Proline-rich Ligands Research Articles

Fluorescent carbazole-derived α-amino acids: structural mimics of tryptophan.

Fluorescent tags are commonly used for imaging of proteins and peptides during biological events; however, the large size of dyes can disrupt protein structure and function, and typically require the use of a chemical spacer. Herein, we report the synthesis of a new class of fluorescent unnatural α-amino acid, containing carbazole side-chains designed to mimic l-tryptophan and thus, readily incorporated into peptides. The amino acids were constructed using a Negishi cross-coupling reaction as the key step and exhibited strong fluorescent emission, with high quantum yields in both organic solvents and water. Compatible with solid phase peptide synthesis, the carbazole amino acids were used to replace tryptophan in a β-hairpin model peptide and shown to be a close structural mimic with retention of conformation. They were also found to be effective fluorescent molecular reporters for biological events. Incorporation into a proline-rich ligand of the WW domain protein demonstrated that the fluorescent properties of a carbazole amino acid could be used to measure the protein-protein binding interaction of this important biological signalling process.

Involvement of Local, Rapid Conformational Dynamics in Binding of Flexible Recognition Motifs.

Flexible protein sequences populate ensembles of rapidly interconverting states differentiated by small-scale fluctuations; however, elucidating whether and how the ensembles determine function experimentally is challenged by the combined high spatial and temporal resolution needed to capture the states. We used carbon-deuterium (C-D) bond vibrations incorporated as infrared probes to characterize with residue-specific detail the heterogeneity of states adopted by proline-rich (PR) sequences and assess their involvement in recognition of Src homology 3 domains. The C-D absorption envelopes provided evidence for two or three sub-populations at all proline residues. The changes in the subpopulations induced by binding generally reflected recognition by conformational selection but depended on the residue and the state of the ligand to illuminate distinct mechanisms among the PR ligands. Notably, the spectral data indicate that greater adaptability among the states is associated with reduced recognition specificity and that perturbation to the ensemble populations contributes to differences in binding entropy. Broadly, the study quantifies rapidly interconverting ensembles with residue-specific detail and implicates them in function.

Engineering protein stability with atomic precision in a monomeric miniprotein.

Miniproteins simplify the protein-folding problem, allowing the dissection of forces that stabilize protein structures. Here we describe PPα-Tyr, a designed peptide comprising an α-helix buttressed by a polyproline II helix. PPα-Tyr is water soluble and monomeric, and it unfolds cooperatively with a midpoint unfolding temperature (TM) of 39 °C. NMR structures of PPα-Tyr reveal proline residues docked between tyrosine side chains, as designed. The stability of PPα is sensitive to modifications in the aromatic residues: replacing tyrosine with phenylalanine, i.e., changing three solvent-exposed hydroxyl groups to protons, reduces the TM to 20 °C. We attribute this result to the loss of CH-π interactions between the aromatic and proline rings, which we probe by substituting the aromatic residues with nonproteinogenic side chains. In analyses of natural protein structures, we find a preference for proline-tyrosine interactions over other proline-containing pairs, and observe abundant CH-π interactions in biologically important complexes between proline-rich ligands and SH3 and similar domains.

Production in Pichia pastoris of complementary protein-based polymers with heterodimer-forming WW and PPxY domains.

BackgroundSpecific coupling of de novo designed recombinant protein polymers for the construction of precisely structured nanomaterials is of interest for applications in biomedicine, pharmaceutics and diagnostics. An attractive coupling strategy is to incorporate specifically interacting peptides into the genetic design of the protein polymers. An example of such interaction is the binding of particular proline-rich ligands by so-called WW-domains. In this study, we investigated whether these domains can be produced in the yeast Pichia pastoris as part of otherwise non-interacting protein polymers, and whether they bring about polymer coupling upon mixing.ResultsWe constructed two variants of a highly hydrophilic protein-based polymer that differ only in their C-terminal extensions. One carries a C-terminal WW domain, and the other a C-terminal proline-rich ligand (PPxY). Both polymers were produced in P.pastoris with a purified protein yield of more than 2 g L−1 of cell-free broth. The proline-rich module was found to be O-glycosylated, and uncommonly a large portion of the attached oligosaccharides was phosphorylated. Glycosylation was overcome by introducing a Ser → Ala mutation in the PPxY peptide. Tryptophan fluorescence monitored during titration of the polymer containing the WW domain with either the glycosylated or nonglycosylated PPxY-containing polymer revealed binding. The complementary polymers associated with a Kd of ~3 µM, regardless of glycosylation state of the PPxY domain. Binding was confirmed by isothermal titration calorimetry, with a Kd of ~9 µM.ConclusionsThis article presents a blueprint for the production in P. pastoris of protein polymers that can be coupled using the noncovalent interaction between WW domains and proline-rich ligands. The availability of this highly specific coupling tool will hereafter allow us to construct various supramolecular structures and biomaterials.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0498-3) contains supplementary material, which is available to authorized users.

Parallel Chemical Protein Synthesis on a Surface Enables the Rapid Analysis of the Phosphoregulation of SH3 Domains.

Analysis of postranslationally modified protein domains is complicated by an availability problem, as recombinant methods rarely allow site-specificity at will. Although total synthesis enables full control over posttranslational and other modifications, chemical approaches are limited to shorter peptides. To solve this problem, we herein describe a method that combines a) immobilization of N-terminally thiolated peptide hydrazides by hydrazone ligation, b) on-surface native chemical ligation with self-purified peptide thioesters, c) radical-induced desulfurization, and d) a surface-based fluorescence binding assay for functional characterization. We used the method to rapidly investigate 20 SH3 domains, with a focus on their phosphoregulation. The analysis suggests that tyrosine phosphorylation of SH3 domains found in Abl kinases act as a switch that can induce both the loss and, unexpectedly, gain of affinity for proline-rich ligands.

Centromeric binding and activity of Protein Phosphatase 4.

The cell division cycle requires tight coupling between protein phosphorylation and dephosphorylation. However, understanding the cell cycle roles of multimeric protein phosphatases has been limited by the lack of knowledge of how their diverse regulatory subunits target highly conserved catalytic subunits to their sites of action. Phosphoprotein phosphatase 4 (PP4) has been recently shown to participate in the regulation of cell cycle progression. We now find that the EVH1 domain of the regulatory subunit 3 of Drosophila PP4, Falafel (Flfl), directly interacts with the centromeric protein C (CENP-C). Unlike other EVH1 domains that interact with proline-rich ligands, the crystal structure of the Flfl amino-terminal EVH1 domain bound to a CENP-C peptide reveals a new target-recognition mode for the phosphatase subunit. We also show that binding of Flfl to CENP-C is required to bring PP4 activity to centromeres to maintain CENP-C and attached core kinetochore proteins at chromosomes during mitosis.

Serine and proline-rich ligands enriched via phage-display technology show preferential binding to BCR/ABL expressing cells

Despite the use of targeted therapy, chronic myelogenous leukemia (CML) currently remains incurable with drug therapy, with patients requiring life-long treatment. Developing either a vaccine to prevent the disease or another novel drug to specifically target and eradicate the CML cell will require the identification of CML-associated cell-surface markers and molecules that can bind specifically to the cell surface. In an attempt to discover peptides that bind specifically to cells in the early chronic phase of the disease, we used phage-display technology to identify heptapeptides that bind specifically to the surface of BCR/ABL-expressing fibroblasts. An in vitro system using NIH3T3 stably transfected with pGD210 (BCR/ABL) was used as a model for the chronic phase of the disease. The cells were panned using a linear heptapeptide phage library (Ph.D 7.0) in a negative/positive panning strategy with NIH3T3 containing only the plasmid vector as the wild type control. We identified four novel peptides that were enriched through this technique. These peptides contained either multiple proline residues or serine/threonine-proline pairs and showed a confirmed binding preference for BCR/ABL+ fibroblasts. The peptide Y-R-A-P-W-P-P also showed a binding affinity for granulocytes from untreated CML patients. We have identified several novel peptides that can be used in future studies to identify specific CML cell-surface antigens or provide a novel drug-delivery mechanism.

Adaptor Proteins Intersectin 1 and 2 Bind Similar Proline-Rich Ligands but Are Differentially Recognized by SH2 Domain-Containing Proteins

BackgroundScaffolding proteins of the intersectin (ITSN) family, ITSN1 and ITSN2, are crucial for the initiation stage of clathrin-mediated endocytosis. These proteins are closely related but have implications in distinct pathologies. To determine how these proteins could be separated in certain cell pathways we performed a comparative study of ITSNs.Methodology/Principal FindingsWe have shown that endogenous ITSN1 and ITSN2 colocalize and form a complex in cells. A structural comparison of five SH3 domains, which mediated most ITSNs protein-protein interactions, demonstrated a similarity of their ligand-binding sites. We showed that the SH3 domains of ITSN2 bound well-established interactors of ITSN1 as well as newly identified ITSNs protein partners. A search for a novel interacting interface revealed multiple tyrosines that could be phosphorylated in ITSN2. Phosphorylation of ITSN2 isoforms but not ITSN1 short isoform was observed in various cell lines. EGF stimulation of HeLa cells enhanced tyrosine phosphorylation of ITSN2 isoforms and enabled their recognition by the SH2 domains of the Fyn, Fgr and Abl1 kinases, the regulatory subunit of PI3K, the adaptor proteins Grb2 and Crk, and phospholipase C gamma. The SH2 domains mentioned were unable to bind ITSN1 short isoform.Conclusions/SignificanceOur results indicate that during evolution of vertebrates ITSN2 acquired a novel protein-interaction interface that allows its specific recognition by the SH2 domains of signaling proteins. We propose that these data could be important to understand the functional diversity of paralogous ITSN proteins.

A direct interaction between DCP1 and XRN1 couples mRNA decapping to 5′ exonucleolytic degradation

The removal of the mRNA 5' cap structure by the decapping enzyme DCP2 leads to rapid 5'→3' mRNA degradation by XRN1, suggesting that the two processes are coordinated, but the coupling mechanism is unknown. DCP2 associates with the decapping activators EDC4 and DCP1. Here we show that XRN1 directly interacts with EDC4 and DCP1 in human and Drosophila melanogaster cells, respectively. In D. melanogaster cells, this interaction is mediated by the DCP1 EVH1 domain and a DCP1-binding motif (DBM) in the XRN1 C-terminal region. The NMR structure of the DCP1 EVH1 domain bound to the DBM reveals that the peptide docks at a conserved aromatic cleft, which is used by EVH1 domains to recognize proline-rich ligands. Our findings reveal a role for XRN1 in decapping and provide a molecular basis for the coupling of decapping to 5'→3' mRNA degradation.

Isolation of monobodies that bind specifically to the SH3 domain of the Fyn tyrosine protein kinase

Fyn is a nonreceptor protein tyrosine kinase that belongs to a highly conserved kinase family, Src family kinases. Fyn plays an important role in inflammatory processes and neuronal functions. To generate a synthetic affinity reagent that can be used to probe Fyn, a phage-display library of fibronectin type III monobodies was affinity selected with the Src Homology 3 (SH3) domain of Fyn and three binders were isolated. One of the three binders, G9, is specific in binding to the SH3 domain of Fyn, but not to the other members of the Src family (i.e. Blk, Fgr, Hck, Lck, Lyn, Src and Yes), even though they share 51-81% amino acid identity. The other two bind principally to the Fyn SH3 domain, with some cross-reactivity to the Yes SH3 domain. The G9 binder has a dissociation constant of 166±6nM, as measured by isothermal titration calorimetry, and binds only to the Fyn SH3 domain out of 150 human SH3 domains examined in an array. Interestingly, although the G9 monobody lacks proline in its randomized BC and FG loops, it binds at the same site on the SH3 domain as proline-rich ligands, as revealed by competition assays. The G9 monobody, identified in this study, may be used as a highly selective probe for detecting and purifying cellular Fyn kinase.

Multivalent Binding of Formin-binding Protein 21 (FBP21)-Tandem-WW Domains Fosters Protein Recognition in the Pre-spliceosome

The high abundance of repetitive but nonidentical proline-rich sequences in spliceosomal proteins raises the question of how these known interaction motifs recruit their interacting protein domains. Whereas complex formation of these adaptors with individual motifs has been studied in great detail, little is known about the binding mode of domains arranged in tandem repeats and long proline-rich sequences including multiple motifs. Here we studied the interaction of the two adjacent WW domains of spliceosomal protein FBP21 with several ligands of different lengths and composition to elucidate the hallmarks of multivalent binding for this class of recognition domains. First, we show that many of the proteins that define the cellular proteome interacting with FBP21-WW1-WW2 contain multiple proline-rich motifs. Among these is the newly identified binding partner SF3B4. Fluorescence resonance energy transfer (FRET) analysis reveals the tandem-WW domains of FBP21 to interact with splicing factor 3B4 (SF3B4) in nuclear speckles where splicing takes place. Isothermal titration calorimetry and NMR shows that the tandem arrangement of WW domains and the multivalency of the proline-rich ligands both contribute to affinity enhancement. However, ligand exchange remains fast compared with the NMR time scale. Surprisingly, a N-terminal spin label attached to a bivalent ligand induces NMR line broadening of signals corresponding to both WW domains of the FBP21-WW1-WW2 protein. This suggests that distinct orientations of the ligand contribute to a delocalized and semispecific binding mode that should facilitate search processes within the spliceosome.

X-ray structure of the SH3 domain of the phosphoinositide 3-kinase p85β subunit.

Src-homology 3 (SH3) domains are involved in extensive protein-protein interactions and constitute key elements of intracellular signal transduction. Three-dimensional structures have been reported for SH3 domains of various proteins, including the 85 kDa regulatory subunit (p85) of phosphoinositide 3-kinase. However, all of the latter structures are of p85 isoform α and no crystal structure of the SH3 domain of the equally important isoform β has been reported to date. In this structural communication, the recombinant production, crystallization and X-ray structure determination at 2.0 Å resolution of the SH3 domain of human p85β is described. The structure reveals a compact β-barrel fold very similar to that of p85α. However, binding studies with two classes of proline-rich ligand peptides demonstrate that the ligand-binding specificity differs slightly between the SH3 domains of human p85β and p85α, despite their high structural similarity.

Conformational choreography of a molecular switch region in myelin basic protein—Molecular dynamics shows induced folding and secondary structure type conversion upon threonyl phosphorylation in both aqueous and membrane-associated environments

The 18.5kDa isoform of myelin basic protein is essential to maintaining the close apposition of myelin membranes in central nervous system myelin, but its intrinsic disorder (conformational dependence on environment), a variety of post-translational modifications, and a diversity of protein ligands (e.g., actin and tubulin) all indicate it to be multifunctional. We have performed molecular dynamics simulations of a conserved central segment of 18.5kDa myelin basic protein (residues Glu80–Gly103, murine sequence numbering) in aqueous and membrane-associated environments to ascertain the stability of constituent secondary structure elements (α-helix from Glu80–Val91 and extended poly-proline type II from Thr92–Gly103) and the effects of phosphorylation of residues Thr92 and Thr95, individually and together. In aqueous solution, all four forms of the peptide bent in the middle to form a hydrophobic cluster. The phosphorylated variants were stabilized further by electrostatic interactions and formation of β-structures, in agreement with previous spectroscopic data. In simulations performed with the peptide in association with a dimyristoylphosphatidylcholine bilayer, the amphipathic α-helical segment remained stable and membrane-associated, although the degree of penetration was less in the phosphorylated variants, and the tilt of the α-helix with respect to the plane of the membrane also changed significantly with the modifications. The extended segment adjacent to this α-helix represents a putative SH3-ligand and remained exposed to the cytoplasm (and thus accessible to binding partners). The results of these simulations demonstrate how this segment of the protein can act as a molecular switch: an amphipathic α-helical segment of the protein is membrane-associated and presents a subsequent proline-rich segment to the cytoplasm for interaction with other proteins. Phosphorylation of threonyl residues alters the degree of membrane penetration of the α-helix and the accessibility of the proline-rich ligand and can stabilize a β-bend. A bend in this region of 18.5kDa myelin basic protein suggests that the N- and C-termini of the proteins can interact with different leaflets of the myelin membrane and explain how a single protein can bring them close together.

Y65C Missense Mutation in the WW Domain of the Golabi-Ito-Hall Syndrome Protein PQBP1 Affects Its Binding Activity and Deregulates Pre-mRNA Splicing

The PQBP1 (polyglutamine tract-binding protein 1) gene encodes a nuclear protein that regulates pre-mRNA splicing and transcription. Mutations in the PQBP1 gene were reported in several X chromosome-linked mental retardation disorders including Golabi-Ito-Hall syndrome. The missense mutation that causes this syndrome is unique among other PQBP1 mutations reported to date because it maps within a functional domain of PQBP1, known as the WW domain. The mutation substitutes tyrosine 65 with cysteine and is located within the conserved core of aromatic amino acids of the domain. We show here that the binding property of the Y65C-mutated WW domain and the full-length mutant protein toward its cognate proline-rich ligands was diminished. Furthermore, in Golabi-Ito-Hall-derived lymphoblasts we showed that the complex between PQBP1-Y65C and WBP11 (WW domain-binding protein 11) splicing factor was compromised. In these cells a substantial decrease in pre-mRNA splicing efficiency was detected. Our study points to the critical role of the WW domain in the function of the PQBP1 protein and provides an insight into the molecular mechanism that underlies the X chromosome-linked mental retardation entities classified globally as Renpenning syndrome.

Role of Interfacial Water Molecules in Proline-rich Ligand Recognition by the Src Homology 3 Domain of Abl

The interaction of Abl-Src homology 3 domain (SH3) with the high affinity peptide p41 is the most notable example of the inconsistency existing between the currently accepted description of SH3 complexes and their binding thermodynamic signature. We had previously hypothesized that the presence of interfacial water molecules is partially responsible for this thermodynamic behavior. We present here a thermodynamic, structural, and molecular dynamics simulation study of the interaction of p41 with Abl-SH3 and a set of mutants designed to alter the water-mediated interaction network. Our results provide a detailed description of the dynamic properties of the interfacial water molecules and a molecular interpretation of the thermodynamic effects elicited by the mutations in terms of the modulation of the water-mediated hydrogen bond network. In the light of these results, a new dual binding mechanism is proposed that provides a better description of proline-rich ligand recognition by Abl-SH3 and that has important implications for rational design.

Evaluation of folding co-operativity of a chimeric protein based on the molecular recognition between polyproline ligands and SH3 domains

In previous work, we designed a chimeric protein, named SPCp41, to evaluate the thermodynamics of the interaction between SH3 domains and proline-rich ligands by combining thermal unfolding measurements and mutagenesis. Here, we have investigated the energetic integrity of the chain extension corresponding to the ligand sequence into the native structure, since the opposite will produce changes in the folding mechanism of the SH3 domain that may give rise to undesirable contributions to the thermodynamic parameters. We have analysed the folding-unfolding kinetics under standard conditions (50 mM phosphate pH 7). Kinetic evolutions are well described by a bi-exponential where, on top of the main kinetic phase, a low-populated slower phase appears as a consequence of cis-trans isomerisation of Pro39, as demonstrated by the influence of prolyl isomerases and by mutational analysis. There is also a burst phase possibly due to a productive formation of some helical ensembles. The main evolution, accounting for the true folding kinetics of SPCp41, can be considered as a two-state process, where the folding transition state produces essentially the same picture shown by the circular permutant S19-P20s (the 'nucleus' of the design) and the ligand will dock at the latter stages of the two-state process. Thus, all conclusions argue in favour of the effectiveness of SPCp41 to study energetic, dynamic and structural aspects of SH3-ligand interactions.

Both actin and polyproline interactions of profilin-1 are required for migration, invasion and capillary morphogenesis of vascular endothelial cells

The objective of the present study was to evaluate how different ligand interactions of profilin-1 (Pfn1), an actin-binding protein that is upregulated during capillary morphogenesis of vascular endothelial cells (VEC), contribute to migration and capillary forming ability of VEC. We adopted a knockdown–knockin experimental system to stably express either fully functional form or mutants of Pfn1 that are impaired in binding to two of its major ligands, actin (H119E mutant) and proteins containing polyproline domains (H133S mutant), in a human dermal microvascular cell line (HmVEC) against near-null endogenous Pfn1 background. We found that silencing endogenous Pfn1 expression in HmVEC leads to slower random migration, reduced velocity of membrane protrusion and a significant impairment in matrigel-induced cord formation. Only re-expression of fully functional but not any of the two ligand-binding deficient mutants of Pfn1 rescues the above defects. We further show that loss of Pfn1 expression in VEC inhibits three-dimensional capillary morphogenesis, MMP2 secretion and ECM invasion. VEC invasion through ECM is also inhibited when actin and polyproline interactions of Pfn1 are disrupted. Together, these experimental data demonstrate that Pfn1 regulates VEC migration, invasion and capillary morphogenesis through its interaction with both actin and proline-rich ligands.

The Interaction of Proline-Rich Ligands with Profilin Probed with an Enzyme-Linked Immunosorbent Assay

To detect interactions of different proline-rich ligands with profilins, the authors developed a simple analytical antibody-based screening method. Profilin I or profilin IIa was coated in microplates, and ligand binding was monitored via antibody detection. Using purified components, the authors show that the assay is very sensitive as nanomolar concentrations of recombinant profilin ligands can be used. They further apply this technique to detect interaction of profilin with various proline-rich partners, either endogenously present or ectopically expressed as tagged fusions, using lysates. With this assay, the authors identify Shootin1 as a novel profilin IIa partner. In addition, they demonstrate that this assay can be used for studying competition or ternary complex formation. In conclusion, they developed a sensitive, easy-to-use, and versatile method for the study of the interaction between profilin and different ligands.

Interactions between the Three CIN85 SH3 Domains and Ubiquitin: Implications for CIN85 Ubiquitination

CIN85 is an adaptor protein linking the ubiquitin ligase Cbl and clathrin-binding proteins in clathrin-mediated receptor endocytosis. The SH3 domains of CIN85 bind to a proline-rich region of Cbl. Here we show that all three SH3 domains of CIN85 bind to ubiquitin. We also present a data-based structural model of the CIN85 SH3-C domain in complex with ubiquitin. In this complex, ubiquitin binds to the canonical interaction surface of the SH3 domain for proline-rich ligands and mimics the PPII helix, and we provide evidence that ubiquitin competes with these ligands for binding. We demonstrate that disruption of ubiquitin binding results in constitutive ubiquitination of CIN85 and an increased level of ubiquitination of EGFR in the absence of EGF stimulation. These results suggest that competition between Cbl and ubiquitin binding to CIN85 regulates Cbl function and EGFR endocytosis.

In silico screening and biological evaluation of inhibitors of Src-SH3 domain interaction with a proline-rich ligand

Src signalling and transduction are directly involved in cell growth, cell cycle, malignant transformation and cell migration, providing therapeutic opportunities through inhibition of Src. Here we report virtual screening for novel compounds that inhibit the Src-SH3 protein-protein interaction with a proline-rich peptide ligand. Computational docking of the ZINC compound database was performed using GOLD. Top-scoring compounds were assayed using a fluorescence polarization-based assay. A benzoquinoline derivative showed micromolar inhibition of binding between Src-SH3 and the proline-rich peptide. Several analogues were subsequently assayed showing the requirement of a linker between the benzoquinoline and phenyl rings, and electron donating substituents on the phenyl ring.