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The paper proposes a key frame extraction method for video copyright protection. The fast and robust method is based on frame difference with low level features, including color feature and structure feature. A two-stage method is used to extract accurate key frames to cover the content for the whole video sequence. Firstly, an alternative sequence is got based on color characteristic difference between adjacent frames from original sequence. Secondly, by analyzing structural characteristic difference between adjacent frames from the alternative sequence, the final key frame sequence is obtained. And then, an optimization step is added based on the number of final key frames in order to ensure the effectiveness of key frame extraction. Compared with the previous methods, the proposed method has advantage in computation complexity and robustness on several video formats, video resolution, and so on. [ABSTRACT FROM AUTHOR]

A multi-domain protein is able to exist as equilibrium of different conformations in solution, which may be critical to its biological function. Besides experimental techniques, computational methods like molecular dynamics (MD) simulations are suitable to study inter-domain motions of the protein and sample different conformational states. A MD simulation usually generates a trajectory containing large amount of protein structures, and a post-processing cluster analysis would be necessary to group similar structures into clusters and identify these typical conformations of the multi-domain protein. In this paper, the widely used k-means clustering algorithm is implemented in the protein essential dynamics (ED) subspace defined by principal component analysis on the MD trajectory. Cluster analysis of the formin binding protein 21 (FBP21) tandem WW domains demonstrate that the k-means clustering results by measuring distances between structures in the ED subspace are superior to those by using other metrics like pairwise inter-domain residue distances. The widely-used k-means clustering method is implemented in the essential dynamics subspace of a multi-domain protein. The structure ensemble generated by a molecular dynamics simulation is clustered into some typical conformational states of the protein, thus inter-domain motions and transitions can be investigated. [ABSTRACT FROM AUTHOR]

LIM homeodomain transcription factor 1‐alpha (LMX1a) is a neuronal lineage‐specific transcription activator that plays an essential role during the development of midbrain dopaminergic (mDA) neurons. LMX1a induces the expression of multiple key genes, which ultimately determine the morphology, physiology, and functional identity of mDA neurons. This function of LMX1a is dependent on its homeobox domain. Here, we determined the structures of the LMX1a homeobox domain in complex with the promoter sequences of the Wnt family member 1 (WNT1) or paired like homeodomain 3 (Pitx3) gene, respectively. The complex structures revealed that the LMX1a homeobox domain employed its α3 helix and an N‐terminal loop to achieve specific target recognition. The N‐terminal loop (loop1) interacted with the minor groove of the double‐stranded DNA (dsDNA), whereas the third α‐helix (α3) was tightly packed into the major groove of the dsDNA. Structure‐based mutations in the α3 helix of the homeobox domain significantly reduced the binding affinity of LMX1a to dsDNA. Moreover, we identified a nonsyndromic hearing loss (NSHL)‐related mutation, R199, which yielded a more flexible loop and disturbed the recognition in the minor groove of dsDNA, consistent with the molecular dynamics (MD) simulations. Furthermore, overexpression of Lmx1a promoted the differentiation of SH‐SY5Y cells and upregulated the transcription of WNT1 and PITX3 genes. Hence, our work provides a detailed elucidation of the specific recognition between the LMX1a homeobox domain and its specific dsDNA targets, which represents valuable information for future investigations of the functional pathways that are controlled by LMX1a during mDA neuron development. [ABSTRACT FROM AUTHOR]

We describe a method for efficient sampling of the energy landscape of a protein in atomic molecular dynamics simulations. A simulation is divided into alternatively occurring relaxation phases and excitation phases. In the relaxation phase (conventional simulation), we use a frequently updated reference structure and deviations from this reference structure to mark whether the system has been trapped in a local minimum. In that case, the simulation enters the excitation phase, during which a few slow collective modes of the system are coupled to a higher temperature bath. After the system has escaped from the minimum (also judged by deviations from the reference structure) the simulation reenters the relaxation phase. The collective modes are obtained from a coarse-grained Gaussian elastic network model. The scheme, which we call ACM-AME (amplified collective motion-assisted minimum escaping), is compared with conventional simulations as well as an alternative scheme that elevates the temperature of all degrees of freedom during the excitation phase (amplified overall motion-assisted minimum escaping, or AOM-AME). Comparison is made using simulations on four peptides starting from non-native extended or all helical structures. In terms of sampling low energy conformations and continuously sampling new conformations throughout a simulation, the ACM-AME scheme demonstrates very good performance while the AOM-AME scheme shows little improvement upon conventional simulations. Limited success is achieved in producing structures close to the native structures of the peptides: for an S-peptide analog, the ACM-AME approach is able to reproduce its native helical structure, and starting from an all-helical structure of the villin headpiece subdomain (HP-36) in implicit solvent, two out of three 150 ns ACM-AME runs are able to sample structures with 3–4 Å backbone root-mean-square deviations from the nuclear magnetic resonance structure of the protein. © 2003 American Institute of Physics. [ABSTRACT FROM AUTHOR]

ZBTB7A, a transcription factor containing a tandem array of four Cys2‐His2 zinc fingers (ZFs), is vital for multiple physiological events through directional binding to different genomic loci. Our previously determined crystal structure of ZBTB7A in complex with a GCCCCTTCCCC sequence revealed that all four ZFs (ZF1–4) are involved in binding to γ‐globin −200 gene element to repress fetal haemoglobin expression. Recently, it has been reported that ZBTB7A drives primed‐to‐naïve transition (PNT) of pluripotent stem cells through binding to a 12‐bp consensus sequence ([AAGGACCCAGAT], referred to as PNT‐associated sequence). Here, we report a crystal structure of ZBTB7A ZF1–3 in complex with the PNT‐associated sequence. The structure shows that ZF1 and ZF2 primarily contribute to recognizing the GACCC core sequence mimicking the half part (GCCCC) of γ‐globin −200 gene element via specific hydrogen bonding and van der Waals contacts. The mutations of key residues in ZF1–2 remarkably reduce their binding affinities for the PNT‐associated sequence in vitro and cannot restore epiblast stem cells to the naïve pluripotent state in vivo. Collectively, our studies demonstrate that ZBTB7A mainly employs its ZF1–2 to recognize the PNT‐associated sequence but recognizes γ‐globin −200 gene element via ZF1–4, providing insights into the molecular mechanism for the diversity of ZBTB7A's genomic localization. [ABSTRACT FROM AUTHOR]

RyhB is a key regulator of iron level in Escherichia coli (E. coli), which assists in conserving iron for life-sustaining cellular functions when cytoplasmic levels of the ferrous form of iron is limited. RyhB affects glucose metabolism. Seventy percent of the genes that are regulated by RyhB are related to metabolism. We demonstrated for the first time that the activity of the pentose phosphate pathway increased upon ryhB activation using a13C stable isotope-based technique called METAFoR (Metabolic flux ratio analysis). U-13C glucose-based studies showed that the reversible exchange activity of serine and glycine was enhanced by flux redistribution, which further favors NADPH formation. In addition, Entner–Doudoroff (ED) pathway activity was inhibited in the ryhB -defective cells. Quantitative physiology-based experiments highlighted a significant increase in the levels of reactive oxygen species (ROS) in ryhB -induced W3100 E. coli cells in batch culture. A simultaneous decrease in NADH/NAD+ and NADPH/NADP+ ratios outlined the potentially direct roles of NADH and NADPH in antagonizing the excess ROS formed after ryhB activation. Our observations offer a new perspective regarding the roles of RyhB and highlight that this small RNA can significantly affect cell metabolism in addition to its role as a regulator of gene expression. • Pentose phosphate pathway activity increased upon ryhB activation. • Reversible exchange of serine and glycine enhanced in ryhB induced expression cells. • Increased intracellular ROS production can be observed upon ryhB activation. • The ED pathway is less active in ryhB KO W3100 cells. • RyhB is not merely a genetic-level regulator, its function can also have profound impacts on cell metabolism. [ABSTRACT FROM AUTHOR]

With the rapid rising of living standard, people gradually developed higher shopping enthusiasm and increasing demand for garment. Nowadays, an increasing number of people pursue fashion. However, facing too many types of garment, consumers need to try them on repeatedly, which is somewhat time- and energy-consuming. Besides, it is difficult for merchants to master the real-time demand of consumers. Herein, there is not enough cohesiveness between consumer information and merchants. Thus, a novel fashion evaluation method on the basis of the appearance weak feature is proposed in this paper. First of all, image database is established and three aspects of appearance weak feature are put forward to characterize the fashion level. Furthermore, the appearance weak features are extracted according to the characters’ facial feature localization method. Last but not least, consumers’ fashion level can be classified through support vector product, and the classification is verified with the hierarchical analysis method. The experimental results show that consumers’ fashion level can be accurately described based on the indexes of appearance weak feature and the approach has higher application value for the clothing recommendation system. [ABSTRACT FROM AUTHOR]

Previous studies indicated that the P-body components, CGH-1 and EDC-3 may play a crucial role in the regulation of lifespan in Caenorhabditis elegans. Homo sapiens DDX6 or Saccharomyces cerevisiae Dhh1p (CGH-1 in C. elegans) could form complexes with EDC3 (Edc3p in yeast), respectively, which is significant for translation inhibition and mRNA decay. However, it is currently unclear how CGH-1 can be recognized by EDC-3 in C. elegans. Here, we provided structural and biochemical insights into the interaction between CGH-1 and EDC-3. Combined with homology modeling, mutation, and ITC assays, we uncovered an interface between CGH-1 RecA2 domain and EDC-3 FDF-FEK. Additionally, GST-pulldown and co-localization experiments confirmed the interaction between CGH-1 and EDC-3 in vitro and in vivo. We also analyzed PATR-1-binding interface on CGH-1 RecA2 by ITC assays. Moreover, we unveiled the similarity and differences of the binding mode between EDC-3 and CAR-1 or PATR-1. Taken together, these findings provide insights into the recognition of DEAD-box protein CGH-1 by EDC-3 FDF-FEK motif, suggesting important functional implications. [ABSTRACT FROM AUTHOR]

A protein-RNA complex containing the RNA helicase CGH-1 and a germline specific RNA-binding protein CAR-1 is involved in various aspects of function in C. elegans. However, the structural basis for the assembly of this protein complex remains unclear. Here, we elucidate the molecular basis of the recognition of CGH-1 by CAR-1. Additionally, we found that the ATPase activity of CGH-1 is stimulated by NTL-1a MIF4G domain in vitro. Furthermore, we determined the structures of the two RecA-like domains of CGH-1 by X-ray crystallography at resolutions of 1.85 and 2.40 Å, respectively. Structural and biochemical approaches revealed a bipartite interface between CGH-1 RecA2 and the FDF-TFG motif of CAR-1. NMR and structure-based mutations in CGH-1 RecA2 or CAR-1 attenuated or disrupted CGH-1 binding to CAR-1, assessed by ITC and GST-pulldown in vitro. These findings provide insights into a conserved mechanism in the recognition of CGH-1 by CAR-1. Together, our data provide the missing physical links in understanding the assembly and function of CGH-1 and CAR-1 in C. elegans. • The crystal structure at 1.85 Å of RNA helicase CGH-1 RecA1 domain. • The crystal structure at 2.40 Å of RNA helicase CGH-1 RecA2 domain. • The interface between CGH-1 RecA2 and CAR-1 identified by NMR methods. • RNA and NTL-1aMIF4G stimulate CGH-1 ATPase activity. [ABSTRACT FROM AUTHOR]

3′ uridylation is an essential modification associated with coding and noncoding RNA degradation in eukaryotes. In Arabidopsis , HESO1 was first identified as the major nucleotidyl transferase that uridylates most unmethylated miRNAs, and URT1 was later reported to play a redundant but important role in miRNA uridylation when HESO1 is absent. Two enzymes work sequentially and collaboratively to tail different forms of the same miRNAs in vivo. For mRNA, however, URT1 becomes the main enzyme to uridylate the majority of mRNA and repairs their deadenylated ends to restore the binding site for Poly(A) Binding Protein (PABP). HESO1, on the other hand, targets mostly the mRNAs with very short oligo(A) tails and fails in fulfilling the same task. To understand the structural basis these two functional homologues possess for their different substrate preferences and catalytic behaviors, we first determined the crystal structures of URT1 in the absence and presence of UTP. Our structures, together with functional assay and sequence analysis, indicated that URT1 has a conserved UTP-recognition mechanism analogue to the terminal uridylyl transferases from other species whereas HESO1 may evolve separately to recognize UTP in a different way. Moreover, URT1 N552 may be an important residue in interacting with 3′ nucleotide of RNA substrate. The URT1 structure we determined represents the first structure of uridylyl transferase from plants, shedding light on the mechanisms of URT1/HESO1-dependent RNA metabolism. • The URT1 structure we determined in this research represents the first structure of 3′ terminal uridylyl transferase of RNAs from plants. • Arabidopsis URT1 as a uridylyl transferase is structurally and functionally similar to Schizosaccharomyces pombe Cid1. • URT1 structure determination paves the path towards the illustration of the molecular mechanism of URT1/HESO1 dual TUTases in plants. [ABSTRACT FROM AUTHOR]

3′ downstream inhibitory stem plays a crucial role in locking rpoS mRNA 5' untranslated region in a self-inhibitory state. Here, we used optical tweezers to study the unfolding/refolding of rpoS inhibitory stem in the absence and presence of Mg2+. We found adding Mg2+ decreased the free energy of the RNA junction without re-arranging its secondary structure, through confirming that this RNA formed a canonical RNA three-way junction. We suspected increased free energy might change the relative orientation of different stems of rpoS and confirmed this by small angle X-ray scattering. Such changed conformation may improve Hfq-bridged annealing between sRNA and rpoS RNA inhibitory stem. We established a convenient route to analyze the changes of RNA conformation and folding dynamics by combining optical tweezers with X-ray scattering methods. This route can be easily applied in the studies of other RNA structure and ligand-RNA. [ABSTRACT FROM AUTHOR]

The origin recognition complex (ORC) is indispensable for the initiation of DNA replication during the cell cycle. The DNA‐binding modes of the human ORC winged helix domain (WHD) remain enigmatic, as the dsDNA recognition sites of archaeal and Saccharomyces cerevisiae ORC WHDs are distinct. Here, we solved the high‐resolution crystal structure of the human ORC2 WHD, although its complex with dsDNA is difficult to crystallize due to its weak binding affinities. The near‐complete NMR backbone assignments and chemical shift perturbations reveal a new dsDNA binding topology in addition to the conserved β‐sheet hairpin region, in which residues show higher dynamics. The key interacting residues (R540, K548, and K549) were validated by mutagenesis studies. Our data suggest that the ORC2 WHD recognizes dsDNA sequences through its flexible β‐sheet hairpin as an anchor point, while the rest of the protein adopts various orientations in different species. This weak but real interaction module identified by NMR is useful for the structural reconstruction of large biomolecular complexes using cryo‐EM. The binding topology and dynamics of ORC2 WHDs were also underpinned by molecular dynamics simulations. [ABSTRACT FROM AUTHOR]

Methyltransferase RlmCD was previously shown to be responsible for the introduction of C5 methylation at both U747 and U1939 of the 23S ribosomal RNA in Streptococcus pneumoniae. Intriguingly, its structural homologue, RumA, can only catalyze the methylation of U1939, while RlmC is the dedicated enzyme for m5U747 in Escherichia coli. In this study, we describe the structure of RlmCD in complex with its cofactor and the RNA substrate containing U747 at 2.00 Å or U1939 at 3.10 Å. We demonstrate that multiple structural features collaborate to establish the dual enzymatic activities of RlmCD. Of them, the side-chain rearrangement of F145 was observed to be an unusual mechanism through which RlmCD can discriminate between U747- and U1939-containing RNA substrate by switching the intermolecular aromatic stacking between protein and RNA on/off. An in-vitro methyltransferase assay and electrophoretic mobility shift assay were performed to validate these findings. Overall, our complex structures allow for a better understanding of the dual-functional mechanism of RlmCD, suggesting useful implications for the evolution of the RumA-type enzyme and the potential development of antibiotic drugs against S. pneumoniae. [ABSTRACT FROM AUTHOR]

The FHA domain‐containing protein Mek1 is a meiosis‐specific kinase that is involved in the regulation of interhomolog recombination in meiosis in Saccharomyces cerevisiae. The recruitment and activation of Mek1 require the phosphorylation of the chromosome axis protein Hop1 at Thr318 (pT318), which is necessary for recognition by the Mek1 FHA domain. Here, crystal structures of the Mek1 FHA domain in the apo state and in complex with the Hop1 pT318 peptide are presented, demonstrating that the hydrophobic residues Phe320 and Val321 at the pT+2 and pT+3 positions in the ligand contribute to the preferential recognition. It was further found that in Schizosaccharomyces pombe Mek1 FHA binds both pT15 in its N‐terminal SQ/TQ cluster domain (SCD) and pT270 in the Hop1 SCD. The results revealed the structural basis for the preferential recognition of phosphorylated Hop1 by Mek1 in S. cerevisiae and facilitate the understanding of the interaction between the S. pombe Mek1 FHA domain and its binding targets. [ABSTRACT FROM AUTHOR]

RlmCD has recently been identified as the S-adenosyl methionine (SAM)-dependent methyltransferase responsible for the formation of m5U at U747 and U1939 of 23S ribosomal RNA in Streptococcus pneumoniae. In this research, we determine the high-resolution crystal structures of apo-form RlmCD and its complex with SAH. Using an in-vitro methyltransferase assay, we reveal the crucial residues for its catalytic functions. Furthermore, structural comparison between RlmCD and its structural homologue RumA, which only catalyzes the m5U1939 in Escherichia coli, implicates that a unique long linker in the central domain of RlmCD is the key factor in determining its substrate selectivity. Its significance in the enzyme activity of RlmCD is further confirmed by in-vitro methyltransferase assay. [ABSTRACT FROM AUTHOR]

Mouse l -threonine dehydrogenase (mTDH), which belongs to the short-chain dehydrogenase/reductase (SDR) superfamily and mediates threonine catabolism, plays pivotal roles in both powerful biosynthesis and signaling in mouse stem cells and has a regulatory residue Arg180. Here we determined three crystal structures of mTDH: wild-type (WT) in the apo form; in complex with NAD + and a substrate analog, glycerol, or with only NAD + ; as well as the R180K variant with NAD + . This is the first description of a structure for mammalian SDR-type TDH. Structural comparison revealed the structural basis for SDR-type TDH catalysis remains strictly conserved in bacteria and mammals. Kinetic enzyme assays, and isothermal titration calorimetry (ITC) measurements indicated the R180K mutation has little effect on NAD + binding affinity, whereas affects the substrate’s affinity for the enzyme. The crystal structure of R180K with NAD + , biochemical and spectroscopic studies suggested that the R180K mutant should bind NAD + in a similar way and have a similar folding to the WT. However, the R180K variant may have difficulty adopting the closed form due to reduced interaction of residue 180 with a loop which connects a key position for mTDH switching between the closed and open forms in mTDH catalysis, and thereby exhibited a significantly decreased k cat / K m value toward the substrate, l -Thr. In sum, our results suggest that activity of GalE-like TDH can be regulated by remote interaction, such as hydrogen bonding and hydrophobic interaction around the Arg180 of mTDH. [ABSTRACT FROM AUTHOR]

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is a key player in regulating the process of excytosis, including insulin secretion. Granuphilin, a tandem C2 domain containing protein, mediates the docking of insulin granules onto plasma membrane. The C2A domain plays key roles in this process through interaction with PI(4,5)P2. In this study, we have investigated the molecular recognition mechanism of granuphilin-C2A domain to PI(4,5)P2 head group, and further to PI(4,5)P2-nanodisc by NMR, ITC, MST and SEC methods. Our results demonstrate that PI(4,5)P2 binds to the concave surface of granuphilin-C2A domain. The key residues involved in the binding were validated by mutation analysis. [ABSTRACT FROM AUTHOR]

Homo sapiens ECT2 is a cell cycle regulator that plays critical roles in cytokinesis. ECT2 activity is restrained during interphase via intra‐molecular interactions that involve its N‐terminal triple‐BRCT‐domain and its C‐terminal DH–PH domain. At anaphase, this self‐inhibitory mechanism is relieved by Plk1‐phosphorylated CYK‐4, which directly engages the ECT2 BRCT domain. To provide a structural perspective for this auto‐inhibitory property, we solved the crystal structure of the ECT2 triple‐BRCT‐domain. In addition, we systematically analyzed the interaction between the ECT2 BRCT domains with phospho‐peptides derived from its binding partner CYK‐4, and have identified Ser164 as the major phospho‐residue that links CYK‐4 to the second ECT2 BRCT domain.We solved the crystal structure of triple‐BRCT‐domain of ECT2. We show that phosphorylation of Ser164 in CYK‐4 is crucial for the interaction with ECT2. A canonical binding pocket in the second BRCT domain of ECT2 is proved to bind to pS164 in CYK‐4. The interactions between ECT2 and CYK‐4 can relieve the auto‐inhibition of ECT2. [ABSTRACT FROM AUTHOR]

Summary: Trypanosoma brucei protein arginine methyltransferase 7 (TbPRMT7) exclusively generates monomethylarginine (MMA), which directs biological consequences distinct from that of symmetric dimethylarginine (SDMA) and asymmetric dimethylarginine (ADMA). However, determinants controlling the strict monomethylation activity are unknown. We present the crystal structure of the TbPRMT7 active core in complex with S-adenosyl-L-homocysteine (AdoHcy) and a histone H4 peptide substrate. In the active site, residues E172, E181, and Q329 hydrogen bond the guanidino group of the target arginine and align the terminal guanidino nitrogen in a position suitable for nucleophilic attack on the methyl group of S-adenosyl-L-methionine (AdoMet). Structural comparisons and isothermal titration calorimetry data suggest that the TbPRMT7 active site is narrower than those of protein arginine dimethyltransferases, making it unsuitable to bind MMA in a manner that would support a second turnover, thus abolishing the production of SDMA and ADMA. Our results present the structural interpretations for the monomethylation activity of TbPRMT7. [ABSTRACT FROM AUTHOR]

Arginine methylation plays vital roles in the cellular functions of the protozoan Trypanosoma brucei. The T. brucei arginine methyltransferase 6 (TbPRMT6) is a type I arginine methyltransferase homologous to human PRMT6. In this study, we report the crystal structures of apo-TbPRMT6 and its complex with the reaction product S-adenosyl-homocysteine (SAH). The structure of apo-TbPRMT6 displays several features that are different from those of type I PRMTs that were structurally characterized previously, including four stretches of insertion, the absence of strand β15, and a distinct dimerization arm. The comparison of the apo-TbPRMT6 and SAH-TbPRMT6 structures revealed the fine rearrangements in the active site upon SAH binding. The isothermal titration calorimetry results demonstrated that SAH binding greatly increases the affinity of TbPRMT6 to a substrate peptide derived from bovine histone H4. The western blotting and mass spectrometry results revealed that TbPRMT6 methylates bovine histone H4 tail at arginine 3 but cannot methylate several T. brucei histone tails. In summary, our results highlight the structural differences between TbPRMT6 and other type I PRMTs and reveal that the active site rearrangement upon SAH binding is important for the substrate binding of TbPRMT6. [ABSTRACT FROM AUTHOR]

Abstract: Plant homeodomain (PHD) finger is found to be a versatile reader that functions in recruiting transcription factors and chromatin modification complexes. Bromodomain- and PHD finger-containing (BRPF) proteins are identified as scaffold component in a couple of histone acetyltransferase (HATs) complexes but the biological function of PHD fingers, composing the motif called PZPM (PHD/Zn-knuckle/PHD Motif), in BRPF proteins is far from being well understood. Here we report the three-dimensional solution structure of the second PHD finger of PZPM in human BRPF2. According to the structure, BRPF2 PHD2 possesses a two-strand β sheet which is different from any other PHD fingers. Functionally, this PHD finger can potentially bind DNA non-specifically with an evolutionarily conserved and positively charged surface. We provide the structural and interaction information of this atypical PHD finger and categorize this BRPF2 PHD2 into a new subset of PHD finger. Moreover our work also shed light on the functional aspect of the PZPM. [Copyright &y& Elsevier]

Abstract: Vav2 is a ubiquitous guanine nucleotide exchange factor (GEF) for the small GTPase Rac1. It regulates processes including cell migration, neuronal development and phagocytosis through interactions with different proteins. In this study, Arap3, a dual GTPase-activating protein (GAP) for RhoA and Arf6, was first identified to be a novel interaction partner for Vav2 both in vitro and in vivo. ITC and NMR chemical shift perturbation experiments demonstrated that Vav2 SH2 domain was able to interact directly with phosphorylated Y1403 and Y1408 within the C-terminal region of Arap3 with high affinities, with the dissociation constants (Kd) of ∼0.27 and ∼1.40μM, respectively. In addition, using different phosphotyrosine peptides, the pY +3 specificity of Vav2 SH2 domain was discovered. The solution structures of Vav2 SH2 domain in free and in complex with the phosphotyrosine peptide pY1408 were therefore determined to understand the structural basis of this recognition specificity. Structural analysis revealed that the presence of a Phe residue in the BG loop (BG6) leads to the formation of a shallow hydrophobic pY +3 pocket on the surface of Vav2 SH2 domain, which determines the pY +3 specificity of Vav2 SH2 domain. [Copyright &y& Elsevier]

Abstract: MCPH1 is especially important for linking chromatin remodeling to DNA damage response. It contains three BRCT (BRCA1-carboxyl terminal) domains. The N-terminal region directly binds with chromatin remodeling complex SWI–SNF, and the C-terminal BRCT2–BRCT3 domains (tandem BRCT domains) are involved in cellular DNA damage response. The MCPH1 gene associates with evolution of brain size, and its variation can cause primary microcephaly. In this study we solve the crystal structures of MCPH1 natural variant (A761) C-terminal tandem BRCT domains alone as well as in complex with γH2AX tail. Compared with other structures of tandem BRCT domains, the most significant differences lie in phosphopeptide binding pocket. Additionally, fluorescence polarization assays demonstrate that MCPH1 tandem BRCT domains show a binding selectivity on pSer +3 and prefer to bind phosphopeptide with free COOH-terminus. Taken together, our research provides new structural insights into BRCT-phosphopeptide recognition mechanism. [Copyright &y& Elsevier]

Abstract: Human Pax2 transactivation domain-interacting protein (hPTIP), containing six BRCT domains, is an essential protein required for the IR induced DDR process with an unclear role. Here we report that the tandem BRCT5–BRCT6 domain of hPTIP recognizes the γH2AX tail, and this interaction depends on the phosphorylation of H2AX Ser139 and binding with the carboxyl ending peptide to the aminoacyl ending peptide. The 2.15Å crystal structure of hPTIP BRCT5/6–γH2AX complex and mutation analysis provide molecular evidence for direct interactions between PTIP and γH2AX. This interaction proffers a new clue to identify the role of PTIP in DDR pathways. Structured summary of protein interactions: PTIP and gamma H2AX bind by fluorescence polarization spectroscopy (View Interaction: 1, 2, 3, 4, 5, 6). PTIP and gamma H2AX bind by X-ray crystallography (View interaction). [Copyright &y& Elsevier]

Abstract: Prp20p is the homolog of mammalian RCC1 (regulator of chromosome condensation 1) in Saccharomyces cerevisiae, which acts as the guanine nucleotide exchange factor (GEF) for Gsp1p (yeast Ran). Prp20p plays multiple roles in mRNA metabolism, nucleocytoplasmic transport and mitosis regulation. Prp20p also functions as a linker between chromatin and nuclear pore complex (NPC) which regulates the NPC-mediated boundary activity (BA). Prp20p contains an N-terminal nuclear localization signal (NLS) and a typical RCC1-like domain (RLD). Here we present the 1.9Å crystal structure of the RCC1-like domain of Prp20p, which exhibits a classical seven-bladed β-propeller. We also proved that the additional β-wedge in Prp20p is essential for the interaction between Prp20p and Gsp1p. Based on this structure, we built a complex model of Prp20p and Gsp1p which was optimized by molecular dynamics (MD) simulations. Our model reveals that Prp20p and RCC1 share similar Ran GTPase binding mode. In addition, we also studied the histone-binding property of Prp20p in vitro. [Copyright &y& Elsevier]

Abstract: Zinc is an important component of many proteins that play key roles in transcription, translation, and catalysis. Kti11p, DESR1, both belonging to a protein family characterized by a CSL zinc finger domain, and the co-catalytic zinc–protein PML containing a Zn2+ binding domain called RING or C3HC4 finger are all structurally determined by NMR although the zinc sites are silent to this spectroscopical method. The comparison of X-ray absorption near-edge spectroscopy (XANES) data for the three proteins demonstrates that fingerprints effect is a reliable method for a primary characterization of ligand species. Ab initio full MS calculations performed by MXAN are applied to obtain chemical and stereo structural information around the Zn ion in Kti11p. For the first time this high-spatial resolution technique confirms the formation of a stable Zn tetrahedral configuration with four sulfur ligands, and returns extremely accurate bond angle information between ligands. [Copyright &y& Elsevier]

Abstract: The solution structure of human MICAL-1 calpolnin homology (CH) domain is composed of six α helices and one 310 helix. To study the unfolding of this domain, we carry out native-state hydrogen exchange, intrinsic fluorescence and far-UV circular dichroism experiments. The free energy of unfolding, ΔG H2O, is calculated to be 7.11±0.58 kcal mol−1 from GuHCl denaturation at pH 6.5. Four cooperative unfolding units are found using native-state hydrogen exchange experiment. Forty-seven slow-exchange residues can be studied by native-state hydrogen exchange experiments. From the concentration dependence of exchange rates, free energy of amide hydrogen with solvent, ΔG HX and m-value (sensitivity of exposure to denaturant) are obtained, which reveal four cooperative unfolding units. The slowest exchanging protons are distributed throughout the whole hydrophobic core of the protein, which might be the folding core. These results will help us understand the structure of MICAL-1 CH domain more deeply. [Copyright &y& Elsevier]

Abstract: BRD7 is an important protein tightly associated with Nasopharyngeal carcinoma (NPC). Overexpression of BRD7 inhibits NPC cell growth and cell cycle by transcriptionally regulating the cell cycle related genes. BRD7 contains a bromodomain that is found in many chromatin-associated proteins and in nearly all known nuclear histone acetyltransferases (HATs) and plays an important role in chromatin remodeling and transcriptional activation. Here, we report the solution structure of BRD7 bromodomain determined by NMR spectroscopy, and its binding specificity revealed by NMR titration with several acetylated histone peptides. We find that BRD7 bromodomain contains the typical left-handed four-helix bundle topology, and can bind with weak affinity to lysine-acetylated peptides derived from histone H3 with K9 or K14 acetylated and from histone H4 with K8, K12 or K16 acetylated. Our results show that BRD7 bromodomain lacks inherent binding specificity when binding to histones in vitro. [Copyright &y& Elsevier]

Abstract: Solution structure of the first Src homology (SH) 3 domain of human vinexin (V_SH3_1) was determined using nuclear magnetic resonance (NMR) method and revealed that it was a canonical SH3 domain, which has a typical β–β–β–β–α–β fold. Using chemical shift perturbation and surface plasmon resonance experiments, we studied the binding properties of the SH3 domain with two different peptides from vinculin hinge regions: P856 and P868. The observations illustrated slightly different affinities of the two peptides binding to V_SH3_1. The interaction between P868 and V_SH3_1 belonged to intermediate exchange with a modest binding affinity, while the interaction between P856 and V_SH3_1 had a low binding affinity. The structure and ligand-binding interface of V_SH3_1 provide a structural basis for the further functional study of this important molecule. [Copyright &y& Elsevier]

Abstract: CMS, cas ligand with multiple Src homology 3 (SH3) domains, belongs to a family of ubiquitously expressed adaptor proteins. Among the CMS binding proteins, c-Cbl has been mostly extensively studied. It was reported that the motif PKPFPR (residues 824–829) of c-Cbl can bind to the N-terminus SH3 domains of CMS. Here we report the solution structure of the second SH3 domain of CMS (CMS_SH3_B), furthermore, we have identified that a peptide from residues 701 to 714 of c-Cbl (Cbl-p), i.e. MTPSSRPLRPLDTS, can specially bind to CMS_SH3_B using NMR chemical shift perturbation, suggesting that the peptide is a new potential CMS binding site. Among the peptide, TPSSRPLR is the core binding motif and Arg709 plays a key role in the interaction. Cbl-p binding interface on CMS_SH3_B along a hydrophobic channel is composed of RT loop, n-Src loop and β4 strand and divided into three pockets. This work indicates the solution structure of CMS_SH3_B bears the canonical β–β–β–β–α–β fold and a new binding site in c-Cbl involved in its interaction with CMS, which probably contributes to the clustering of CMS. All the information provided here should be beneficial for the future functional study of CMS. [Copyright &y& Elsevier]

Abstract: Human coactosin-like protein (CLP) is a small (MW ∼17 kDa) evolutionarily conserved actin-binding protein. It can bind to actin filaments but not globular actin and belongs to the fourth class of ADF-H-domain-containing proteins. Human CLP can also bind to 5LO, which plays an important role in cellular leukotriene synthesis. Although the structure of hCLP has been determined by both NMR and X-ray experiments, how hCLP binds to the actin filament is still a controversial question. To obtain insights into the structure of the complex, we studied the three-dimensional structure and backbone dynamics of hCLP using multidimensional NMR spectroscopy. Guided by the solution structure of the protein, a series of site-directed mutants were generated and their F-actin-binding activities were measured by high-speed cosedimentation assays. Furthermore, the structure model of the hCLP-F-actin complex was proposed using computational docking with the docking results filtered by the mutation data. Several previously untested residues (including T66, L89, R91, K102, D116 and E119) in hCLP were found important for the F-actin-binding activity. The extended region of β4–β5 of hCLP (residue 66–75) was found very flexible and very important for F-actin binding. The C-terminal residues of hCLP were not involved in F-actin binding, which was different from UNC-60B. Based on our hCLP-F-actin-binding model, different affinities of the four classes of ADF-H domain containing proteins for F-actin were explained. [Copyright &y& Elsevier]

Abstract: The mutations in the gene encoding copper–zinc superoxide dismutase (SOD1) cause approximately 20% cases of familial amyotrophic lateral sclerosis (FALS), characterized by selective loss of motor neurons. Mutant SOD1 forms inclusions in tissues from FALS patients. However, the precise mechanism of the accumulation of mutant SOD1 remains unclear. Here we show that human SOD1 is a substrate modified by SUMO-1. A conversion of lysine 75 to an arginine within a SUMO consensus sequence in SOD1 completely abolishes SOD1 sumoylation. We further show that SUMO-1 modification, on both wild-type and mutant SOD1, increases SOD1 steady state level and aggregation. Moreover, SUMO-1 co-localizes onto the aggregates formed by SOD1. These findings imply that SUMO-1 modification on lysine 75 may participate in regulating SOD1 stability and its aggregation process. Thus, our results suggest that sumoylation of SOD1 may be involved in the pathogenesis of FALS associated with mutant SOD1. [Copyright &y& Elsevier]

Abstract: SH3 domain binding glutamic acid-rich protein like 3 (SH3BGRL3) is the new member of thioredoxin (TRX) super family, whose posttranslational modified form was identified as tumor necrosis factor α (TNF-α) inhibitory protein, TIP-B1. In this paper, we determined its solution structure by multi-dimensional nuclear magnetic resonance spectroscopy. The overall structure of human SH3BGRL3 conformed to a TRX-like fold. To understand its function in vivo, the upregulated expression in acute promyelocytic leukemia cell line NB4 at both mRNA and protein level was elucidated. Immunofluorescence and immunohistochemistry staining with monoclonal antibody against SH3BGRL3 demonstrated that it was a cytoplasmic protein in both NB4 cell and human tissues. These results, as a whole, indicate that SH3BGRL3 may function as a regulator in all-trans retinoic acid-induced pathway. [Copyright &y& Elsevier]

Abstract: Using far and near-UV CD, ANS fluorescence and 2D NMR spectroscopy, an acid-induced partly folded state (A state) at extremely low pH for hUBF HMG Box1 was identified and characterized. As compared to the native state (N), the A state has similar secondary structure, less compact pack with larger amounts of exposed hydrophobic surface, and narrower chemical shift dispersion in 1H–15N HSQC spectrum, which implies that it is a molten globule (MG)-like species. On the other hand, substantial tertiary contacts and cooperative thermal denaturing transition indicate that the A state is closer–relative to the classic MG–to the native folded state. In addition, when the solution pH is adjusted to neutrality, the protein in the A state refolds to the native state easily. All these data suggest that the A state of hUBF HMG Box1 could represent a potential folding intermediate on protein folding pathway. [Copyright &y& Elsevier]

Small ubiquitin-related modifier SUMO-3 is a member of a growing family of ubiquitin-like proteins (Ubls). So far, four isoforms of SUMO have been identified in humans. It is generally known that SUMO modification regulates protein localization and activity. Previous structure and function studies have been mainly focused on SUMO-1. The sequence of SUMO-3 is 46% identical with that of SUMO- 1; nevertheless, functional heterogeneity has been found between the two homologues. Here we report the solution structure of SUMO-3 C47S (residues 14-92) featuring the β-β-α-β-β-α-β ubiquitin fold. Structural comparison shows that SUMO-3 C47S resembles ubiquitin more than SUMO-1. On the helix- sheet interface, a strong hydrophobic interaction contributes to formation of the globular and compact fold. A Gly-Gly motif at the C-terminal tail, extending away from the core structure, is accessible to enzymes and substrates. In vivo, SUMO modification proceeds via a multistep pathway, and Ubc9 plays an indispensable role as the SUMO conjugating enzyme (E2) in this process. To develop a better understanding of SUMO-3 conjugation, the Ubc9 binding surface on SUMO-3 C47S has been detected by chemical shift perturbation using NMR spectroscopy. The binding site mainly resides on the hydrophilic side of the β-sheet. Negatively charged and hydrophobic residues of this region are highly or moderately conserved among SUMO family members. Notably, the negatively charged surface of SUMO-3 C47S is highly complementary in its electrostatic potentials and hydrophobicity to the positively charged surface of Ubc9. This work indicates dissimilarities between SUMO-3 and SUMO-1 in tertiary structure and provides insight into the specific interactions of SUMO-3 with its modifying enzyme. [ABSTRACT FROM AUTHOR]

Abstract: Scorpion venom is a rich source of bioactive peptides. From the venom of Chinese scorpion Buthus martensi Karsch (BmK), a novel short chain peptide BmKX of 31-amino acid residues was purified, and its amino acid sequence and gene structure were determined. The gene of BmKX was composed of two exons interrupted by an 86-bp intron at the codon-7 upstream of the mature peptide. Although its gene structure is similar to those of other known scorpion toxins, its amino acid sequence, especially the cysteine framework, is different from those of all other known subfamilies of short-chain scorpion toxins. The solution structure of BmKX, determined with two-dimensional NMR spectroscopy, shows that BmKX also forms a typical cysteine-stabilized α/β scaffold adopted by most short-chain scorpion toxins, consisting of a short 310-helix and a two-stranded antiparallel β-sheet, and the short N-terminal segment forms a pseudo-strand of the β-sheet. However, the orientation between the helix and the β-sheet is significantly different from the others, which might be the reason for its unique but still unclear physiological function. [Copyright &y& Elsevier]

Four 10-ns molecular dynamics (MD) simulations of the human prion protein domain (HuPrP 125–228) in explicit water solution have been performed. Each of the simulations mimicked a different environment of the protein: the neutral pH environment was simulated with all histidine residues neutral and bearing a ND proton and with other titratable side chains charged, the weakly acidic environment was simulated with all titratable side chains charged, the strongly acidic environment was simulated with all titratable side chains protonated. The protein in neutral pH environment was simulated at both ambient (298 K) and higher (350 K) temperatures. The native fold is stable in the neutral pH/ambient temperature simulation. Through out all other simulations, a quite stable core consisted of 10–20 residues around the disulfide bond retain their initial conformations. However, the secondary structures of the protein show changes of various degrees compared to the native fold, parts of the helices unfolded and the β-sheets extended. Our simulations indicated that the heat-induced unfolding and acid-induced unfolding of HuPrP might follow different pathways: the initial stage of the acid-induced unfolding may include not only changes in secondary structures, but also changes in the tertiary structures. Under the strongly acidic condition, obvious tertiary structure changes take place after 10-ns simulation, the secondary structure elements and the loops becoming more parallel to each other, resulting in a compact state, which was stabilized by a large number of new, non-native side chain-side chain contacts. Such tertiary structure changes were not observed in the higher temperature simulation, and intuitively, they may favor the further extension of the β-sheets and eventually the agglomeration of multiple protein molecules. The driving forces for this tertiary structure changes are discussed. Two additional 10-ns MD simulations, one with Asp202 protonated and the other with Glu196 protonated compared to the neutral pH simulation, were carried out. The results showed that the stability of the native fold is very subtle and can be strongly disturbed by eliminating a single negative charge at one of such key sites. Correlations of our results with previous experimental and theoretical studies are discussed. [Copyright &y& Elsevier]

Human upstream binding factor (hUBF) belonging to a family of protein containing DNA binding domain-HMG box, is important in the activation of rRNA gene transcription. It contains six tandemly arranged HMG box domains, each of which is thought to be as a basic architectural unit in the interaction of DNA and protein. Here the DNA binding domain of hUBF HMG box 1 was cloned and heterologously expressed in Escherichia coli. Through a single purification step using a Ni2+-chelating column, the highly purified recombinant protein could be obtained. This recombinant protein contains 99 amino acids with a hexahistidine tag added to the C-terminus. It was expressed as a monomer, which was determined by gel filtration. Circular dischroism studies show that it comprises approximately 54.3% α-helix and 43.6% random coil at pH 7. This result is in good agreement with that of FTIR, which are 59.9% α-helix and 40.1% random coil. There is no obvious change for the secondary structure of the recombinant protein as increasing pH from 5.0 to 12.0. But denaturation occurs at pH 3.0. Like many HMG box domains that were found in other proteins, it could bind to four-way DNA junction, a putative intermediate in DNA recombination, in a structure-specific manner. Magnesium ion has no effect on this binding activity, which is determined by both gel mobility shift assays and surface plasmon resonance (SPR). Since Mg2+ is present in the nucleus and RNA polymerase I is Mg2+-stimulated, we believe that this property is relevant for hUBF in vivo. SPR research shows that the recombinant hUBF HMG box 1 also has a strong binding ability to a GC-rich fragment within the rRNA gene core promoter. [Copyright &y& Elsevier]

In eukaryotes, nucleosome assembly is crucial for genome integrity. The histone chaperone NAP1 plays an important role in histone folding, storage, and transport, as well as histone exchange and nucleosome assembly. At present, the molecular basis of these activities is not fully understood. We have solved high-resolution crystal structures of Caenorhabditis elegans NAP1 (ceNAP1) in complex with its cognate substrates: the C. elegans H2A-H2B dimer (ceH2A-H2B) and the H2A.Z-H2B dimer (ceH2A.Z-H2B). Our structural and biochemical data reveals the acidic concave surface is relevant to tetramerization, and uncovers how a ceNAP1 homodimer uses its concave surface to asymmetrically recognize a ceH2A-H2B or ceH2A.Z-H2B heterodimer. Intriguingly, an "acidic strip" within the concave surface of ceNAP1 is crucial for binding histones, including H2A-H2B, H3-H4, and histone variants. Thus, our results provide insight into the molecular mechanisms of NAP1 histone chaperone activity. • High-resolution crystal structure of ceNAP1-H2A-H2B • ceNAP1-H2A-H2B mode is relevant to nucleosome assembly • Molecular basis of NAP1's broad recognition of histone folding • Acidic concave surface of ceNAP1 is important for oligomerization Liu et al. describe crystal structures of ceNAP1 in complex with its cognate substrates: H2A-H2B and H2A.Z-H2B. The structural and biochemical studies provide insight into a mechanism by which NAP1 functions as a histone chaperone in nucleosome assembly. [ABSTRACT FROM AUTHOR]

The TDP-43 is originally a nuclear protein but translocates to the cytoplasm in the pathological condition. TDP-43, as an RNA-binding protein, consists of two RNA Recognition Motifs (RRM1 and RRM2). RRMs are known to involve both protein-nucleotide and protein-protein interactions and mediate the formation of stress granules. Thus, they assist the entire TDP-43 protein with participating in neurodegenerative and cancer diseases. Consequently, they are potential therapeutic targets. Protein-observed and ligand-observed nuclear magnetic resonance (NMR) spectroscopy were used to uncover the small molecule inhibitors against the tandem RRM of TDP-43. We identified three hits weakly binding the tandem RRMs using the ligand-observed NMR fragment-based screening. The binding topology of these hits is then depicted by chemical shift perturbations (CSP) of the 15N-labeled tandem RRM and RRM2, respectively, and modeled by the CSP-guided High Ambiguity Driven biomolecular DOCKing (HADDOCK). These hits mainly bind to the RRM2 domain, which suggests the druggability of the RRM2 domain of TDP-43. These hits also facilitate further studies regarding the hit-to-lead evolution against the TDP-43 RRM domain. [ABSTRACT FROM AUTHOR]

Summary CsdA has been proposed to be essential for the biogenesis of ribosome and gene regulation after cold shock. However, the structure of CsdA and the function of its long C-terminal regions are still unclear. Here, we solved all of the domain structures of CsdA and found two previously uncharacterized auxiliary domains: a dimerization domain (DD) and an RNA-binding domain (RBD). Small-angle X-ray scattering experiments helped to track the conformational flexibilities of the helicase core domains and C-terminal regions. Biochemical assays revealed that DD is indispensable for stabilizing the CsdA dimeric structure. We also demonstrate for the first time that CsdA functions as a stable dimer at low temperature. The C-terminal regions are critical for RNA binding and efficient enzymatic activities. CsdA_RBD could specifically bind to the regions with a preference for single-stranded G-rich RNA, which may help to bring the helicase core to unwind the adjacent duplex. [ABSTRACT FROM AUTHOR]