Structure-function analysis of amino acid 74 of human RAMP1 and RAMP3 and its role in peptide interactions with adrenomedullin and calcitonin gene-related peptide receptors

The receptors for calcitonin gene-related peptide (CGRP) and adrenomedullin (AM) are complexes of the calcitonin receptor-like receptor (CLR) and receptor activity-modifying proteins (RAMP). The CGRP receptor is a CLR/RAMP1 pairing whereas CLR/RAMP2 and CLR/RAMP3 constitute two subtypes of AM receptor: AM(1) and AM(2), respectively. Previous studies identified Glu74 in RAMP3 to be important for AM binding and potency. To further understand the importance of this residue and its equivalent in RAMP1 (Trp74) we substituted the native amino acids with several others. In RAMP3, these were Trp, Phe, Tyr, Ala, Ser, Thr, Arg and Asn; in RAMP1, Glu, Phe, Tyr, Ala and Asn substitutions were made. The mutant RAMPs were co-expressed with CLR in Cos7 cells; receptor function in response to AM, AM(2)/intermedin and CGRP was measured in a cAMP assay and cell surface expression was determined by ELISA. Phe reduced AM potency in RAMP3 but had no effect in RAMP1. In contrast, Tyr had no effect in RAMP3 but enhanced AM potency in RAMP1. Most other substitutions had a small effect on AM potency in both receptors whereas there was little impact on CGRP or AM(2) potency. Overall, these data suggest that the geometry and charge of the residue at position 74 contribute to how AM interacts with the AM(2) and CGRP receptors and confirms the role of this position in dictating differential AM pharmacology at the AM(2) and CGRP receptors.

Structure-activity relationships for α-calcitonin gene-related peptide

Calcitonin gene-related peptide (CGRP) is a member of the calcitonin (CT) family of peptides. It is a widely distributed neuropeptide implicated in conditions such as neurogenic inflammation. With other members of the CT family, it shares an N-terminal disulphide-bonded ring which is essential for biological activity, an area of potential α-helix, and a C-terminal amide. CGRP binds to the calcitonin receptor-like receptor (CLR) in complex with receptor activity-modifying protein 1 (RAMP1), a member of the family B (or secretin-like) GPCRs. It can also activate other CLR or calcitonin-receptor/RAMP complexes. This 37 amino acid peptide comprises the N-terminal ring that is required for receptor activation (residues 1-7); an α-helix (residues 8-18), a region incorporating a β-bend (residues 19-26) and the C-terminal portion (residues 27-37), that is characterized by bends between residues 28-30 and 33-34. A few residues have been identified that seem to make major contributions to receptor binding and activation, with a larger number contributing either to minor interactions (which collectively may be significant), or to maintaining the conformation of the bound peptide. It is not clear if CGRP follows the pattern of other family B GPCRs in binding largely as an α-helix. Linked Articles This article is part of a themed section on Neuropeptides. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2013.170.issue-7 © 2012 The Authors. British Journal of Pharmacology © 2012 The British Pharmacological Society.

Structure-activity relationships of the N-terminus of calcitonin gene-related peptide:key roles of alanine-5 and threonine-6 in receptor activation

Background and purpose - The N-terminus of calcitonin gene-related peptide (CGRP) is important for receptor activation, especially the disulphide-bonded ring (residues 1-7). However, the roles of individual amino acids within this region have not been examined and so the molecular determinants of agonism are unknown. This study has examined the role of residues 1, 3-6 and 8-9, excluding Cys-2 and Cys-7. Experimental approach - CGRP derivatives were substituted with either cysteine or alanine; further residues were introduced at position 6. Their affinity was measured by radioligand binding and their efficacy by measuring cAMP production in SK-N-MC cells and ß-arrestin 2 translocation in CHO-K1 cells at the CGRP receptor. Key results - Substitution of Ala-5 by cysteine reduced affinity 270-fold and reduced efficacy for production of cAMP in SK-N-MCs. Potency at ß-arrestin translocation was reduced by 9-fold. Substitution of Thr-6 by cysteine destroyed all measurable efficacy of both cAMP and ß-arrestin responses; substitution with either alanine or serine impaired potency. Substitutions at positions 1, 4, 8 and 9 resulted in approximately 10-fold reductions in potency at both responses. Similar observations were made at a second CGRP-activated receptor, the AMY1(a) receptor. Conclusions and implications - Ala-5 and Thr-6 are key determinants of agonist activity for CGRP. Ala-5 is also very important for receptor binding. Residues outside of the 1-7 ring also contribute to agonist activity.

Structure-activity relationship of calcitonin gene-related peptide and its receptor

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Toward the prediction of class I and II mouse major histocompatibility complex-peptide-binding affinity:in silico bioinformatic step-by-step guide using quantitative structure-activity relationships

Quantitative structure-activity relationship (QSAR) analysis is a cornerstone of modern informatics. Predictive computational models of peptide-major histocompatibility complex (MHC)-binding affinity based on QSAR technology have now become important components of modern computational immunovaccinology. Historically, such approaches have been built around semiqualitative, classification methods, but these are now giving way to quantitative regression methods. We review three methods--a 2D-QSAR additive-partial least squares (PLS) and a 3D-QSAR comparative molecular similarity index analysis (CoMSIA) method--which can identify the sequence dependence of peptide-binding specificity for various class I MHC alleles from the reported binding affinities (IC50) of peptide sets. The third method is an iterative self-consistent (ISC) PLS-based additive method, which is a recently developed extension to the additive method for the affinity prediction of class II peptides. The QSAR methods presented here have established themselves as immunoinformatic techniques complementary to existing methodology, useful in the quantitative prediction of binding affinity: current methods for the in silico identification of T-cell epitopes (which form the basis of many vaccines, diagnostics, and reagents) rely on the accurate computational prediction of peptide-MHC affinity. We have reviewed various human and mouse class I and class II allele models. Studied alleles comprise HLA-A*0101, HLA-A*0201, HLA-A*0202, HLA-A*0203, HLA-A*0206, HLA-A*0301, HLA-A*1101, HLA-A*3101, HLA-A*6801, HLA-A*6802, HLA-B*3501, H2-K(k), H2-K(b), H2-D(b) HLA-DRB1*0101, HLA-DRB1*0401, HLA-DRB1*0701, I-A(b), I-A(d), I-A(k), I-A(S), I-E(d), and I-E(k). In this chapter we show a step-by-step guide into predicting the reliability and the resulting models to represent an advance on existing methods. The peptides used in this study are available from the AntiJen database (http://www.jenner.ac.uk/AntiJen). The PLS method is available commercially in the SYBYL molecular modeling software package. The resulting models, which can be used for accurate T-cell epitope prediction, will be made are freely available online at the URL http://www.jenner.ac.uk/MHCPred.

Flexibility of short-strand RNA in aqueous solution as revealed by molecular dynamics simulation:are A′-RNA and A-RNA distinct conformational structures?

We use molecular dynamics simulations to compare the conformational structure and dynamics of a 21-base pair RNA sequence initially constructed according to the canonical A-RNA and A'-RNA forms in the presence of counterions and explicit water. Our study aims to add a dynamical perspective to the solid-state structural information that has been derived from X-ray data for these two characteristic forms of RNA. Analysis of the three main structural descriptors commonly used to differentiate between the two forms of RNA namely major groove width, inclination and the number of base pairs in a helical twist over a 30 ns simulation period reveals a flexible structure in aqueous solution with fluctuations in the values of these structural parameters encompassing the range between the two crystal forms and more. This provides evidence to suggest that the identification of distinct A-RNA and A'-RNA structures, while relevant in the crystalline form, may not be generally relevant in the context of RNA in the aqueous phase. The apparent structural flexibility observed in our simulations is likely to bear ramifications for the interactions of RNA with biological molecules (e.g. proteins) and non-biological molecules (e.g. non-viral gene delivery vectors). © CSIRO 2009.

Structure-function analysis of RAMP1 by Alanine Mutagenesis

Receptor activity modifying protein 1 (RAMP1) is an integral component of several receptors including the calcitonin gene-related peptide (CGRP) receptor. It forms a complex with the calcitonin receptor-like receptor (CLR) and is required for receptor trafficking and ligand binding. The N-terminus of RAMP1 comprises three helices. The current study investigated regions of RAMP1 important for CGRP or CLR interactions by alanine mutagenesis. Modeling suggested the second and third helices were important in protein-protein interactions. Most of the conserved residues in the N-terminus (M48, W56, Y66, P85, N66, H97, F101, D113, P114, P115), together with a further 13 residues spread throughout three helices of RAMP1, were mutated to alanine and coexpressed with CLR in Cos 7 cells. None of the mutations significantly reduced RAMP expression. Of the nine mutants from helix 1, only M48A had any effect, producing a modest reduction in trafficking of CLR to the cell surface. In helix 2 Y66A almost completely abolished CLR trafficking; L69A and T73A reduced the potency of CGRP to produce cAMP. In helix 3, H97A abolished CLR trafficking; P85A, N86A, and F101A had caused modest reductions in CLR trafficking and also reduced the potency of CGRP on cAMP production. F93A caused a modest reduction in CLR trafficking alone and L94A increased cAMP production. The data are consistent with a CLR recognition site particularly involving Y66 and H97, with lesser roles for adjacent residues in helix 3. L69 and T73 may contribute to a CGRP recognition site in helix 2 also involving nearby residues.

Structure and dynamics of multiple cationic vectors-siRNA complexation by all-atomic molecular dynamics simulations

Understanding the molecular mechanism of gene condensation is a key component to rationalizing gene delivery phenomena, including functional properties such as the stability of the gene-vector complex and the intracellular release of the gene. In this work, we adopt an atomistic molecular dynamics simulation approach to study the complexation of short strand duplex RNA with four cationic carrier systems of varying charge and surface topology at different charge ratios. At lower charge ratios, polymers bind quite effectively to siRNA, while at high charge ratios, the complexes are saturated and there are free polymers that are unable to associate with RNA. We also observed reduced fluctuations in RNA structures when complexed with multiple polymers in solution as compared to both free siRNA in water and the single polymer complexes. These novel simulations provide a much better understanding of key mechanistic aspects of gene-polycation complexation and thereby advance progress toward rational design of nonviral gene delivery systems.

Structure-function analysis of RAMP1-RAMP3 chimeras

The role of receptor activity modifying protein 1 (RAMP1) in forming receptors with the calcitonin receptor-like receptor (CLR) and the calcitonin receptor (CTR) was examined by producing chimeras between RAMP1 and RAMP3. RAMPs have three extracellular helices. Exchange of helix 1 of the RAMPs or residues 62-69 in helix 2 greatly reduced CLR trafficking (a marker for CLR association). Modeling suggests that these exchanges alter the CLR recognition site on RAMP1, which is more exposed than on RAMP3. Exchange of residues 86-89 of RAMP1 had no effect on the trafficking of CLR but reduced the potency of human (h) alphaCGRP and adrenomedullin. However, these alterations to RAMP1 had no effect on the potency of hbetaCGRP. These residues of RAMP1 lie at the junction of helix 3 and its connecting loop with helix 2. Modeling suggests that the loop is more exposed in RAMP1 than RAMP3; it may play an important role in peptide binding, either directly or indirectly. Exchange of residues 90-94 of RAMP1 caused a modest reduction in CLR expression and a 15-fold decrease in CGRP potency. It is unlikely that the decrease in expression is enough to explain the reduction in potency, and so these may have dual roles in recognizing CLR and CGRP. For CTR, only 6 out of 26 chimeras covering the extracellular part of RAMP1 did not reduce agonist potency. Thus the association of CTR with RAMP1 seems more sensitive to changes in RAMP1 structure induced by the chimeras than is CLR.

Tethering a laminin peptide to a crosslinked collagen scaffold for biofunctionality

Cell adhesion peptide regulates various cellular functions like proliferation, attachment, and spreading. The cellular response to laminin peptide (PPFLMLLKGSTR), a motif of laminin-5 alpha3 chain, tethered to type I collagen, crosslinked using microbial transglutaminase (mTGase) was investigated. mTGase is an enzyme that initiates crosslinking by reacting with the glutamine and lysine residues on the collagen fibers stabilizing the molecular structure. In this study that tethering of the laminin peptide in a mTGase crosslinked collagen scaffold enhanced cell proliferation and attachment. Laminin peptide tethered crosslinked scaffold showed unaltered cell morphology of 3T3 fibroblasts when compared with collagen and crosslinked scaffold. The triple helical structure of collagen remained unaltered by the addition of laminin peptide. In addition a dose-dependent affinity of the laminin peptide towards collagen was seen. The degree of crosslinking was measured by amino acid analysis, differential scanning calorimeter and fourier transform infrared spectroscopy. Increased crosslinking was observed in mTGase crosslinked group. mTGase crosslinking showed higher shrinkage temperature. There was alteration in the fibrillar architecture due to the crosslinking activity of mTGase. Hence, the use of enzyme-mediated linking shows promise in tethering cell adhesive peptides through biodegradable scaffolds.

Hairpin vortex solution in planar Couette flow: a tapestry of knotted vortices

A numerical continuation method has been carried out seeking solutions for two distinct flow configurations, planar Couette flow (PCF) and laterally heated flow in a vertical slot (LHF). We found that the spanwise vortex solution in LHF identifies a new solution in PCF. The vortical structure of our new solution has the shape of a hairpin observed ubiquitously in high-Reynolds-number turbulent flow, and we believe this discovery may provide the paradigm for a hierarchical organization of coherent structures in turbulent shear layers.

Soft hydrogels from nanotubes of poly(ethylene oxide)−tetraphenylalanine conjugates prepared by click chemistry

A new poly(ethylene oxide)-tetraphenylalanine polymer-peptide conjugate has been prepared via a “click” reaction between an alkyne-modified peptide and an azide-terminated PEO oligomer. Self-assembled nanotubes are formed after dialysis of a THF solution of this polymer-peptide conjugate against water. The structure of these nanotubes has been probed by circular dichroism, IR, TEM, and SAXS. From these data, it is apparent that self-assembly involves the formation of antiparallel ß-sheets and p-p-stacking. Nanotubes are formed at concentrations between 2 and 10 mg mL-1. Entanglement between adjacent nanotubes occurs at higher concentrations, resulting in the formation of soft hydrogels. Gel strength increases at higher polymer-peptide conjugate concentration, as expected.

Peptide conjugate hydrogelators

Molecular gelators are currently receiving a great deal of attention. These are small molecules which, under the appropriate conditions, assemble in solution to, in the majority of cases, give long fibrillar structures which entangle to form a three-dimensional network. This immobilises the solvent, resulting in a gel. Such gelators have potential application in a number of important areas from drug delivery to tissue engineering. Recently, the use of peptide-conjugates has become prevalent with oligopeptides (from as short as two amino acids in length) conjugated to a polymer, alkyl chain or aromatic group such as naphthalene or fluorenylmethoxycarbonyl (Fmoc) being shown to be effective molecular gelators. The field of gelation is extremely large; here we focus our attention on the use of these peptide-conjugates as molecular hydrogelators.

Peptide binding prediction for the human class II MHC allele HLA-DP2:a molecular docking approach

MHC class II proteins bind oligopeptide fragments derived from proteolysis of pathogen antigens, presenting them at the cell surface for recognition by CD4+ T cells. Human MHC class II alleles are grouped into three loci: HLA-DP, HLA-DQ and HLA-DR. In contrast to HLA-DR and HLA-DQ, HLA-DP proteins have not been studied extensively, as they have been viewed as less important in immune responses than DRs and DQs. However, it is now known that HLA-DP alleles are associated with many autoimmune diseases. Quite recently, the X-ray structure of the HLA-DP2 molecule (DPA*0103, DPB1*0201) in complex with a self-peptide derived from the HLA-DR a-chain has been determined. In the present study, we applied a validated molecular docking protocol to a library of 247 modelled peptide-DP2 complexes, seeking to assess the contribution made by each of the 20 naturally occurred amino acids at each of the nine binding core peptide positions and the four flanking residues (two on both sides).

Protein deposition at polymer surfaces:the bulk and surface structure-property effects of hydrogels

The primary objective of this research has been to investigate the interfacial phenomenon of protein adsorption in relation to the bulk and surface structure-property effect s of hydrogel polymers. In order to achieve this it was first necessary to characterise the bulk and surface properties of the hydrogels, with regard to the structural chemistry of their component monomers. The bulk properties of the hydrogels were established using equilibrium water content measurements, together with water-binding studies by differential scanning calorimetry (D.S.C.). Hamilton and captive air bubble-contact angle techniques were employed to characterise the hydrogel-water interface and from which by a mathematical derivation, the interfacial free energy (ðsw) and the surface free energy components (ð psv, ðdsv, ðsv) were obtained. From the adsorption studies using the radio labelled iodinated (125I) proteins of human serum albumin (H.S.A.) and human fibrinogen (H.Fb.), it was Found that multi-layered adsorption was occurring and that the rate and type of this adsorption was dependent on the physico-chemical behaviour of the adsorbing protein (and its bulk concentration in solution), together with the surface energetics of the adsorbent polymer. A potential method for the invitro evaluation of a material's 'biocompatibility' was also investigated, based on an empirically observed relationship between the adsorption of albumin and fibrinogen and the 'biocompatibility' of polymeric materials. Furthermore, some consideration was also given to the biocompatibility problem of proteinaceous deposit formation on hydrophilic soft' contact lenses and in addition a number of potential continual wear contact lens formulations now undergoing clinical trials,were characterised by the above techniques.