Exclusion of a transmembrane-type peptide from ordered-lipid domains (rafts) detected by fluorescence quenching: extension of quenching analysis to account for the effects of domain size and domain boundaries

Sphingolipid/cholesterol-rich rafts are membrane domains thought to exist in the liquid-ordered state. To understand the rules governing the association of proteins with rafts, the behavior of a model membrane-inserted hydrophobic polypeptide (LW peptide, acetyl-K(2)W(2)L(8)AL(8)W(2)K(2)-amide) was examined. The distribution of LW peptide between coexisting ordered and disordered lipid domains was probed by measuring the amount of LW Trp fluorescence quenched by a nitroxide-labeled phospholipid that concentrated in disordered lipid domains. Strong quenching of the Trp fluorescence (relative to quenching in model membranes lacking domains) showed that LW peptide was concentrated in quencher-rich disordered domains and was largely excluded from ordered domains. Exclusion of LW peptide from the ordered domains was observed both in the absence and in the presence of 25-33 mol % cholesterol, indicating that the peptide is relatively excluded both from gel-state domains (which form in the absence of cholesterol) and from liquid-ordered-state domains (which form at high cholesterol concentrations). Because exclusion was also observed when ordered domains contained sphingomyelin in place of DPPC, or ergosterol in place of cholesterol, it appeared that this behavior was not strongly dependent on lipid structure. In both the absence and the presence of 25 mol % cholesterol, exclusion was also not strongly dependent upon the fraction of the bilayer in the form of ordered domains. To evaluate LW peptide behavior in more detail, an analysis of the effects of domain size and edges upon quenching was formulated. This analysis showed that quenching can be affected both by domain size and by whether a fluorescent molecule localized at domain edges. Its application to the quenching of LW peptide indicated that the peptide did not preferentially reside at the boundaries between ordered and disordered domains.     

Tracking peptide-membrane interactions: insights from in situ coupled confocal-atomic force microscopy imaging of NAP-22 peptide insertion and assembly

Elucidating the role that charged membrane proteins play in determining cell membrane structure and dynamics is an area of active study. We have applied in situ correlated atomic force and confocal microscopies to characterize the interaction of the NAP-22 peptide with model membranes prepared as supported planar bilayers containing both liquid-ordered and liquid-disordered domains. Our results demonstrated that the NAP-22 peptide interacts with membranes in a concentration-dependent manner, preferentially inserting into DOPC (ld) domains. While at low peptide concentrations, the NAP-22 peptide formed aggregate-like structures within the ld domains, at high peptide concentrations, it appeared to sequester cholesterol into the ld domains and recruited phosphatidyl-myo-inositol 4,5-bisphosphate by inducing a blending effect that homogenizes the phase-segregated domains into one liquid-ordered domain. This study describes a possible mechanism by which the NAP-22 peptide can affect neuronal morphology.     

Comparative structural and energetic analysis of WW domain-peptide interactions

WW domains are small globular protein interaction modules found in a wide spectrum of proteins. They recognize their target proteins by binding specifically to short linear peptide motifs that are often proline-rich. To infer the determinants of the ligand binding propensities of WW domains, we analyzed 42 WW domains. We built models of the 3D structures of the WW domains and their peptide complexes by comparative modeling supplemented with experimental data from peptide library screens. The models provide new insights into the orientation and position of the peptide in structures of WW domain-peptide complexes that have not yet been determined experimentally. From a protein interaction property similarity analysis (PIPSA) of the WW domain structures, we show that electrostatic potential is a distinguishing feature of WW domains and we propose a structure-based classification of WW domains that expands the existent ligand-based classification scheme. Application of the comparative molecular field analysis (CoMFA), GRID/GOLPE and comparative binding energy (COMBINE) analysis methods permitted the derivation of quantitative structure-activity relationships (QSARs) that aid in identifying the specificity-determining residues within WW domains and their ligand-recognition motifs. Using these QSARs, a new group-specific sequence feature of WW domains that target arginine-containing peptides was identified. Finally, the QSAR models were applied to the design of a peptide to bind with greater affinity than the known binding peptide sequences of the yRSP5-1 WW domain. The prediction was verified experimentally, providing validation of the QSAR models and demonstrating the possibility of rationally improving peptide affinity for WW domains. The QSAR models may also be applied to the prediction of the specificity of WW domains with uncharacterized ligand-binding properties.     

Mechanisms of antimicrobial peptide action: studies of indolicidin assembly at model membrane interfaces by in situ atomic force microscopy

We report here on an in situ atomic force microscopy study of the interaction of indolicidin, a tryptophan-rich antimicrobial peptide, with phase-segregated zwitterionic DOPC/DSPC supported planar bilayers. By varying the peptide concentration and bilayer composition through the inclusion of anionic lipids (DOPG or DSPG), we found that indolicidin interacts with these model membranes in one of two concentration-dependent manners. At low peptide concentrations, indolicidin forms an amorphous layer on the fluid domains when these domains contain anionic lipids. At high peptide concentrations, indolicidin appears to initiate a lowering of the gel-phase domains independent of the presence of an anionic lipid. Similar studies performed using membrane-raft mimetic bilayers comprising 30mol% cholesterol/1:1 DOPC/egg sphingomyelin revealed that indolicidin does not form a carpet-like layer on the zwitterionic DOPC domains at low peptide concentrations and does not induce membrane lowering of the liquid-ordered sphingomyelin/cholesterol-rich domains at high peptide concentration. Simultaneous AFM-confocal microscopy imaging did however reveal that indolicidin preferentially inserts into the fluid-phase DOPC domains. These data suggest that the indolicidin-membrane association is influenced greatly by specific electrostatic interactions, lipid fluidity, and peptide concentration. These insights provide a glimpse into the mechanism of the membrane selectivity of antibacterial peptides and suggest a powerful correlated approach for characterizing peptide-membrane interactions.     

Analysis of engineered multifunctional peptide synthetases. Enzymatic characterization of surfactin synthetase domains in hybrid bimodular systems.

The combinatorial reorganization of distinct modules of multimodular peptide synthetases is of increasing interest for the generation of new peptides with optimized bioactive properties. Each module is at least composed of enzymatic domains responsible for the adenylation, thioester formation, and condensation of an amino acid residue of the final peptide product. We analyzed various possible fusion sites for the recombination of peptide synthetases and evaluated the impact of different recombination strategies on the amino acid adenylation and acyl-thioester formation activities of peptide synthetase modules. Hybrid bimodular peptide synthetases were generated by recombination of the corresponding reading frames encoding for L-glutamic acid- and L-leucine-specific modules of surfactin synthetase SrfA-A at presumed inner- and intradomainic regions. We demonstrate that fusions at a previously postulated hinge region, dividing the amino acid adenylating domains of peptide synthetase modules into two subdomains, and at the highly conserved 4'-phosphopantetheine binding motif in acyl-thioester forming domains resulted in enzymatically active hybrid domains. By contrast, most manipulations in condensation domains like deletions, the complete exchange or the construction of chimeric domains considerably reduced or completely abolished the amino acid adenylation and thioester formation activity of the hybrid module.     

Ligand binding by TPR domains

Tetratricopeptide repeat (TPR) domains bind specific peptide ligands and are thought to mediate protein-protein interactions in a variety of biological systems. Here we compare peptide ligand-binding by several different TPR domains. We present specific examples that demonstrate that TPR domains typically undergo little or no structural rearrangement upon ligand binding. Our data suggest that, contrary to a recent proposal, coupled folding and binding is not the common mechanism of ligand recognition by TPR domains.     

Biological implications of SNPs in signal peptide domains of human proteins

Proteins destined for secretion or membrane compartments possess signal peptides for insertion into the membrane. The signal peptide is therefore critical for localization and function of cell surface receptors and ligands that mediate cell-cell communication. About 4% of all human proteins listed in UniProt database have signal peptide domains in their N terminals. A comprehensive literature survey was performed to retrieve functional and disease associated genetic variants in the signal peptide domains of human proteins. In 21 human proteins we have identified 26 disease associated mutations within their signal peptide domains, 14 mutations of which have been experimentally shown to impair the signal peptide function and thus influence protein transportation. We took advantage of SignalP 3.0 predictions to characterize the signal peptide prediction score differences between the mutant and the wild-type alleles of each mutation, as well as 189 previously uncharacterized single nucleotide polymorphisms (SNPs) found to be located in the signal peptide domains of 165 human proteins. Comparisons of signal peptide prediction outcomes of mutations and SNPs, have implicated SNPs potentially impacting the signal peptide function, and thus the cellular localization of the human proteins. The majority of the top candidate proteins represented membrane and secreted proteins that are associated with molecular transport, cell signaling and cell to cell interaction processes of the cell. This is the first study that systematically characterizes genetic variation occurring in the signal peptides of all human proteins. This study represents a useful strategy for prioritization of SNPs occurring within the signal peptide domains of human proteins. Functional evaluation of candidates identified herein may reveal effects on major cellular processes including immune cell function, cell recognition and adhesion, and signal transduction.     

ATP-independent activation of natriuretic peptide receptors

Natriuretic peptide receptor A (NPR-A) is an essential cardiovascular regulator that is stimulated by atrial natriuretic peptide and B-type natriuretic peptide, whereas natriuretic peptide receptor B (NPR-B) stimulates long bone growth in a C-type natriuretic peptide-dependent manner. Many reports indicate that ATP is essential for NPR-A and NPR-B activation. Current models suggest that natriuretic peptide binding to receptor extracellular domains causes ATP binding to intracellular kinase homology domains, which derepresses adjacent catalytic domains. Here, we report 100-fold activations of natriuretic peptide receptors in the absence of ATP. The addition of a nonhydrolyzable ATP analog had no effect at early time periods (measured in seconds) but increased cGMP production about 2-fold after longer incubations (measured in minutes), consistent with a stabilization, not activation, mechanism. These data indicate that ATP does not activate natriuretic peptide receptors as has been repeatedly reported. Instead, ATP increases activity primarily by maintaining proper receptor phosphorylation status but also serves a previously unappreciated enzyme stabilizing function.     

Reorganization of lipid domain structure in membranes by a transmembrane peptide: an ESR spin label study on the effect of the Escherichia coli outer membrane protein A signal peptide on the fluid lipid domain connectivity in binary mixtures of dimyristoyl phosphatidylcholine and distearoyl phosphatidylcholine.

The effect of a transmembrane peptide on the domain structure of a two-component, two-phase lipid bilayer composed of dimyristoyl phosphatidylcholine (DMPC) and distearoyl phosphatidylcholine (DSPC) was examined by spin label electron spin resonance (ESR) spectroscopy. The peptide, pOmpA, is the hydrophobic, 25-residue signal sequence of the outer membrane protein A from Escherichia coli. Nitroxide derivatives of the phospholipid DSPC, 16-DSPCSL, and of the pOmpA signal peptide, pOmpA-IASL, were used as probes. The first-derivative lineshapes of the ESR spectra were analyzed using a normalized intensity ratio, R, that gives information on the average sizes of the disconnected fluid domains and their point of connectivity (Sankaram, M.B., D. Marsh, and T.E. Thompson. 1992. Biophys. J. 63:340-349). In the absence of the peptide, the number of fluid lipid domains does not vary with the fraction of lipid that is in the fluid phase, and phase conversion is accomplished solely by changes in the domain size. The phase boundaries of the lipid mixture remain largely unchanged by the presence of the peptide at mole fractions up to 0.02, but both the size and number of the fluid domains is changed, and the point at which they become connected is shifted to lower fractions of the fluid phase. In addition, the number of domains in the presence of the peptide no longer remains constant but increases from a domain density at low fractions of the fluid phase that is much lower than that in the absence of peptide to one that is comparable to the natural state in the absence of peptide at the point of domain connectivity. A simple model is presented for the process of domain fission, where the latter is determined by a balance between the effects of peptide concentration in the fluid domains, the line tension at the domain boundaries, and the distributional entropy of the domains.     

Identification of the bacteria-binding peptide domain on salivary agglutinin (gp-340/DMBT1), a member of the scavenger receptor cysteine-rich superfamily

Salivary agglutinin is encoded by DMBT1 and identical to gp-340, a member of the scavenger receptor cysteine-rich (SRCR) superfamily. Salivary agglutinin/DMBT1 is known for its Streptococcus mutans agglutinating properties. This 300-400 kDa glycoprotein is composed of conserved peptide motifs: 14 SRCR domains that are separated by SRCR-interspersed domains (SIDs), 2 CUB (C1r/C1s Uegf Bmp1) domains, and a zona pellucida domain. We have searched for the peptide domains of agglutinin/DMBT1 responsible for bacteria binding. Digestion with endoproteinase Lys-C resulted in a protein fragment containing exclusively SRCR and SID domains that binds to S. mutans. To define more closely the S. mutans-binding domain, consensus-based peptides of the SRCR domains and SIDs were designed and synthesized. Only one of the SRCR peptides, designated SRCRP2, and none of the SID peptides bound to S. mutans. Strikingly, this peptide was also able to induce agglutination of S. mutans and a number of other bacteria. The repeated presence of this peptide in the native molecule endows agglutinin/DMBT1 with a general bacterial binding feature with a multivalent character. Moreover, our studies demonstrate for the first time that the polymorphic SRCR domains of salivary agglutinin/DMBT1 mediate ligand interactions.     

Preferential accumulation of Abeta(1-42) on gel phase domains of lipid bilayers: an AFM and fluorescence study

Peptide-membrane interactions have been implicated in both the toxicity and aggregation of beta-amyloid (Abeta) peptides. Recent studies have provided evidence for the involvement of liquid-ordered membrane domains known as lipid rafts in the formation and aggregation of Abeta. As a model, we have examined the interaction of Abeta(1-42) with phase separated DOPC/DPPC lipid bilayers using a combination of atomic force microscopy (AFM) and total internal reflection fluorescence microscopy (TIRF). AFM images show that addition of Abeta to preformed supported bilayers leads to accumulation of small peptide aggregates exclusively on the gel phase DPPC domains. Initial aggregates are observed approximately 90 min after peptide addition and increase in diameter to 45-150 nm within 24 h. TIRF studies with a mixture of Abeta and Abeta-Fl demonstrate that accumulation of the peptide on the gel phase domains occurs as early as 15 min after Abeta addition and is maintained for over 24 h. By contrast, Abeta is randomly distributed throughout both fluid and gel phases when the peptide is reconstituted into DOPC/DPPC vesicles prior to formation of a supported bilayer. The preferential accumulation of Abeta on DPPC domains suggests that rigid domains may act as platforms to concentrate peptide and enhance its aggregation and may be relevant to the postulated involvement of lipid rafts in modulating Abeta activity in vivo.     

Promiscuous binding nature of SH3 domains to their target proteins

SH3 domains are small but important domains in cell-signaling and function through protein-protein interactions. Their promiscuous nature in binding to polyproline peptides makes them much more important because many SH3 domains from different proteins bind to different proteins having polyproline template on their surface. Very subtle changes in the sequence of SH3 domains and the binding peptides determine the specificity of the peptide binding. Recent observation that SH3 domains bind to non- proline peptides makes the scenario of peptide binding involving SH3 domains complicated. If domain swapped dimerization as observed in Eps8-SH3 domain also binds different peptides, it proves the versatility of the SH3 domains in binding to peptides in various ways. An overview of the promiscuity of SH3 domains has been discussed.     

Ubiquitin binds to and regulates a subset of SH3 domains

SH3 domains are modules of 50-70 amino acids that promote interactions among proteins, often participating in the assembly of large dynamic complexes. These domains bind to peptide ligands, which usually contain a core Pro-X-X-Pro (PXXP) sequence. Here we identify a class of SH3 domains that bind to ubiquitin. The yeast endocytic protein Sla1, as well as the mammalian proteins CIN85 and amphiphysin, carry ubiquitin-binding SH3 domains. Ubiquitin and peptide ligands bind to the same hydrophobic groove on the SH3 domain surface, and ubiquitin and a PXXP-containing protein fragment compete for binding to SH3 domains. We conclude that a subset of SH3 domains constitutes a distinct type of ubiquitin-binding domain and that ubiquitin binding can negatively regulate interaction of SH3 domains with canonical proline-rich ligands.     

Transmembrane domain-mediated colocalization of HLA-DM and HLA-DR is required for optimal HLA-DM catalytic activity.

HLA-DM catalyzes peptide loading and exchange reactions by MHC class II molecules. Soluble recombinant DM, lacking transmembrane and cytoplasmic domains, was observed to have 200- to 400-fold less activity compared with the full-length protein in assays measuring DM-catalyzed peptide dissociation from purified HLA-DR1 in detergent solutions. Additional studies with truncated soluble DR1 demonstrated that transmembrane domains in DR1 molecules are also required for optimal activity. The potential requirement for specific interaction between the transmembrane domains of DM and DR was ruled out in experiments with chimeric DR1 molecules containing transmembrane domains from either DM or the unrelated protein CD80. These results suggested that the major role of the transmembrane domains is to facilitate colocalization of DM and DR in detergent micelles. The latter conclusion was further supported by the observation that HLA-DM-catalyzed peptide binding to certain murine class II proteins is increased by reducing the volume of detergent micelles. The importance of membrane colocalization was directly demonstrated in experiments in which DM and DR were reconstituted separately or together into membrane bilayers in unilamellar liposomes. Our findings demonstrate the importance of membrane anchoring in DM activity and underscore the potential importance of membrane localization in regulating peptide exchange by class II molecules.     

The molecular basis of FHA domain:phosphopeptide binding specificity and implications for phospho-dependent signaling mechanisms

Forkhead-associated (FHA) domains are a class of ubiquitous signaling modules that appear to function through interactions with phosphorylated target molecules. We have used oriented peptide library screening to determine the optimal phosphopeptide binding motifs recognized by several FHA domains, including those within a number of DNA damage checkpoint kinases, and determined the X-ray structure of Rad53p-FHA1, in complex with a phospho-threonine peptide, at 1.6 A resolution. The structure reveals a striking similarity to the MH2 domains of Smad tumor suppressor proteins and reveals a mode of peptide binding that differs from SH2, 14-3-3, or PTB domain complexes. These results have important implications for DNA damage signaling and CHK2-dependent tumor suppression, and they indicate that FHA domains play important and unsuspected roles in S/T kinase signaling mechanisms in prokaryotes and eukaryotes.     

Independence of metal binding between tandem Cys2His2 zinc finger domains.

Most Cys2His2 zinc finger proteins contain tandem arrays of metal binding domains. The tandem nature of these arrays suggests that metal binding by these domains may not be independent but rather that metal binding may occur in a cooperative manner. This is especially true in light of the crystal structure of a three zinc finger array bound to DNA that revealed several types of interactions between domains. To address this question, peptides containing two tandem domains have been prepared. While metal binding studies do show that the two finger peptide has a metal ion affinity about threefold higher than that for a single domain peptide with the same sequence, additional studies reveal that this behavior is due to increased single site affinities in the context of the two domain peptide rather than to cooperativity. These studies indicate that domains of this type are independent of one another with regard to metal binding, at least in the absence of DNA. This observation has implications with regard to the question of whether the activities of proteins of this class might be modulated by available zinc concentrations.     

Screening for PTB domain binding partners and ligand specificity using proteome-derived NPXY peptide arrays

Modular interaction domains that recognize peptide motifs in target proteins can impart selectivity in signaling pathways. Phosphotyrosine binding (PTB) domains are components of cytoplasmic docking proteins that bind cell surface receptors through NPXY motifs. We have employed a library of human proteome-derived NXXY sequences to explore PTB domain specificity and function. SPOTS peptide arrays were used to create a comprehensive matrix of receptor motifs that were probed with a set of 10 diverse PTB domains. This approach confirmed that individual PTB domains have selective and distinct recognition properties and provided a means to explore over 2,500 potential PTB domain-NXXY interactions. The results correlated well with previously known associations between full-length proteins and predicted novel interactions, as well as consensus binding data for specific PTB domains. Using the Ret, MuSK, and ErbB2 receptor tyrosine kinases, we show that interactions of these receptors with PTB domains predicted to bind by the NXXY arrays do occur in cells. Proteome-based peptide arrays can therefore identify networks of receptor interactions with scaffold proteins that may be physiologically relevant.     

The multiple‐specificity landscape of modular peptide recognition domains

Modular protein interaction domains form the building blocks of eukaryotic signaling pathways. Many of them, known as peptide recognition domains, mediate protein interactions by recognizing short, linear amino acid stretches on the surface of their cognate partners with high specificity. Residues in these stretches are usually assumed to contribute independently to binding, which has led to a simplified understanding of protein interactions. Conversely, we observe in large binding peptide data sets that different residue positions display highly significant correlations for many domains in three distinct families (PDZ, SH3 and WW). These correlation patterns reveal a widespread occurrence of multiple binding specificities and give novel structural insights into protein interactions. For example, we predict a new binding mode of PDZ domains and structurally rationalize it for DLG1 PDZ1. We show that multiple specificity more accurately predicts protein interactions and experimentally validate some of the predictions for the human proteins DLG1 and SCRIB. Overall, our results reveal a rich specificity landscape in peptide recognition domains, suggesting new ways of encoding specificity in protein interaction networks.     

Modular structure of genes encoding multifunctional peptide synthetases required for non-ribosomal peptide synthesis

Abstract Peptide synthetases are large multienzyme complexes that catalyze the non-ribosomal synthesis of a structurally diverse family of bioactive peptides. They possess a multidomain structure and employ the thiotemplate mechanism to activate, modify and link together by amide or ester bonds the constituent amino acids of the peptide product. The domains, which represent the functional building units of peptide synthetases, appear to act as independent enzymes whose specific linkage order forms the protein-template that defines the sequence of the incorporated amino acids. Two types of domains have been characterized in peptide synthetases of bacterial and fungal origin: type I comprises about 600 amino acids and contains at least two modules involved in substrate recognition, adenylation and thioester formation, whereas type II domains carry in addition an insertion of about 430 amino acids that may function as a N-methyltransferase module. The role of other genes associated with bacterial opérons encoding peptide synthetases is also discussed.     

Constraint-based, homology model of the extracellular domain of the epithelial Na+ channel α subunit reveals a mechanism of channel activation by proteases

The epithelial Na(+) channel (ENaC) mediates Na(+) transport across high resistance epithelia. This channel is assembled from three homologous subunits with the majority of the protein's mass found in the extracellular domains. Acid-sensing ion channel 1 (ASIC1) is homologous to ENaC, but a key functional domain is highly divergent. Here we present molecular models of the extracellular region of α ENaC based on a large data set of mutations that attenuate inhibitory peptide binding in combination with comparative modeling based on the resolved structure of ASIC1. The models successfully rationalized the data from the peptide binding screen. We engineered new mutants that had not been tested based on the models and successfully predict sites where mutations affected peptide binding. Thus, we were able to confirm the overall general fold of our structural models. Further analysis suggested that the α subunit-derived inhibitory peptide affects channel gating by constraining motions within two major domains in the extracellular region, the thumb and finger domains.