Comparison of mRNA-display-based selections using synthetic peptide and natural protein libraries

mRNA display is a genotype-phenotype conjugation method that allows the amplification-based, iterative rounds of in vitro selection to be applied to peptides and proteins. Compared to prior protein selection techniques, mRNA display can be used to select functional sequences from both long natural protein and short combinatorial peptide libraries with much higher complexities. To investigate the basic features and problems of using mRNA display in studying conditional protein-protein interactions, we compared the target-binding selections against calmodulin (CaM) using both a natural protein library and a combinatorial peptide library. The selections were efficient in both cases and required only two rounds to isolate numerous Ca2+/CaM-binding natural proteins and synthetic peptides with a wide range of affinities. Many known and novel CaM-binding proteins were identified from the natural human protein library. More than 2000 CaM-binding peptides were selected from the combinatorial peptide library. Unlike sequences from prior CaM-binding selections that correlated poorly with naturally occurring proteins, synthetic peptides homologous to the Ca2+/CaM-binding motifs in natural proteins were isolated. Interestingly, a large number of synthetic peptides that lack the conventional CaM-binding secondary structures bound to CaM tightly and specifically, suggesting the presence of other interaction modes between CaM and its downstream binding targets. Our results indicate that mRNA display is an ideal approach to the identification of Ca2+-dependent protein-protein interactions, which are important in the regulation of numerous signaling pathways.     

Different conformational switches underlie the calmodulin-dependent modulation of calcium pumps and channels

We have used fluorescence spectroscopy to investigate the structure of calmodulin (CaM) bound with CaM-binding sequences of either the plasma membrane Ca-ATPase or the skeletal muscle ryanodine receptor (RyR1) calcium release channel. Following derivatization with N-(1-pyrene)maleimide at engineered sites (T34C and T110C) within the N- and C-domains of CaM, contact interactions between these opposing domains of CaM resulted in excimer fluorescence that permits us to monitor conformational states of bound CaM. Complementary measurements take advantage of the unique conserved Trp within CaM-binding sequences that functions as a hydrophobic anchor in CaM binding and permits measurements of both a local and global peptide structure. We find that CaM binds with high affinity in a collapsed structure to the CaM-binding sequences of both the Ca-ATPase and RyR1, resulting in excimer formation that is indicative of contact interactions between the N- and the C-domains of CaM in complex with these CaM-binding peptides. There is a 4-fold larger amount of excimer formation for CaM bound to the CaM-binding sequence of the Ca-ATPase in comparison to RyR1, indicating a closer structural coupling between CaM domains in this complex. Prior to CaM association, the CaM-binding sequences of the Ca-ATPase and RyR1 are conformationally disordered. Upon CaM association, the CaM-binding sequence of the Ca-ATPase assumes a highly ordered structure. In comparison, the CaM-binding sequence of RyR1 remains conformationally disordered irrespective of CaM binding. These results suggest an important role for interdomain contact interactions between the opposing domains of CaM in stabilizing the structure of the peptide complex. The substantially different structural responses associated with CaM binding to Ca-ATPase and RyR1 indicates a plasticity in their respective binding mechanisms that accomplishes different physical mechanisms of allosteric regulation, involving either the dissociation of a C-terminal regulatory domain necessary for pump activation or the modulation of intersubunit interactions to diminish RyR1 channel activity.     

The cilia of Paramecium tetraurelia contain both Ca2+-dependent and Ca2+-inhibitable calmodulin-binding proteins.

To identify protein targets for calmodulin (CaM) in the cilia of Paramecium tetraurelia, we employed a 125I-CaM blot assay after resolution of ciliary proteins on SDS/polyacrylamide gels. Two distinct types of CaM-binding proteins were detected. One group bound 125I-CaM at free Ca2+ concentrations above 0.5-1 microM and included a major binding activity of 63 kDa (C63) and activities of 126 kDa (C126), 96 kDa (C96), and 36 kDa (C36). CaM bound these proteins with high (nanomolar) affinity and specificity relative to related Ca2+ receptors. The second type of protein bound 125I-CaM only when the free Ca2+ concentration was below 1-2 microM and included polypeptides of 95 kDa (E95) and 105 kDa (E105). E105 may also contain Ca2+-dependent binding sites for CaM. Both E95 and E105 exhibited strong specificity for Paramecium CaM over bovine CaM. Ciliary subfractionation experiments suggested that C63, C126, C96, E95, and E105 are bound to the axoneme, whereas C36 is a soluble and/or membrane-associated protein. Additional Ca2+-dependent CaM-binding proteins of 63, 70, and 120 kDa were found associated with ciliary membrane vesicles. In support of these results, filtration binding assays also indicated high-affinity binding sites for CaM on isolated intact axonemes and suggested the presence of both Ca2+-dependent and Ca2+-inhibitable targets. Like E95 and E105, the Ca2+-inhibitable CaM-binding sites showed strong preference for Paramecium CaM over vertebrate CaM and troponin C. Together, these results suggest that CaM has multiple targets in the cilium and hence may regulate ciliary motility in a complex and pleiotropic fashion.     

Ca(2+)/Calmodulin-binding proteins from the C. elegans proteome

Calmodulin (CaM) is the primary Ca(2+)-sensor that regulates a wide variety of cellular processes in eukaryotes. Although many Ca(2+)/CaM-binding proteins have been identified, very few such proteins could be found from the genome-wide protein-protein interaction maps of Caenorhabditis elegans constructed by yeast two-hybrid screening. Using a genotype-phenotype conjugation method called mRNA-display, we performed a selection for Ca(2+)/CaM-binding proteins from a proteome library of C. elegans. The method allowed the identification of 9 known and 47 previously uncharacterized Ca(2+)-dependent CaM-binding proteins from the adult worm proteome. The Ca(2+)/CaM-binding properties of these proteins were characterized and their binding motifs were identified. The availability of such information could facilitate our understanding of the signaling pathways mediated by Ca(2+)/CaM in C. elegans. Due to its simplicity and efficiency, the method could be readily applied to examine the Ca(2+)-dependent binding partners of numerous other Ca(2+)-binding proteins, which may play important roles in many signaling pathways in C. elegans.     

Using Bayesian multinomial classifier to predict whether a given protein sequence is intrinsically disordered

Intrinsically disordered proteins (IDPs) lack a well-defined three-dimensional structure under physiological conditions. Intrinsic disorder is a common phenomenon, particularly in multicellular eukaryotes, and is responsible for important protein functions including regulation and signaling. Many disease-related proteins are likely to be intrinsically disordered or to have disordered regions. In this paper, a new predictor model based on the Bayesian classification methodology is introduced to predict for a given protein or protein region if it is intrinsically disordered or ordered using only its primary sequence. The method allows to incorporate length-dependent amino acid compositional differences of disordered regions by including separate statistical representations for short, middle and long disordered regions. The predictor was trained on the constructed data set of protein regions with known structural properties. In a Jack-knife test, the predictor achieved the sensitivity of 89.2% for disordered and 81.4% for ordered regions. Our method outperformed several reported predictors when evaluated on the previously published data set of Prilusky et al. [2005. FoldIndex: a simple tool to predict whether a given protein sequence is intrinsically unfolded. Bioinformatics 21 (16), 3435-3438]. Further strength of our approach is the ease of implementation.     

The Roles of Intrinsic Disorder in Orchestrating the Wnt-Pathway

Abstract

The canonical Wnt-pathway plays a number of crucial roles in the development of organism. Malfunctions of this pathway lead to various diseases including cancer. In the inactivated state, this pathway involves five proteins, Axin, CKI-α, GSK-3β, APC, and β-catenin. We analyzed these proteins by a number of computational tools, such as PONDR®VLXT, PONDR®VSL2, MoRF-II predictor and Hydrophobic Cluster Analysis (HCA) to show that each of the Wnt-pathway proteins contains several intrinsically disordered regions. Based on a comprehensive analysis of published data we conclude that these disordered regions facilitate protein-protein interactions, post-translational modifications, and signaling. The scaffold protein Axin and another large protein, APC, act as flexible concentrators in gathering together all other proteins involved in the Wnt-pathway, emphasizing the role of intrinsically disordered regions in orchestrating the complex protein-protein interactions. We further explore the intricate roles of highly disordered APC in regulation of β-catenin function. Intrinsically disordered APC helps the collection of β-catenin from cytoplasm, facilitates the β-catenin delivery to the binding sites on Axin, and controls the final detachment of β-catenin from Axin.     

Erratum to: Structural analysis of the complex between calmodulin and full-length myelin basic protein, an intrinsically disordered molecule

Myelin basic protein (MBP) is present between the cytoplasmic leaflets of the compact myelin membrane in both the peripheral and central nervous systems, and characterized to be intrinsically disordered in solution. One of the best-characterized protein ligands for MBP is calmodulin (CaM), a highly acidic calcium sensor. We pulled down MBP from human brain white matter as the major calcium-dependent CaM-binding protein. We then used full-length brain MBP, and a peptide from rodent MBP, to structurally characterize the MBP–CaM complex in solution by small-angle X-ray scattering, NMR spectroscopy, synchrotron radiation circular dichroism spectroscopy, and size exclusion chromatography. We determined 3D structures for the full-length protein–protein complex at different stoichiometries and detect ligand-induced folding of MBP. We also obtained thermodynamic data for the two CaM-binding sites of MBP, indicating that CaM does not collapse upon binding to MBP, and show that CaM and MBP colocalize in myelin sheaths. In addition, we analyzed the post-translational modifications of rat brain MBP, identifying a novel MBP modification, glucosylation. Our results provide a detailed picture of the MBP–CaM interaction, including a 3D model of the complex between full-length proteins.     

Structural analysis of the complex between calmodulin and full-length myelin basic protein, an intrinsically disordered molecule

Myelin basic protein (MBP) is present between the cytoplasmic leaflets of the compact myelin membrane in both the peripheral and central nervous systems, and characterized to be intrinsically disordered in solution. One of the best-characterized protein ligands for MBP is calmodulin (CaM), a highly acidic calcium sensor. We pulled down MBP from human brain white matter as the major calcium-dependent CaM-binding protein. We then used full-length brain MBP, and a peptide from rodent MBP, to structurally characterize the MBP–CaM complex in solution by small-angle X-ray scattering, NMR spectroscopy, synchrotron radiation circular dichroism spectroscopy, and size exclusion chromatography. We determined 3D structures for the full-length protein–protein complex at different stoichiometries and detect ligand-induced folding of MBP. We also obtained thermodynamic data for the two CaM-binding sites of MBP, indicating that CaM does not collapse upon binding to MBP, and show that CaM and MBP colocalize in myelin sheaths. In addition, we analyzed the post-translational modifications of rat brain MBP, identifying a novel MBP modification, glucosylation. Our results provide a detailed picture of the MBP–CaM interaction, including a 3D model of the complex between full-length proteins.     

钙不依赖性钙调素结合蛋白的研究进展

钙调素是普遍存在于真核生物细胞中、发挥多种生物学调控作用的信号组分.钙调素不仅在有Ca2 情况下通过与钙依赖性钙调素结合蛋白作用而传递信号,也能在相对无Ca2 条件下直接结合钙不依赖性钙调素结合蛋白而传递信号.综述了无钙离子结合钙调素及钙不依赖性钙调素结合蛋白的结构特性、钙不依赖性钙调素结合蛋白的种类及其可能的生物学作用,这将有助于我们深入认识钙调素介导信号途径的特异性、复杂性和多样性.     

The roles of intrinsic disorder in orchestrating the Wnt-pathway

The canonical Wnt-pathway plays a number of crucial roles in the development of organism. Malfunctions of this pathway lead to various diseases including cancer. In the inactivated state, this pathway involves five proteins, Axin, CKI-α, GSK-3β, APC, and β-catenin. We analyzed these proteins by a number of computational tools, such as PONDR(r)VLXT, PONDR(r)VSL2, MoRF-II predictor and Hydrophobic Cluster Analysis (HCA) to show that each of the Wnt-pathway proteins contains several intrinsically disordered regions. Based on a comprehensive analysis of published data we conclude that these disordered regions facilitate protein-protein interactions, post-translational modifications, and signaling. The scaffold protein Axin and another large protein, APC, act as flexible concentrators in gathering together all other proteins involved in the Wnt-pathway, emphasizing the role of intrinsically disordered regions in orchestrating the complex protein-protein interactions. We further explore the intricate roles of highly disordered APC in regulation of β-catenin function. Intrinsically disordered APC helps the collection of β-catenin from cytoplasm, facilitates the b-catenin delivery to the binding sites on Axin, and controls the final detachment of β-catenin from Axin.     

Novel Mechanism of Regulation of Protein 4.1G Binding Properties Through Ca2+/Calmodulin-Mediated Structural Changes

Protein 4.1G (4.1G) is a widely expressed member of the protein 4.1 family of membrane skeletal proteins. We have previously reported that Ca²⁺-saturated calmodulin (Ca²⁺/CaM) modulates 4.1G interactions with transmembrane and membrane-associated proteins through binding to Four.one-ezrin–radixin–moesin (4.1G FERM) domain and N-terminal headpiece region (GHP). Here we identify a novel mechanism of Ca²⁺/CaM-mediated regulation of 4.1G interactions using a combination of small-angle X-ray scattering, nuclear magnetic resonance spectroscopy, and circular dichroism spectroscopy analyses. We document that GHP intrinsically disordered coiled structure switches to a stable compact structure upon binding of Ca²⁺/CaM. This dramatic conformational change of GHP inhibits in turn 4.1G FERM domain interactions due to steric hindrance. Based upon sequence homologies with the Ca²⁺/CaM-binding motif in protein 4.1R headpiece region, we establish that the 4.1G S⁷¹RGISRFIPPWLKKQKS peptide (pepG) mediates Ca²⁺/CaM binding. As observed for GHP, the random coiled structure of pepG changes to a relaxed globular shape upon complex formation with Ca²⁺/CaM. The resilient coiled structure of pepG, maintained even in the presence of trifluoroethanol, singles it out from any previously published CaM-binding peptide. Taken together, these results show that Ca²⁺/CaM binding to GHP, and more specifically to pepG, has profound effects on other functional domains of 4.1G.     

Calmodulin binding to the Fas death domain. Regulation by Fas activation

Fas (APO-1/CD95) is a cell surface receptor that initiates apoptotic pathways, and its cytoplasmic domain interacts with various molecules suggesting that Fas signaling is complex and regulated by multiple proteins. Calmodulin (CaM) is an intracellular Ca(2+)-binding protein, and it mediates many of the effects of Ca2+. Here, we demonstrate that CaM binds to Fas directly and identify the CaM-binding site on the cytoplasmic death domain (DD) of Fas. Fas binds to CaM-Sepharose and is co-immunoprecipitated with CaM. Other death receptors, such as tumor necrosis factor receptor, DR4, and DR5 do not bind to CaM. The interaction between Fas and CaM is Ca(2+)-dependent. Deletion mapping analysis with various GST-fused Fas cytoplasmic domain fragments revealed that the fragment containing helices 1, 2, and 3 of the Fas DD has the CaM-binding ability. Sequence analysis of this fragment predicted a potential CaM-binding site in helix 2 and connected loops. A valine 254 to asparagine mutation in this region, which is analogous to the identified mutant allele of Fas in lpr mice that have a deficiency in Fas-mediated apoptosis, showed reduced CaM binding. Computer modeling of the interaction between CaM and helix 2 of the Fas DD predicted that amino acids, which are important for Fas-CaM binding, and point mutations of these amino acids caused reduced Fas-CaM binding. The interaction between Fas and CaM is increased approximately 2-fold early upon Fas activation (at 30 min) and is decreased to approximately 50% of control at 2 h. These findings suggest a novel function of CaM in Fas-mediated apoptosis.     

Calcineurin B- and calmodulin-binding preferences identified with phage-displayed peptide libraries

Calcineurin B (CnB) and calmodulin (CaM) are two structurally similar but functionally distinct 'EF-hand' Ca2+-binding proteins. CnB is the regulatory subunit of the CaM-stimulated protein phosphatase, calcineurin. CaM is a unique multifunctional protein that interacts with and modulates the activity of many target proteins. CnB and CaM are both required for the full activation of the phosphatase activity of calcineurin and are not interchangeable. The two proteins recognize distinct binding sites on calcineurin A subunit (CnA) and perform different functions. Phage-displayed peptide libraries (pIII and pVIII libraries) were screened with CnB and CaM to isolate peptides that could then be compared to determine if there were binding preferences of the two proteins. The Ca2+-dependent binding of phage-displayed peptides to CnB and CaM is specifically blocked by synthetic peptides derived from the CnB-binding domain of CnA and the CaM-binding domain of myosin light chain kinase respectively. Both CnB- and CaM-binding peptides have a high content of tryptophan and leucine, but CnB-binding peptides are more hydrophobic than CaM-binding peptides. CnB-binding peptides are negatively charged with clusters of hydrophobic residues rich in phenylalanine, whereas the CaM-binding peptides are positively charged and often contain an Arg/Lys-Trp motif. The binding preferences identified with peptide libraries are consistent with the features of the CnB-binding domains of all CnA isoforms and the CaM-binding domains of CaM targets.     

Structural determinants of calmodulin binding to the intracellular C-terminal domain of the metabotropic glutamate receptor 7A

Calmodulin (CaM) binds in a Ca2+-dependent manner to the intracellular C-terminal domains of most group III metabotropic glutamate receptors (mGluRs). Here we combined mutational and biophysical approaches to define the structural basis of CaM binding to mGluR 7A. Ca2+/CaM was found to interact with mGluR 7A primarily via its C-lobe at a 1:1 CaM:C-tail stoichiometry. Pulldown experiments with mutant CaM and mGluR 7A C-tail constructs and high resolution NMR with peptides corresponding to the CaM binding region of mGluR 7A allowed us to define hydrophobic and ionic interactions required for Ca2+/CaM binding and identified a 1-8-14 CaM-binding motif. The Ca2+/CaM.mGluR 7A peptide complex displays a classical wraparound structure that closely resembles that formed by Ca2+/CaM upon binding to smooth muscle myosin light chain kinase. Our data provide insight into how Ca2+/CaM regulates group III mGluR signaling via competition with intracellular proteins for receptor-binding sites.     

Identification of non-specific lipid transfer protein-1 as a calmodulin-binding protein in Arabidopsis

Although non-specific lipid transfer proteins (nsLTPs) are widely present in plants, their functions and regulations have not been fully understood. In this report, Arabidopsis nsLTP1 was cloned and expressed to investigate its binding to calmodulin (CaM). Gel overlay assays revealed that recombinant nsLTP1 bound to CaM in a calcium-independent manner. The association of nsLTP1 and CaM was corroborated using CaM-Sepharose beads to specifically isolate recombinant nsLTP1 from crude bacterial lysate. The CaM-binding site was mapped in nsLTP1 to the region of 69-80 amino acids. This region is highly conserved among plant nsLTPs, implicating that nsLTPs are a new family of CaM-binding proteins whose functions may be mediated by CaM signaling.     

Isolation and characterization of a novel calmodulin-binding protein from potato.

Tuberization in potato is controlled by hormonal and environmental signals. Ca(2+), an important intracellular messenger, and calmodulin (CaM), one of the primary Ca(2+) sensors, have been implicated in controlling diverse cellular processes in plants including tuberization. The regulation of cellular processes by CaM involves its interaction with other proteins. To understand the role of Ca(2+)/CaM in tuberization, we have screened an expression library prepared from developing tubers with biotinylated CaM. This screening resulted in isolation of a cDNA encoding a novel CaM-binding protein (potato calmodulin-binding protein (PCBP)). Ca(2+)-dependent binding of the cDNA-encoded protein to CaM is confirmed by (35)S-labeled CaM. The full-length cDNA is 5 kb long and encodes a protein of 1309 amino acids. The deduced amino acid sequence showed significant similarity with a hypothetical protein from another plant, Arabidopsis. However, no homologs of PCBP are found in nonplant systems, suggesting that it is likely to be specific to plants. Using truncated versions of the protein and a synthetic peptide in CaM binding assays we mapped the CaM-binding region to a 20-amino acid stretch (residues 1216-1237). The bacterially expressed protein containing the CaM-binding domain interacted with three CaM isoforms (CaM2, CaM4, and CaM6). PCBP is encoded by a single gene and is expressed differentially in the tissues tested. The expression of CaM, PCBP, and another CaM-binding protein is similar in different tissues and organs. The predicted protein contained seven putative nuclear localization signals and several strong PEST motifs. Fusion of the N-terminal region of the protein containing six of the seven nuclear localization signals to the reporter gene beta-glucuronidase targeted the reporter gene to the nucleus, suggesting a nuclear role for PCBP.     

Interaction of calmodulin with the serotonin 5-hydroxytryptamine2A receptor. A putative regulator of G protein coupling and receptor phosphorylation by protein kinase C

The 5-hydroxytryptamine2A (5-HT2A) receptor is a G(q/11)-coupled serotonin receptor that activates phospholipase C and increases diacylglycerol formation. In this report, we demonstrated that calmodulin (CaM) co-immunoprecipitates with the 5-HT2A receptor in NIH-3T3 fibroblasts in an agonist-dependent manner and that the receptor contains two putative CaM binding regions. The putative CaM binding regions of the 5-HT2A receptor are localized to the second intracellular loop and carboxyl terminus. In an in vitro binding assay peptides encompassing the putative second intracellular loop (i2) and carboxyl-terminal (ct) CaM binding regions bound CaM in a Ca2+-dependent manner. The i2 peptide bound with apparent higher affinity and shifted the mobility of CaM in a nondenaturing gel shift assay. Fluorescence emission spectral analyses of dansyl-CaM showed apparent K(D) values of 65 +/- 30 nM for the i2 peptide and 168 +/- 38 nM for the ct peptide. The ct CaM-binding domain overlaps with a putative protein kinase C (PKC) site, which was readily phosphorylated by PKC in vitro. CaM binding and phosphorylation of the ct peptide were found to be antagonistic, suggesting a putative role for CaM in the regulation of 5-HT2A receptor phosphorylation and desensitization. Finally, we showed that CaM decreases 5-HT2A receptor-mediated [35S]GTPgammaS binding to NIH-3T3 cell membranes, supporting a possible role for CaM in regulating receptor-G protein coupling. These data indicate that the serotonin 5-HT2A receptor contains two high affinity CaM-binding domains that may play important roles in signaling and function.     

Calmodulin interacts with the third intracellular loop of the serotonin 5-hydroxytryptamine1A receptor at two distinct sites: putative role in receptor phosphorylation by protein kinase C

The serotonin 5-HT(1A) receptor couples to heterotrimeric G proteins and intracellular second messengers, yet no studies have investigated the possible role of additional receptor-interacting proteins in 5-HT(1A) receptor signaling. We have found that the ubiquitous Ca(2+)-sensor calmodulin (CaM) co-immunoprecipitates with the 5-HT(1A) receptor in Chinese hamster ovary fibroblasts. The human 5-HT(1A) receptor contains two putative CaM binding motifs, located in the N- and C-terminal juxtamembrane regions of the third intracellular loop of the receptor. Peptides encompassing both the N-terminal (i3N) and C-terminal (i3C) CaM-binding domains were tested for CaM binding. Using in vitro binding assays in combination with gel shift analysis, we demonstrated Ca(2+)-dependent formation of complexes between CaM and both peptides. We determined kinetic data using a combination of BIAcore surface plasmon resonance (SPR) and dansyl-CaM fluorescence. SPR analysis gave an apparent K(D) of approximately 110 nm for the i3N peptide and approximately 700 nm for the i3C peptide. Both peptides also caused characteristic shifts in the fluorescence emission spectrum of dansyl-CaM, with apparent affinities of 87 +/- 23 nm and 1.70 +/- 0.16 microm. We used bioluminescence resonance energy transfer to show that CaM interacts with the 5-HT(1A) receptor in living cells, representing the first in vivo evidence of a G protein-coupled receptor interacting with CaM. Finally, we showed that CaM binding and phosphorylation of the 5-HT(1A) receptor i3 loop peptides by protein kinase C are antagonistic in vitro, suggesting a possible role for CaM in the regulation of 5-HT(1A) receptor phosphorylation and desensitization. These data suggest that the 5-HT(1A) receptor contains high and moderate affinity CaM binding regions that may play important roles in receptor signaling and function.