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A key function of reversible protein phosphorylation is to regulate protein-protein interactions, many of which involve short linear motifs (3-12 amino acids). Motif-based interactions are difficult to capture because of their often low-to-moderate affinities. Here, we describe phosphomimetic proteomic peptide-phage display, a powerful method for simultaneously finding motif-based interaction and pinpointing phosphorylation switches. We computationally designed an oligonucleotide library encoding human C-terminal peptides containing known or predicted Ser/Thr phosphosites and phosphomimetic variants thereof. We incorporated these oligonucleotides into a phage library and screened the PDZ (PSD-95/Dlg/ZO-1) domains of Scribble and DLG1 for interactions potentially enabled or disabled by ligand phosphorylation. We identified known and novel binders and characterized selected interactions through microscale thermophoresis, isothermal titration calorimetry, and NMR. We uncover site-specific phospho-regulation of PDZ domain interactions, provide a structural framework for how PDZ domains accomplish phosphopeptide binding, and discuss ligand phosphorylation as a switching mechanism of PDZ domain interactions. The approach is readily scalable and can be used to explore the potential phospho-regulation of motif-based interactions on a large scale. [ABSTRACT FROM AUTHOR]

The spliceosomal protein SF3b49, a component of the splicing factor 3b (SF3b) protein complex in the U2 small nuclear ribonucleoprotein, contains two RNA recognition motif (RRM) domains. In yeast, the first RRM domain (RRM1) of Hsh49 protein (yeast orthologue of human SF3b49) reportedly interacts with another component, Cus1 protein (orthologue of human SF3b145). Here, we solved the solution structure of the RRM1 of human SF3b49 and examined its mode of interaction with a fragment of human SF3b145 using NMR methods. Chemical shift mapping showed that the SF3b145 fragment spanning residues 598-631 interacts with SF3b49 RRM1, which adopts a canonical RRM fold with a topology of β1-α1-β2-β3-α2-β4. Furthermore, a docking model based on NOESY measurements suggests that residues 607-616 of the SF3b145 fragment adopt a helical structure that binds to RRM1 predominantly via α1, consequently exhibiting a helix-helix interaction in almost antiparallel. This mode of interaction was confirmed by a mutational analysis using GST pull-down assays. Comparison with structures of all RRM domains when complexed with a peptide found that this helix-helix interaction is unique to SF3b49 RRM1. Additionally, all amino acid residues involved in the interaction are well conserved among eukaryotes, suggesting evolutionary conservation of this interaction mode between SF3b49 RRM1 and SF3b145. [ABSTRACT FROM AUTHOR]

The inclusion of peptoid monomers into antimicrobial peptides (AMPs) increases their proteolytic resistance, but introduces conformational flexibility (reduced hydrogen bonding ability and cis/trans isomerism). We here use NMR spectroscopy to answer how the insertion of a peptoid monomer influences the structure of a regular α‐helical AMP upon interaction with a dodecyl phosphocholine (DPC) micelle. Insertion of [(2‐methylpropyl)amino]acetic acid in maculatin‐G15 shows that the structural change and conformational flexibility depends on the site of insertion. This is governed by the micelle interaction of the amphipathic helices flanking the peptoid monomer and the side chain properties of the peptoid and its preceding residue.A high‐resolution structure of a peptoid–peptide hybrid (“peptomer”) is presented. The peptoid residue breaks the helix and displays cis/trans isomerism. Maculatin‐Nleu11 displays only the trans isomer. Maculatin‐Nleu13 displays almost equimolar amounts of cis and trans conformers. Peptide amphipathicity and peptoid hydrophobicity govern the cis/trans isomerism. [ABSTRACT FROM AUTHOR]

Abstract: The WWE domain is often identified in proteins associated with ubiquitination or poly-ADP-ribosylation. Structural information about WWE domains has been obtained for the ubiquitination-related proteins, such as Deltex and RNF146, but not yet for the poly-ADP-ribose polymerases (PARPs). Here we determined the solution structures of the WWE domains from PARP11 and PARP14, and compared them with that of the RNF146 WWE domain. NMR perturbation experiments revealed the specific differences in their ADP-ribose recognition modes that correlated with their individual biological activities. The present structural information sheds light on the ADP-ribose recognition modes by the PARP WWE domains. [Copyright &y& Elsevier]

The TAR RNA-binding Protein (TRBP) is a double-stranded RNA (dsRNA)-binding protein, which binds to Dicer and is required for the RNA interference pathway. TRBP consists of three dsRNA-binding domains (dsRBDs). The first and second dsRBDs (dsRBD1 and dsRBD2, respectively) have affinities for dsRNA, whereas the third dsRBD (dsRBD3) binds to Dicer. In this study, we prepared the single domain fragments of human TRBP corresponding to dsRBD1 and dsRBD2 and solved the crystal structure of dsRBD1 and the solution structure of dsRBD2. The two structures contain an α−β−β−β−α fold, which is common to the dsRBDs. The overall structures of dsRBD1 and dsRBD2 are similar to each other, except for a slight shift of the first α helix. The residues involved in dsRNA binding are conserved. We examined the small interfering RNA (siRNA)-binding properties of these dsRBDs by isothermal titration colorimetry measurements. The dsRBD1 and dsRBD2 fragments both bound to siRNA, with dissociation constants of 220 and 113 n M, respectively. In contrast, the full-length TRBP and its fragment with dsRBD1 and dsRBD2 exhibited much smaller dissociation constants (0.24 and 0.25 n M, respectively), indicating that the tandem dsRBDs bind simultaneously to one siRNA molecule. On the other hand, the loop between the first α helix and the first β strand of dsRBD2, but not dsRBD1, has a Trp residue, which forms hydrophobic and cation-π interactions with the surrounding residues. A circular dichroism analysis revealed that the thermal stability of dsRBD2 is higher than that of dsRBD1 and depends on the Trp residue. | PDB Code(s): , [ABSTRACT FROM AUTHOR]

In eukaryotic replication licensing, Cdt1 plays a key role by recruiting the MCM2-7 complex onto the origin of chromosome. The C-terminal domain of mouse Cdt1 (mCdt1C), the most conserved region in Cdt1, is essential for licensing and directly interacts with the MCM2-7 complex. We have determined the structures of mCdt1CS (mCdt1C_small; residues 452 to 557) and mCdt1CL (mCdt1C_large; residues 420 to 557) using X-ray crystallography and solution NMR spectroscopy, respectively. While the N-terminal 31 residues of mCdt1CL form a flexible loop with a short helix near the middle, the rest of mCdt1C folds into a winged helix structure. Together with the middle domain of mouse Cdt1 (mCdt1M, residues 172-368), this study reveals that Cdt1 is formed with a tandem repeat of the winged helix domain. The winged helix fold is also conserved in other licensing factors including archaeal ORC and Cdc6, which supports an idea that these replication initiators may have evolved from a common ancestor. Based on the structure of mCdt1C, in conjunction with the biochemical analysis, we propose a binding site for the MCM complex within the mCdt1C. [ABSTRACT FROM AUTHOR]

Fn14 is the smallest member of the tumor necrosis factor (TNF) receptor superfamily, and specifically binds to its ligand, TWEAK (TNF-like weak inducer of apoptosis), which is a member of the TNF superfamily. The receptor-ligand recognition between Fn14 and TWEAK induces a variety of cellular processes for tissue remodeling and is also involved in the pathogenesis of some human diseases, such as cancer, chronic autoimmune diseases, and acute ischaemic stroke. The extracellular ligand-binding region of Fn14 is composed of 53 amino acid residues and forms a single, cysteine-rich domain (CRD). In this study, we determined the solution structure of the Fn14 CRD (Glu28-Ala70) by heteronuclear NMR, with a 13C-/15N-labeled sample. The tertiary structure of the CRD comprises a β-sheet with two strands, followed by a 310 helix and a C-terminal α-helix, and is stabilized by three disulfide bonds connecting Cys36-Cys49, Cys52-Cys67, and Cys55-Cys64. Comparison of the disulfide bond connectivities and the tertiary structures with those of other CRDs revealed that the Fn14 CRD is similar to the fourth CRD of TNF receptor 1 (A1-C2 module type), but not to the CRD of B-cell maturation antigen and the second CRD of transmembrane activator and CAML (calcium modulator and cyclophilin ligand) interactor (A1-D2 module type). This is the first structural report about the A1-C2 type CRD that could bind to the known target. [ABSTRACT FROM AUTHOR]

The muscleblind-like (MBNL) proteins 1, 2, and 3, which contain four CCCH zinc finger motifs (ZF1-4), are involved in the differentiation of muscle inclusion by controlling the splicing patterns of several pre-mRNAs. Especially, MBNL1 plays a crucial role in myotonic dystrophy. The CCCH zinc finger is a sequence motif found in many RNA binding proteins and is suggested to play an important role in the recognition of RNA molecules. Here, we solved the solution structures of both tandem zinc finger (TZF) motifs, TZF12 (comprising ZF1 and ZF2) and TZF34 (ZF3 and ZF4), in MBNL2 from Homo sapiens. In TZF12 of MBNL2, ZF1 and ZF2 adopt a similar fold, as reported previously for the CCCH-type zinc fingers in the TIS11d protein. The linker between ZF1 and ZF2 in MBNL2 forms an antiparallel β-sheet with the N-terminal extension of ZF1. Furthermore, ZF1 and ZF2 in MBNL2 interact with each other through hydrophobic interactions. Consequently, TZF12 forms a single, compact global fold, where ZF1 and ZF2 are approximately symmetrical about the C2 axis. The structure of the second tandem zinc finger (TZF34) in MBNL2 is similar to that of TZF12. This novel three-dimensional structure of the TZF domains in MBNL2 provides a basis for functional studies of the CCCH-type zinc finger motifs in the MBNL protein family. [ABSTRACT FROM AUTHOR]

The product of gene At3g16450.1 from Arabidopsis thaliana is a 32 kDa, 299-residue protein classified as resembling a myrosinase-binding protein (MyroBP). MyroBPs are found in plants as part of a complex with the glucosinolate-degrading enzyme myrosinase, and are suspected to play a role in myrosinase-dependent defense against pathogens. Many MyroBPs and MyroBP-related proteins are composed of repeated homologous sequences with unknown structure. We report here the three-dimensional structure of the At3g16450.1 protein from Arabidopsis, which consists of two tandem repeats. Because the size of the protein is larger than that amenable to high-throughput analysis by uniform 13C/15N labeling methods, we used stereo-array isotope labeling (SAIL) technology to prepare an optimally 2H/13C/15N-labeled sample. NMR data sets collected using the SAIL protein enabled us to assign 1H, 13C and 15N chemical shifts to 95.5% of all atoms, even at a low concentration (0.2 mm) of protein product. We collected additional NOESY data and determined the three-dimensional structure using thecyana software package. The structure, the first for a MyroBP family member, revealed that the At3g16450.1 protein consists of two independent but similar lectin-fold domains, each composed of three β-sheets. [ABSTRACT FROM AUTHOR]

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Abstract: The WW domain is known as one of the smallest protein modules with a triple-stranded β-sheet fold. Here, we present the solution structure of the second WW domain from the mouse salvador homolog 1 protein. This WW domain forms a homodimer with a β-clam-like motif, as evidenced by size exclusion chromatography, analytical ultracentrifugation and NMR spectroscopy. While typical WW domains are believed to function as monomeric modules that recognize proline-rich sequences, by using conserved aromatic and hydrophobic residues that are solvent-exposed on the surface of the β-sheet, this WW domain buries these residues in the dimer interface. [Copyright &y& Elsevier]

The structure of the C-terminal antifreeze-like (AFL) domain of human sialic acid synthase was determined by NMR spectroscopy. The structure comprises one α- and two single-turn 310-helices and two β-strands, and is similar to those of the type III antifreeze proteins. Evolutionary trace analyses of the type III antifreeze protein family suggested that the class-specific residues in the human and bacterial AFL domains are important for their substrate binding, while the class-specific residues of the fish antifreeze proteins are gathered on the ice-binding surface. [ABSTRACT FROM AUTHOR]

Among the many PWWP-containing proteins, the largest group of homologous proteins is related to hepatoma-derived growth factor (HDGF). Within a well-conserved region at the extreme N-terminus, HDGF and five HDGF-related proteins (HRPs) always have a PWWP domain, which is a module found in many chromatin-associated proteins. In this study, we determined the solution structure of the PWWP domain of HDGF-related protein-3 (HRP-3) by NMR spectroscopy. The structure consists of a five-stranded β-barrel with a PWWP-specific long loop connecting β2 and β3 (PR-loop), followed by a helical region including two α-helices. Its structure was found to have a characteristic solvent-exposed hydrophobic cavity, which is composed of an abundance of aromatic residues in the β1/β2 loop (β-β arch) and the β3/β4 loop. A similar ligand binding cavity occurs at the corresponding position in the Tudor, chromo, and MBT domains, which have structural and probable evolutionary relationships with PWWP domains. These findings suggest that the PWWP domains of the HDGF family bind to some component of chromatin via the cavity. [ABSTRACT FROM AUTHOR]

The three-dimensional structure of the rhodanese homology domain At4g01050(175-195) from Arabidopsis thaliana has been determined by solution nuclear magnetic resonance methods based on 3043 upper distance limits derived from NOE intensities measured in three-dimensional NOESY spectra. The structure shows a backbone root mean square deviation to the mean coordinates of 0.43 Å for the structured residues 7-125. The fold consists of a central parallel β-sheet with five strands in the order 1-5-4-2-3 and arranged in the conventional counterclockwise twist, and helices packing against each side of the β-sheet. Comparison with the sequences of other proteins with a rhodanese homology domain in Arabidopsis thaliana indicated residues that could play an important role in the scaffold of the rhodanese homology domain. Finally, a three-dimensional structure comparison of the present noncatalytic rhodanese homology domain with the noncatalytic rhodanese domains of sulfurtransferases from other organisms discloses differences in the length and conformation of loops that could throw light on the role of the noncatalytic rhodanese domain in sulfurtransferases. [ABSTRACT FROM AUTHOR]

The nuclear magnetic resonance structure of the unliganded pheromone-binding protein (PBP) from Bombyx mori at pH above 6.5, BmPBPB, consists of seven helices with residues 3–8, 16–22, 29–32, 46–59, 70–79, 84–100, and 107–124, and contains the three disulfide bridges 19–54, 50–108, and 97–117. This polypeptide fold encloses a large hydrophobic cavity, with a sufficient volume to accommodate the natural ligand bombykol. The polypeptide folds in free BmPBPB and in crystals of a BmPBP–bombykol complex are nearly identical, indicating that the B-form of BmPBP in solution represents the active conformation for ligand binding. [Copyright &y& Elsevier]

NMR studies of amyloid β-peptides (Aβ) in aqueous solution provide a novel way in which to characterize the apparent Alzheimer’s disease-related conformational polymorphism of Aβ. In the aqueous medium, neither of the polypeptides Aβ(1–40)ox or Aβ(1–42)ox (both of which contain a methionine sulfoxide at position 35) is folded into a globular structure, but they both deviate from random coil behavior by local conformational preferences of several short segments along the amino-acid sequence. Differences between the solution structures of Aβ(1–40)ox and Aβ(1–42)ox are indicated only by decreased flexibility of the region from about residue 32 to the C-terminus in Aβ(1–42)ox when compared to Aβ(1–40)ox. The lack of the observation of more extensive conformational differences between the two molecules is intriguing, considering that Aβ(1–42)ox in aqueous solution has much higher plaque-competence than Aβ(1–40)ox. [ABSTRACT FROM AUTHOR]

NMR studies of amyloid β-peptides (Aβ) in aqueous solution provide a novel way in which to characterize the apparent Alzheimer’s disease-related conformational polymorphism of Aβ. In the aqueous medium, neither of the polypeptides Aβ(1–40)ox or Aβ(1–42)ox (both of which contain a methionine sulfoxide at position 35) is folded into a globular structure, but they both deviate from random coil behavior by local conformational preferences of several short segments along the amino-acid sequence. Differences between the solution structures of Aβ(1–40)ox and Aβ(1–42)ox are indicated only by decreased flexibility of the region from about residue 32 to the C-terminus in Aβ(1–42)ox when compared to Aβ(1–40)ox. The lack of the observation of more extensive conformational differences between the two molecules is intriguing, considering that Aβ(1–42)ox in aqueous solution has much higher plaque-competence than Aβ(1–40)ox. [ABSTRACT FROM AUTHOR]