) s-1 respectively), but not in their rate of formation of the toxin-receptor complex (k + 1 = 11.7 x 10(6) M-1 s-1). From these rate constants the same two values of dissociation constant were deduced (Kd1 = 2 pM and Kd2 = 53 pM). All the specific binding was prevented by the cholinergic antagonists alpha-bungarotoxin and d-tubocurarine. In addition, a biphasic competition phenomenon allowed us to differentiate between two d-tubocurarine sites (Kda = 103 nM and Kdb = 13.7 microM respectively). Evidence is provided indicating that these two sites are shared by d-tubocurarine and alpha-neurotoxin I, with inverse affinities. Fairly conclusive agreement between our equilibrium, kinetic and competition data demonstrates that the two high-affinity binding sites for this short alpha-neurotoxin are selectively distinguishable.     

XRCC1 interaction with the REV1 C-terminal domain suggests a role in post replication repair

The function of X-ray cross complementing group 1 protein (XRCC1), a scaffold that binds to DNA repair enzymes involved in single-strand break and base excision repair, requires that it be recruited to sites of damaged DNA. However, structural insights into this recruitment are currently limited. Sequence analysis of the first unstructured linker domain of XRCC1 identifies a segment consistent with a possible REV1 interacting region (X1RIR) motif. The X1RIR motif is present in translesion polymerases that can be recruited to the pol ζ/REV1 DNA repair complex via a specific interaction with the REV1 C-terminal domain. NMR and fluorescence titration studies were performed on XRCC1-derived peptides containing this putative RIR motif in order to evaluate the binding affinity for the REV1 C-terminal domain. These studies demonstrate an interaction of the XRCC1-derived peptide with the human REV1 C-terminal domain characterized by dissociation constants in the low micromolar range. Ligand competition studies comparing the XRCC1 RIR peptide with previously studied RIR peptides were found to be inconsistent with the NMR based K_d values. These discrepancies were resolved using a fluorescence assay for which the RIR–REV1 system is particularly well suited. The structure of a REV1-XRCC1 peptide complex was determined by using NOE restraints to dock the unlabeled XRCC1 peptide with a labeled REV1 C-terminal domain. The structure is generally homologous with previously determined complexes with the pol κ and pol η RIR peptides, although the helical segment in XRCC1 is shorter than was observed in these cases. These studies suggest the possible involvement of XRCC1 and its associated repair factors in post replication repair.     

Development of a functional backbone cyclic mimetic of the HIV-1 Tat arginine-rich motif

We have used the backbone cyclic proteinomimetics approach to develop peptides that functionally mimic the arginine-rich motif (ARM) of the HIV-1 Tat protein. This consensus sequence serves both as a nuclear localization signal (NLS) and as an RNA binding domain. Based on the NMR structure of Tat, we have designed and synthesized a backbone cyclic ARM mimetic peptide library. The peptides were screened for their ability to mediate nuclear import of the corresponding BSA conjugates in permeabilized cells. One peptide, designated "Tat11," displayed active NLS properties. Nuclear import of Tat11-BSA was found to proceed by the same distinct pathway used by the Tat-NLS and not by the common importin alpha pathway, which is used by the SV40-NLS. Most of the Tat-derived backbone cyclic peptides display selective inhibitory activity as demonstrated by the inhibition of the nuclear import mediated by the Tat-NLS and not by the SV40-NLS. The Tat-ARM-derived peptides, including Tat-11, also inhibited binding of the HIV-1 Rev-ARM to its corresponding RNA element (Rev response element) with inhibition constants of 5 nm. Here we have shown for the first time (a) a functional mimetic of a protein sequence, which activates a nuclear import receptor and (b) a mimetic of a protein sequence with a dual functionality. Tat11 is a lead compound which can potentially inhibit the HIV-1 life cycle by a dual mechanism: inhibition of nuclear import and of RNA binding.     

Design and synthesis of cyclic disulfide-bonded antibacterial peptides on the basis of the alpha helical domain of Tenecin 1, an insect defensin

We synthesized cyclic disulfide-bonded (i, i+4) peptides with various net positive charges (+2-+5) from linear peptides derived from the alpha helical domain of Tenecin 1, an insect defensin, and investigated the effect of the intradisulfide bridge (i, i+4) on hydrophobicity, secondary structure, leakage activity and binding activity for large unilamellar vesicles, antimicrobial activity, and hemolytic activity. Intradisulfide bridge formation of the peptides resulted in the increase of amphiphilicity and hydrophobicity. Cyclic forms of the peptides did not deeply penetrate into PG/PC (1:1, mole ratio) large unilamellar vesicles and had a decreased lipid membrane perturbation activity for PG/PC LUVs. When the peptides interacted with PG/CL (2:1, mole ratio) LUVs, cyclic peptides with a high net positive charge (+4-+5) showed similar binding affinities and leakage activities for vesicles to those of linear forms, whereas cyclic peptides with a low net positive charge (+2-+3) exhibited lower leakage activity than their linear forms. CD spectra indicate that the intradisulfide bridge (i, i+4) provided little conformational constraint to linear peptides in buffer solution but resulted in the decrease of alpha helicity of the peptides in lipid membrane mimic conditions. The cyclic peptide with the highest net positive charge had a similar antibacterial activity to that of the linear peptide, whereas the cyclic peptides with a low net positive charge (+3-+4) exhibited lower antibacterial activity than their linear forms. The cyclic peptides of an appropriate net charge showed more potent activities against some bacteria than those of linear forms under high salt conditions.     

Characterisation of human dipeptidyl peptidase IV expressed in Pichia pastoris. A structural and mechanistic comparison between the recombinant human and the purified porcine enzyme

Dipeptidyl peptidase IV/CD26 (DP IV) is a multifunctional serine protease cleaving off dipeptides from the N-terminus of peptides. The enzyme is expressed on the surface of epithelial and endothelial cells as a type II transmembrane protein. However, a soluble form of DP IV is also present in body fluids. Large scale expression of soluble human recombinant His(6)-37-766 DP IV, using the methylotrophic yeast Pichia pastoris, yielded 1.7 mg DP IV protein per litre of fermentation supernatant. The characterisation of recombinant DP IV confirmed proper folding and glycosylation similar to DP IV purified from porcine kidney. Kinetic comparison of both proteins using short synthetic substrates and inhibitors revealed similar characteristics. However, interaction analysis of both proteins with the gastrointestinal hormone GLP-1(7-36) resulted in significantly different binding constants for the human and the porcine enzyme (Kd = 153.0 +/- 17.0 microM and Kd = 33.4 +/- 2.2 microM, respectively). In contrast, the enzyme adenosine deaminase binds stronger to human than to porcine DP IV (Kd = 2.15 +/- 0.18 nM and Kd = 7.38 +/- 0.54 nM, respectively). Even though the sequence of porcine DP IV, amplified by RT-PCR, revealed 88% identity between both enzymes, the species-specific variations between amino acids 328 to 341 are likely to be responsible for the differences in ADA-binding.     

Solution structure of a phage-derived peptide antagonist in complex with vascular endothelial growth factor

Vascular endothelial growth factor (VEGF) is a potent endothelial cell-specific mediator of angiogenesis and vasculogenesis. VEGF is involved pathologically in cancer, proliferative retinopathy and rheumatoid arthritis, and as such represents an important therapeutic target. Three classes of disulfide-constrained peptides that antagonize binding of the VEGF dimer to its receptors, KDR and Flt-1, were identified previously using phage display methods. NMR studies of a representative peptide from the most potent class of these peptide antagonists, v107 (GGNECDAIRMWEWECFERL), were undertaken to characterize its interactions with VEGF. v107 has no defined structure free in solution, but binding to VEGF induces folding of the peptide. The solution structure of the VEGF receptor-binding domain-v107 complex was determined using 3940 (1970 per VEGF monomer) internuclear distance and 476 (238 per VEGF monomer) dihedral angle restraints derived from NMR data obtained using samples containing either (13)C/(15)N-labeled protein plus excess unlabeled peptide or (13)C/(15)N-labeled peptide plus excess unlabeled protein. Residual dipolar coupling restraints supplemented the structure determination of the complex and were found to increase significantly both the global precision of VEGF in the complex and the agreement with available crystal structures of VEGF. The calculated ensemble of structures is of high precision and is in excellent agreement with the experimental restraints. v107 has a turn-helix conformation with hydrophobic residues partitioned to one face of the peptide and polar or charged residues at the other face. Contacts between two v107 peptides and the VEGF dimer are mediated by primarily hydrophobic side-chain interactions. The v107-binding site on VEGF overlaps partially with the binding site of KDR and is similar to that for domain 2 of Flt-1. The structure of the VEGF-v107 complex provides new insight into how binding to VEGF can be achieved that may be useful for the design of small molecule antagonists.     

Reps2: a cellular signaling and molecular trafficking nexus

Since its discovery in the late 1990s as a signaling molecule in the Ras/Ral pathway, Reps2 has emerged as an important player in receptor-mediated endocytosis. Reps2 contains Eps15 homology (EH) domains, proline-rich regions, and a coiled-coil domain that engage in several protein-protein interactions to coordinate the internalization of various receptors with molecular signaling. Reps2 has clinical importance as it suppresses the ability of its binding partner RalBP1 to transport chemotherapeutic drugs, such as doxorubicin, out of a cell. Reps2 is also dysregulated during the progression of prostate cancer and is a potential biomarker for breast and prostate cancer.