FEBS Letters

Dages, Samantha; Zhi, Xiaoduo; Leng, Fenfei

doi: 10.1002/1873-3468.13643pmid: 31639222

Previously, we demonstrated that transcription‐coupled DNA supercoiling (TCDS) potently activated or inhibited nearby promoters in Escherichia coli even in the presence of all four DNA topoisomerases, suggesting that DNA topoisomerases are not the only factors regulating TCDS. A different mechanism exists to confine this localized DNA supercoiling. Using an in vivo system containing the TCDS‐activated leu‐500 promoter (Pleu‐500), we find that the nucleoid‐associated Fis protein potently inhibits the TCDS‐mediated activation of Pleu‐500. We also find that deletion of the fis gene significantly enhances TCDS‐mediated inhibition of transcription of three genes purH, yieP, and yrdA divergently coupled to different rrn operons in the early log phase. These results suggest that Fis protein forms DNA topological barriers upon binding to its recognition sites, blocks TCDS diffusion, and potently inhibits the TCDS‐activated Pleu‐500.

Wang, Xinye; Jing, Xiaoran; Deng, Yi; Nie, Yao; Xu, Fei; Xu, Yan; Zhao, Yi‐Lei; Hunt, John F.; Montelione, Gaetano T.; Szyperski, Thomas

doi: 10.1002/1873-3468.13652pmid: 31665817

Pullulanases are well‐known debranching enzymes hydrolyzing α‐1,6‐glycosidic linkages. To date, engineering of pullulanase is mainly focused on catalytic pocket or domain tailoring based on structure/sequence information. Saturation mutagenesis‐involved directed evolution is, however, limited by the low number of mutational sites compatible with combinatorial libraries of feasible size. Using Bacillus naganoensis pullulanase as a target protein, here we introduce the ‘evolutionary coupling saturation mutagenesis’ (ECSM) approach: residue pair covariances are calculated to identify residues for saturation mutagenesis, focusing directed evolution on residue pairs playing important roles in natural evolution. Evolutionary coupling (EC) analysis identified seven residue pairs as evolutionary mutational hotspots. Subsequent saturation mutagenesis yielded variants with enhanced catalytic activity. The functional pairs apparently represent distant sites affecting enzyme activity.

Müller, Christian; Lukas, Phil; Böhmert, Michel; Hildebrandt, Jan‐Peter

doi: 10.1002/1873-3468.13683pmid: 31733150

The hirudin‐like factor 1 (HLF1) of Hirudo medicinalis belongs to a new class of leech‐derived factors. In previous investigations, HLF1 did not exhibit anticoagulatory activities. Here, we describe the analysis of natural and synthetic variants of HLF1 and HLF‐Hyb, a yet uncharacterized member of the HLF family. Modifications within the N terminus of HLF1 have a strong impact on its activity. Some variants of HLF1 exhibit thrombin‐inhibiting activity comparable to hirudins, whereas others have reduced or no activity. The analyses of HLF‐Hyb variants revealed a strong impact of the central globular domain on activity. Our results indicate a comparable mode of action of hirudins and thrombin‐inhibiting HLF variants. Finally, we propose and discuss criteria for classifying hirudins and HLFs.

Kitagawa, Yoshinori; Yamaguchi, Mayu; Kohno, Miki; Sakai, Madoka; Itoh, Masae; Gotoh, Bin

doi: 10.1002/1873-3468.13670pmid: 31705658

Respirovirus C protein blocks the type I interferon (IFN)‐stimulated activation of the JAK‐STAT pathway. It has been reported that C protein inhibits IFN‐α‐stimulated tyrosine phosphorylation of STATs, but the underlying mechanism is poorly understood. Here, we show that the C protein of Sendai virus (SeV), a member of the Respirovirus genus, binds to the IFN receptor subunit IFN‐α/β receptor subunit (IFNAR)2 and inhibits IFN‐α‐stimulated tyrosine phosphorylation of the upstream receptor‐associated kinases, JAK1 and TYK2. Analysis of various SeV C mutant (Cm) proteins demonstrates the importance of the inhibitory effect on receptor‐associated kinase phosphorylation for blockade of JAK‐STAT signaling. Furthermore, this inhibitory effect and the IFNAR2 binding capacity are observed for all the respirovirus C proteins examined. Our results suggest that respirovirus C protein inhibits activation of the receptor‐associated kinases JAK1 and TYK2 possibly through interaction with IFNAR2.

Kovács, Bertalan; Zajácz‐Epresi, Nóra; Gáspári, Zoltán

doi: 10.1002/1873-3468.13626pmid: 31562775

The postsynaptic density protein‐95 (PSD‐95) regulates synaptic plasticity through interactions mediated by its peptide‐binding PDZ domains. The two N‐terminal PDZ domains of PSD‐95 form an autonomous structural unit, and their interdomain orientation and dynamics depend on ligand binding. To understand the mechanistic details of the effect of ligand binding, we generated conformational ensembles using available experimentally determined nuclear Overhauser effect interatomic distances and S2 order parameters. In our approach, the fast dynamics of the two domains is treated independently. We find that intradomain structural changes induced by ligand binding modulate the probability of the occurrence of specific domain–domain orientations. Our results suggest that the β2‐β3 loop in the PDZ domains is a key regulatory region, which influences both intradomain motions and supramodular rearrangement.

Kawade, Raiji; Kuroda, Daisuke; Tsumoto, Kouhei

doi: 10.1002/1873-3468.13674pmid: 31713840

Physicochemical properties of proteins are controlled mainly by post‐translational modifications such as amino acid phosphorylation. Although molecular dynamics simulations have been shown to be a valuable tool for studying the effects of phosphorylation on protein structure and dynamics, most of the previous studies assumed that the phosphate group was in the unprotonated (PO32-) state, even though the protonation state could in fact vary at physiological pH. In this study, we performed molecular dynamics simulations of four different protein‐phosphorylated peptide complexes both in the PO32- and PO3H− states. Our simulations delineate different dynamics and energetics between the two states, suggesting importance of the protonation state of a phosphorylated amino acid in molecular recognition.

Fukuda, Atsushi; Motosugi, Nami; Ando, Mikiko; Kimura, Minoru; Umezawa, Akihiro; Akutsu, Hidenori

doi: 10.1002/1873-3468.13676pmid: 31721177

Epigenetic and transcriptome alterations are essential for lineage specification, represented by imprinted X‐chromosome inactivation (iXCI) in female mouse preimplantation embryos. However, how various factors affect transcriptome states and lineage commitment remains unclear. We found that in vitro culture duration strongly influences transcriptional variation compared to iXCI loss. Single‐cell analysis of the inner cell mass (ICM) for major transcription and epigenomic factors revealed that sex‐specific differences in expression are diminished by loss of iXCI in the primitive endoderm (PrE) but not in the epiblast. Females had a higher proportion of ICM compared to that in males, and PrE development was affected by iXCI states in female embryos. Our findings provide insight into sex differences and iXCI function in lineage specification.

Zhao, Cuiping; Lyu, Zhe; Long, Feng; Akinyemi, Taiwo; Manakongtreecheep, Kasidet; Söll, Dieter; Whitman, William B.; Vinyard, David J.; Liu, Yuchen

doi: 10.1002/1873-3468.13673pmid: 31709520

The nucleotide binding protein 35 (Nbp35)/cytosolic Fe‐S cluster deficient 1 (Cfd1)/alternative pyrimidine biosynthetic protein C (ApbC) protein homologs have been identified in all three domains of life. In eukaryotes, the Nbp35/Cfd1 heterocomplex is an essential Fe‐S cluster assembly scaffold required for the maturation of Fe‐S proteins in the cytosol and nucleus, whereas the bacterial ApbC is an Fe‐S cluster transfer protein only involved in the maturation of a specific target protein. Here, we show that the Nbp35/ApbC homolog MMP0704 purified from its native archaeal host Methanococcus maripaludis contains a [4Fe‐4S] cluster that can be transferred to a [4Fe‐4S] apoprotein. Deletion of mmp0704 from M. maripaludis does not cause growth deficiency under our tested conditions. Our data indicate that Nbp35/ApbC is a nonessential [4Fe‐4S] cluster transfer protein in methanogenic archaea.

Stach, Lasse; Morgan, Rhodri Marc; Makhlouf, Linda; Douangamath, Alice; Delft, Frank; Zhang, Xiaodong; Freemont, Paul S.

doi: 10.1002/1873-3468.13667pmid: 31701538

Al‐Hilaly, Youssra K.; Foster, Bronwen E.; Biasetti, Luca; Lutter, Liisa; Pollack, Saskia J.; Rickard, Janet E.; Storey, John M. D.; Harrington, Charles R.; Xue, Wei‐Feng; Wischik, Claude M.; Serpell, Louise C.

doi: 10.1002/1873-3468.13675pmid: 31721178

The constituent paired helical filaments (PHFs) in neurofibrillary tangles are insoluble intracellular deposits central to the development of Alzheimer’s disease (AD) and other tauopathies. Full‐length tau requires the addition of anionic cofactors such as heparin to enhance assembly. We have shown that a fragment from the proteolytically stable core of the PHF, tau 297‐391 known as ‘dGAE’, spontaneously forms cross‐β‐containing PHFs and straight filaments under physiological conditions. Here, we have analysed and compared the structures of the filaments formed by dGAE in vitro with those deposited in the brains of individuals diagnosed with AD. We show that dGAE forms PHFs that share a macromolecular structure similar to those found in brain tissue. Thus, dGAEs may serve as a model system for studying core domain assembly and for screening for inhibitors of tau aggregation.