Three-dimensional structure of the mammalian tachykinin peptide neurokinin A bound to lipid micelles

The solution structure of NKA, a decapeptide of mammalian origin, has been characterized by CD spectropolarimetry and 2D proton nuclear magnetic resonance (2D 1H-NMR) spectroscopy in both aqueous and membrane mimetic solvents. Unambiguous NMR assignments of protons have been made with the aid of correlation spectroscopy (DQF-COSY and TOCSY) experiments and nuclear Overhauser effect spectroscopy (NOESY and ROESY) experiments. The distance constraints obtained from the NMR data have been utilized to generate a family of structures, which have been refined using restrained energy minimization and dynamics. These data show that in water NKA prefers to be in an extended chain conformation whereas a helical conformation is induced in the central core and the C-terminal region (D4-M10) of the peptide in the presence of perdeuterated dodecylphosphocholine (DPC) micelles, a membrane model system. Though less defined the N-terminus also displays some degree of order and a possible turn structure. The conformation adopted by NKA in the presence of DPC micelles represents a structural motif typical of neurokinin-2 selective agonists and is similar to that reported for eledoisin in hydrophobic environment.     

Membrane‐induced structure of novel human tachykinin hemokinin‐1 (hHK1)

PPT‐C encoded hemokinin‐1(hHK‐1) of Homo sapiens (TGKASQFFGLM) is a structurally distinct neuropeptide among the tachykinin family that participate in the NK‐1 receptor downstream signaling processes. Subsequently, signal transduction leads to execution of various effector functions which includes aging, immunological, and central nervous system (CNS) regulatory actions. However the conformational pattern of ligand receptor binding is unclear. The three‐dimensional structure of the hemokinin‐1 in aqueous and micellar environment has been studied by one and two‐dimensional proton nuclear magnetic resonance (2D 1H‐NMR spectroscopy) and distance geometry calculations. Data shows that hemokinin‐1 was unstructured in aqueous environment; anionic detergent SDS induces α‐helix formation. Proton NMR assignments have been carried out with the aid of correlation spectroscopy (gradient‐COSY and TOCSY) and nuclear Overhauser effect spectroscopy (NOESY and ROESY) experiments. The inter proton distances and dihedral angle constraints obtained from the NMR data have been used in torsion angle dynamics algorithm for NMR applications (CYANA) to generate a family of structures, which have been refined using restrained energy minimization and dynamics. Helical conformation is observed from residue K3‐M11. The conformational range of the peptide revealed by NMR studies has been analyzed in terms of characteristic secondary features. Observed conformational features have been compared to that of Substance P potent NK1 agonist. Thus the report provides a structural insight to study hHK‐1‐NK1 interaction that is essential for hHK1 based signaling events. © 2015 Wiley Periodicals, Inc. Biopolymers 103: 702–710, 2015.     

Different subtypes of tachykinin NK(1) receptor binding sites are present in the rat brain

(2-[(125)I]iodohistidyl(1))Neurokinin A ([(125)I]NKA), which labels "septide-sensitive" but not classic NK(1) binding sites in peripheral tissues, was used to determine whether septide-sensitive binding sites are also present in the rat brain. Binding studies were performed in the presence of SR 48968 (NK(2) antagonist) and senktide (NK(3) agonist) because [(125)I]NKA also labels peripheral NK(2) binding sites and, as shown in this study, central NK(3) binding sites. [(125)I]NKA was found to label not only septide-sensitive binding sites but also a new subtype of NK(1) binding site distinct from classic NK(1) binding sites. Both subtypes of [(125)I]NKA binding sites were sensitive to tachykinin NK(1) antagonists and agonists but also to the endogenous tachykinins NKA, neuropeptide K (NPK), and neuropeptide gamma (NPgamma). However, compounds of the septide family such as substance P(6-11) [SP(6-11)] and propionyl-[Met(O(2))(11)]SP(7-11) and some NK(1) antagonists, GR 82334, RP 67580, and CP 96345, had a much lower affinity for the new NK(1)-sensitive sites than for the septide-sensitive sites. The hypothalamus and colliculi possess only this new subtype of NK(1) site, whereas both types of [(125)I]NKA binding sites were found in the amygdala and some other brain structures. These results not only explain the central effects of septide or SP(6-11), but also those of NKA, NPK, and NPgamma, which can be selectively blocked by NK(1) receptor antagonists.     

Structural polymorphism of chromodomains in Chd1

Chromodomain from heterochromatin protein 1 and polycomb protein is known to be a lysine-methylated histone H3 tail-binding module. Chromo-helicase/ATPase DNA-binding protein 1 (CHD1) is an ATP-dependent chromatin remodeling factor, containing two tandem chromodomains. In human CHD1, both chromodomains are essential for specific binding to a K4 methylated histone H3 (H3 MeK4) peptide and are found to bind cooperatively in the crystal structure. For the budding yeast homologue, Chd1, the second but not the first chromodomain was once reported to bind to an H3 MeK4 peptide. Here, we reveal that neither the second chromodomain nor a region containing tandem chromodomains from yeast Chd1 bind to any lysine-methylated or arginine-methylated histone peptides that we examined. In addition, we examined the structures of the chromodomains from Chd1 by NMR. Although the tertiary structure of the region containing tandem chromodomains could not be obtained, the secondary structure deduced from NMR is well conserved in the tertiary structures of the corresponding first and second chromodomains determined individually by NMR. Both chromodomains of Chd1 demonstrate a structure similar to that of the corresponding part of CHD1, consisting of a three-stranded beta-sheet followed by a C-terminal alpha-helix. However, an additional helix between the first and second beta-strands, which is found in both of the first chromodomains of Chd1 and CHD1, is positioned in an entirely different manner in Chd1 and CHD1. In human CHD1 this helix forms the peptide-binding site. The amino acid sequences of the chromodomains could be well aligned on the basis of these structures. The alignment showed that yeast Chd1 lacks several key functional residues, which are responsible for specific binding to a methylated lysine residue in other chromodomains. Chd1 is likely to have no binding affinity for any H3 MeK peptide, as found in other chromodomain proteins.     

Membrane-induced structure of the mammalian tachykinin neuropeptide gamma

Neuropeptide gamma (NPgamma) is a neurokinin-2 (NK-2) receptor selective agonist, which plays an important role in mediation of asthma and elicits a wide range of biological responses like bronchoconstriction, vasodepression and regulation of endocrine functions. The structure determination of this peptide agonist is important in understanding the molecular basis of peptide ligand recognition by the receptor and for rational drug design. In the present study we report the solution structure of NPgamma characterized by circular dichroism (CD) spectropolarimetry and 2D (1)H NMR spectroscopy in both aqueous and membrane mimetic solvents. Effect of calcium ions on the conformation of NPgamma was also studied using CD spectropolarimetry. Sequence-specific resonance assignments of protons have been made with the aid of correlation spectroscopy experiments and nuclear Overhauser effect spectroscopy experiments. The distance constraints obtained from the NMR data have been utilized to generate a family of structures, which have been refined using restrained energy minimization and dynamics. These data show that in water NPgamma prefers to be in an extended chain conformation whereas a helical conformation is induced in the central core and the C-terminal region of the peptide (K13-M21) in the presence of perdeuterated dodecylphosphocholine micelles, a membrane model system. A type II' beta turn from H9 to R11 precedes the helical core in the C-terminus of NPgamma. N-terminus of NPgamma also displays some degree of order and a possible turn structure. Conformation adopted by NPgamma in presence of lipid micelles represents a structural motif typical of NK-2 selective agonists and is similar to that observed for Neurokinin A in hydrophobic environment. The observed conformational features have been correlated to the binding ability and biological activity of NPgamma.     

Solving the structure of PTB in complex with pyrimidine tracts: an NMR study of protein-RNA complexes of weak affinities

NMR spectroscopy has proven to be a powerful tool for the structure determination of protein/RNA complexes. However, the quality of these structures depends critically on the number of unambiguous intermolecular and intra-RNA nuclear Overhauser effect (NOE) constraints that can be derived. This number is often limited due to exchange phenomena that can cause signal line broadening and the fact that unambiguous NOE assignments are challenging in systems that exchange between different conformations in the intermediate to fast exchange limit. These exchange processes can include exchange between free and bound form, as well as exchange of the ligand between different binding sites on the protein. Furthermore, for the large class of RNA metabolizing proteins that bind repetitive low-complexity RNA sequences in multiple register, exchange of the protein between these overlapping binding sites introduces additional exchange pathways. Here, we describe the strategy we used to overcome these exchange processes and to reduce significantly the line width of the RNA resonances in complexes of the RNA recognition motifs (RRMs) of the polypyrimidine tract-binding protein (PTB) in complex with pyrimidine tracts and hence allowed a highly precise structure determination. This method could be employed to derive structures of other protein/single-stranded nucleic acid complexes by NMR spectroscopy. Furthermore, we have determined the affinities of the individual RRMs of PTB for pyrimidine tracts of different length and sequence. These measurements show that PTB binds preferentially to long pyrimidine tracts that contain cytosine and hence confirm the structure of PTB in complex with RNA. Furthermore, they provide quantitative insight into the question of which pyrimidine sequences within alternatively spliced pre-mRNAs will be preferentially bound by PTB.     

Conformational Studies of Tachykinin Peptides Using NMR Spectroscopy

Summary A conformational analysis in water and DMSO of two tachykinin family peptides (scyliorhinin I (ScyI) and scyliorhinin II (ScyII)) was carried out by 1D and 2D NMR (DQF-COSY, TOCSY, HMQC, HMBC, NOESY and ROESY) and molecular dynamics calculation methods. In DMSO, two groups of conformations (major and minor) were obtained for both peptides based on the experimental data. The conformations proposed for ScyI represent a folded structure, which shows certain similarities to the structures reported for other NK-1 and NK-2 tachykinin agonists. In water ScyII displays a flexible, extended structure, whereas in DMSO the structure is more compact and, in the fragment from the centre to the C-terminus, several β-turns may be present.     

Gelatin binding to the 8F19F1 module pair of human fibronectin requires site-specific N-glycosylation

The gelatin (denatured collagen) binding domain of the extracellular matrix protein fibronectin contains three potential N-glycosylation sites. Complete deglycosylation of this domain is known to reduce the thermal stability of the eighth type 1 (8F1) module. We have conducted a site-specific analysis of the structural and functional consequences of N-linked glycosylation in the 8F19F1 module pair. Three glycoforms have been identified by mass spectrometry and nuclear magnetic resonance spectroscopy. Chemical shift differences between the glycoforms have revealed an intimate interaction between one N-linked sugar and the polypeptide that is critical for gelatin binding, as shown by affinity chromatography.     

Solution structure of the tachykinin peptide eledoisin

Both the aqueous and the lipid-induced structure of eledoisin, an undecapeptide of mollusk origin, have been studied by two-dimensional proton nuclear magnetic resonance spectroscopy and distance geometry calculations. Unambiguous nuclear magnetic resonance assignments of protons have been made with the aid of correlation spectroscopy experiments and nuclear Overhauser effect spectroscopy experiments. The distance constraints obtained from the nuclear magnetic resonance data have been utilized in a distance geometry algorithm to generate a family of structures, which have been refined using restrained energy minimization and dynamics. These data show that, while in water and dimethyl sulfoxide, eledoisin prefers to be in an extended chain conformation, whereas in the presence of perdeuterated dodecylphosphocholine micelles, a membrane model system, helical conformation is induced in the central core and C-terminal region (K4-M11) of the peptide. N terminus, though less defined, also displays some degree of order and a possible turn structure. The conformation adopted by eledoisin in the presence of dodecylphosphocholine micelles is similar to the structural motif typical of neurokinin-2 selective agonists and with that reported for kassinin in hydrophobic environment.     

How to Arm a Supervillin: Designing F-Actin Binding Activity into Supervillin Headpiece

Villin-type headpiece domains are compact motifs that have been used extensively as model systems for protein folding. Although the majority of headpiece domains bind actin, there are some that lack this activity. Here, we present the first NMR solution structure and ¹⁵N-relaxation analysis of a villin-type headpiece domain natively devoid of F-actin binding activity, that of supervillin headpiece (SVHP). The structure was found to be similar to that of other headpiece domains that bind F-actin. Our NMR analysis demonstrates that SVHP lacks a conformationally flexible region (V-loop) present in all other villin-type headpiece domains and which is essential to the phosphoryl regulation of dematin headpiece. In comparing the electrostatic surface potential map of SVHP to that of other villin-type headpiece domains with significant affinity for F-actin, we identified a positive surface potential conserved among headpiece domains that bind F-actin but absent from SVHP. A single point mutation (L38K) in SVHP, which creates a similar positive surface potential, endowed SVHP with specific affinity for F-actin that is within an order of magnitude of the tightest binding headpiece domains. We propose that this effect is likely conferred by a specific buried salt bridge between headpiece and actin. As no high-resolution structural information exists for the villin-type headpiece F-actin complex, our results demonstrate that through positive mutagenesis, it is possible to design binding activity into homologous proteins without structural information of the counterpart's binding surface.     

Characterization of the bioactive conformation of the C-terminal tripeptide Gly-Leu-Met-NH2 of substance P using [3-prolinoleucine10]SP analogues.

Residue Leu10 of substance P (SP) is critical for NK-1 receptor recognition and agonist activity. In order to probe the bioactive conformation of this residue, cis- and trans-3-substituted prolinoleucines were introduced in position 10 of SP. The substituted SP analogues were tested for their affinity to human NK-1 receptor specific binding sites (NK-1M and NK-1m) and their potency to stimulate adenylate cyclase and phospholipase C in CHO cells transfected with the human NK-1 receptor. [trans-3-prolinoleucine10]SP retained affinity and potency similar to SP whereas [cis-3-prolinoleucine10]SP shows dramatic loss of affinity and potency. To analyze the structural implications of these biological results, the conformational preferences of the SP analogues were analyzed by NMR spectroscopy and minimum-energy conformers of Ac-cis-3-prolinoleucine-NHMe, Ac-trans-3-prolinoleucine-NHMe and model dipeptides were generated by molecular mechanics calculations. From NMR and modeling studies it can be proposed that residue Leu10 of SP adopts a gauche(+) conformation around the chi1 angle and a trans conformation around the chi2 angle in the bioactive conformation. Together with previously published results, our data indicate that the C-terminal SP tripeptide should preferentially adopt an extended conformation or a PPII helical structure when bound to the receptor.     

Mapping the ADF/cofilin binding site on monomeric actin by competitive cross-linking and peptide array: evidence for a second binding site on monomeric actin

The binding sites for actin depolymerising factor (ADF) and cofilin on G-actin have been mapped by competitive chemical cross-linking using deoxyribonuclease I (DNase I), gelsolin segment 1 (G1), thymosin beta4 (Tbeta4), and vitamin D-binding protein (DbP). To reduce ADF/cofilin induced actin oligomerisation we used ADP-ribosylated actin. Both vitamin D-binding protein and thymosin beta4 inhibit binding by ADF or cofilin, while cofilin or ADF and DNase I bind simultaneously. Competition was observed between ADF or cofilin and G1, supporting the hypothesis that cofilin preferentially binds in the cleft between sub-domains 1 and 3, similar to or overlapping the binding site of G1. Because the affinity of G1 is much higher than that of ADF or cofilin, even at a 20-fold excess of the latter, the complexes contained predominantly G1. Nevertheless, cross-linking studies using actin:G1 complexes and ADF or cofilin showed the presence of low concentrations of ternary complexes containing both ADF or cofilin and G1. Thus, even with monomeric actin, it is shown for the first time that binding sites for both G1 and ADF or cofilin can be occupied simultaneously, confirming the existence of two separate binding sites. Employing a peptide array with overlapping sequences of actin overlaid by cofilin, we have identified five sequence stretches of actin able to bind cofilin. These sequences are located within the regions of F-actin predicted to bind cofilin in the model derived from image reconstructions of electron microscopical images of cofilin-decorated filaments. Three of the peptides map to the cleft region between sub-domains 1 and 3 of the upper actin along the two-start long-pitch helix, while the other two are in the DNase I loop corresponding to the site of the lower actin in the helix. In the absence of any crystal structures of ADF or cofilin in complex with actin, these studies provide further information about the binding sites on F-actin for these important actin regulatory proteins.     

Conformational Studies of Tachykinin Peptides Using NMR Spectroscopy

A conformational analysis in water and DMSO of two tachykinin family peptides (scyliorhinin I (ScyI) and scyliorhinin II (ScyII)) was carried out by 1D and 2D NMR (DQF-COSY, TOCSY, HMQC, HMBC, NOESY and ROESY) and molecular dynamics calculation methods. In DMSO, two groups of conformations (major and minor) were obtained for both peptides based on the experimental data. The conformations proposed for ScyI represent a folded structure, which shows certain similarities to the structures reported for other NK-1 and NK-2 tachykinin agonists. In water ScyII displays a flexible, extended structure, whereas in DMSO the structure is more compact and, in the fragment from the centre to the C-terminus, several -turns may be present.     

Structural characterization of a new binding motif and a novel binding mode in group 2 WW domains

Formin homology 1 (FH1), is a long proline-rich region of formins, shown to bind to five WW containing proteins named formin binding proteins (FBPs). FH1 has several potential binding regions but only the PPLPx motif and its interaction with FBP11WW1 has been characterized structurally. To detect whether additional motifs exist in FH1, we synthesized five peptides and investigated their interaction with FBP28WW2, FBP11WW1 and FBP11WW2 domains. Peptides of sequence PTPPPLPP (positive control), PPPLIPPPP and PPLIPPPP (new motifs) interact with the domains with micromolar affinity. We observed that FBP28WW2 and FBP11WW2 behave differently from FBP11WW1 in terms of motif selection and affinity, since they prefer a doubly interrupted proline stretch of sequence PPLIPP. We determined the NMR structure of three complexes involving the FBP28WW2 domain and the three ligands. Depending on the peptide under study, the domain interacts with two proline residues accommodated in either the XP or the XP2 groove. This difference represents a one-turn displacement of the domain along the ligand sequence. To understand what drives this behavior, we performed further structural studies with the FBP11WW1 and a mutant of FBP28WW2 mimicking the XP2 groove of FBP11WW1. Our observations suggest that the nature of the XP2 groove and the balance of flexibility/rigidity around loop 1 of the domain contribute to the selection of the final ligand positioning in fully independent domains. Additionally, we analyzed the binding of a double WW domain region, FBP11WW1-2, to a long stretch of FH1 using fluorescence spectroscopy and NMR titrations. With this we show that the presence of two consecutive WW domains may also influence the selection of the binding mode, particularly if both domains can interact with consecutive motifs in the ligand. Our results represent the first observation of protein-ligand recognition where a pair of WW and two consecutive motifs in a ligand participate simultaneously.     

Solution structure of amphibian tachykinin Uperolein bound to DPC micelles

Uperolein, a physalaemin-like endecapeptide, has been shown to be selective for Neurokinin 1 receptor. As a first step towards understanding the structure-activity relationship, we report the membrane-induced structure of Uperolein with the aid of circular dichroism and 2D (1)H NMR spectroscopy. Sequence-specific resonance assignments of protons have been made using correlation spectroscopy (TOCSY, DQF-COSY) and NOESY spectroscopy. The interproton distance constraints and dihedral angle constraints have been utilized to generate a family of structures using torsion angle molecular dynamics within program DYANA. The conformational range of the peptide revealed by NMR and CD studies has been analysed in terms of characteristic secondary features. Analysis of NMR data indicates that the global fold of Uperolein can be explained in terms of equilibrium between 3(10)-helix and alpha-helix from residues 5 to 11. An extended highly flexible N-terminus displays some degree of order and a possible turn structure. A comparison between the structures of Uperolein and Substance P, a prototype and endogenous Neurokinin 1 receptor agonist, indicates several common features in the distribution of hydrophobic and hydrophilic residues. Both the peptides show an amphiphilic character towards the middle region. The similarities suggest that the molecules interact with the receptor in an analogous manner.     

Interaction of local anesthetics with a peptide encompassing the IV/S4-S5 linker of the Na+ channel

The peptide pIV/S4-S5 encompasses the cytoplasmic linker between helices S4-S5 in domain IV of the voltage-gated Na+ channel, residues 1644-1664. The interaction of two local anesthetics (LA), lidocaine and benzocaine, with pIV/S4-S5 has been studied by DOSY, heteronuclear NMR 1H-15N-HSQC spectroscopy and computational methods. DOSY indicates that benzocaine, a neutral ester, exhibits stronger interaction with pIV/S4-S5 than lidocaine, a charged amine-amide. Weighted average chemical shifts, Deltadelta(1H-15N), show that benzocaine affects residues L1653, M1655 and S1656 while lidocaine slightly perturbs residues I1646, L1649 and A1659, L1660, near the N- and C-terminus, respectively. Computational methods confirmed the stability of the benzocaine binding and the existence of two binding sites for lidocaine. Even considering that the approach of studying the peptide in the presence of a co-solvent (TFE/H2O, 30%/70% v/v) has an inherently limited implication, our data strongly support the existence of multiple LA binding sites in the IV/S4-S5 linker, as suggested in the literature. In addition, we consider that LA can bind to the S4-S5 linker with diverse binding modes and strength since this linker is part of the receptor for the "inactivation gate particle". Conditions for devising new functional studies, aiming to better understand Na+ channel functionality as well as the various facets of LA pharmacological activity are proposed in this work.     

Rolling adhesion of alphaL I domain mutants decorrelated from binding affinity

Activated lymphocyte function-associated antigen-1 (LFA-1, alphaLbeta2 integrin) found on leukocytes facilitates firm adhesion to endothelial cell layers by binding to intercellular adhesion molecule-1 (ICAM-1), which is up-regulated on endothelial cells at sites of inflammation. Recent work has shown that LFA-1 in a pre-activation, low-affinity state may also be involved in the initial tethering and rolling phase of the adhesion cascade. The inserted (I) domain of LFA-1 contains the ligand-binding epitope of the molecule, and a conformational change in this region during activation increases ligand affinity. We have displayed wild-type I domain on the surface of yeast and validated expression using I domain specific antibodies and flow cytometry. Surface display of I domain supports yeast rolling on ICAM-1-coated surfaces under shear flow. Expression of a locked open, high-affinity I domain mutant supports firm adhesion of yeast, while yeast displaying intermediate-affinity I domain mutants exhibit a range of rolling phenotypes. We find that rolling behavior for these mutants fails to correlate with ligand binding affinity. These results indicate that unstressed binding affinity is not the only molecular property that determines adhesive behavior under shear flow.     

Presence of NK2 binding sites in the rat brain

Attempts were made to label tachykinin NK2 binding sites in the adult rat brain using [125I]neurokinin A (NKA) as ligand in the presence of NK1 and NK3 agonist or antagonist to avoid labelling of NK1 and NK3 binding sites, respectively. A high-affinity, specifically NK2-sensitive, [125I]NKA-binding, temperature-dependent, reversible, sensitive to GTPgammaS and correspondence to a single population of binding sites (K(D) and B(max) values: 2.2 nM and 7.3 fmol/mg protein) was demonstrated on hippocampal membranes. Competition studies performed with tachykinins and tachykinin-related compounds indicated that the pharmacological properties of these NK2-sensitive [125I]NKA binding sites were identical to those identified in the rat urinary bladder and duodenum. NKA, neuropeptide K, and neuropeptide gamma, as well as the potent and selective NK2 antagonists SR 144190, SR 48968 and MEN 10627, presented a nanomolar affinity for these sites. The regional distribution of these NK2-sensitive [125I]NKA binding sites differs markedly from those of NK1 and NK3 binding sites, with the largest labeling being found in the hippocampus, the thalamus and the septum. Binding in other brain structures was low or negligible. A preliminary autoradiographic analysis confirmed [125I]NKA selective binding in hippocampal CA1 and CA3 areas, particularly, and in several thalamic nuclei.     

Full assignment of the 1H and 13C spectra and revision of the O-acetylation site of the capsular polysaccharide of Streptococcus pneumoniae Type 33F, a component of the current pneumococcal polysaccharide vaccine

The structure of the capsular polysaccharide from Streptococcus pneumoniae Type 33F was originally determined by a combination of chemical methods and limited use of NMR spectroscopy [Can. J. Biochem. Cell Biol.1984, 62, 666-677]. We report full 1H and 13C assignments and confirm the structure of the saccharide repeat unit, but find that the site of O-acetylation is O-2 of the -->5)-beta-D-Galf, rather than the -->3)-beta-D-Galf residue. We find that a slightly higher percentage of the repeat units are O-acetylated: [carbohydrate: see text].