The solution structure of human leptin reveals a conformational plasticity important for receptor recognition.

Leptin is a multi-potency cytokine that regulates various physiological functions, including weight control and energy homeostasis. Signaling of leptin is also important in many aging-related diseases. Leptin is required for the noncovalent crosslinking of different extracellular domains of leptin receptors, which is critical for receptor activation and downstream signaling. Nevertheless, the structure of intact apo-form leptin and the structural transition leptin undergoes upon receptor binding are not fully understood yet. Here, we determined the monomeric structure of wild-type human leptin by solution-state nuclear magnetic resonance spectroscopy. Leptin contains an intrinsically disordered region (IDR) in the internal A-B loop and the flexible helix E in the C-D loop, both of which undergo substantial local structural changes when leptin binds to its receptor. Our findings provide further insights into the molecular mechanisms of leptin signaling.

Chemical shift assignments of wildtype human leptin.

Leptin is an adipose tissue-expressed 16-kDa hormone encoded by the ob/ob gene. It serves a crucial role in regulating diverse physiological processes, including body weight control, energy homeostasis regulation, promotion of cell proliferation, and more. Emerging research has also revealed potential implications of leptin in various aging-related diseases, suggesting multifaceted physiological roles of leptin. Structural investigation of wild-type leptin in apo form is of particular importance to understand its conformational plasticity for receptor interaction and recognition. Here, we report backbone and side-chain resonance assignments of wild-type human leptin as a basis for structural and functional studies on leptin-mediated signaling.

Induction of Transcriptional Inhibitor HES1 and the Related Repression of Tumor-Suppressor TXNIP Are Important Components of Cell-Transformation Program Imposed by Oncogenic Kinase NPM-ALK.

This study reports that hairy and enhancer of split homolog-1 (HES1), known to repress gene transcription in progenitor cells of several cell lineages, was strongly expressed in cells and tissues of T-cell lymphoma expressing the oncogenic chimeric tyrosine kinase nucleophosmin (NPM)-anaplastic lymphoma kinase [ALK; ALK+ T-cell lymphoma (TCL)]. The structural analysis of the Orange domain of HES1 indicated that HES1 formed a highly stable homodimer. Of note, repression of HES1 expression led to inhibition of ALK+ TCL cell growth in vivo. The expression of the HES1 gene was induced by NPM-ALK through activation of STAT3, which bound to the gene's promoter and induced the gene's transcription. NPM-ALK also directly phosphorylated HES1 protein. In turn, HES1 up-regulated and down-regulated in ALK+ TCL cells, the expression of numerous genes, protein products of which are involved in key cell functions, such as cell proliferation and viability. Among the genes inhibited by HES1 was thioredoxin-interacting protein (TXNIP), encoding a protein implicated in promotion of cell death in various types of cells. Accordingly, ALK+ TCL cells and tissues lacked expression of TXNIP, and its transcription was co-inhibited by HES1 and STAT3 in an NPM-ALK-dependent manner. Finally, the induced expression of TXNIP induced massive apoptotic cell death of ALK+ TCL cells. The results reveal a novel NPM-ALK-controlled pro-oncogenic regulatory network and document an important role of HES and TXNIP in the NPM-ALK-driven oncogenesis, with the former protein displaying oncogenic and the latter tumor suppressor properties.

C-Terminal Domains of Spider Silk Proteins Having Divergent Structures but Conserved Functional Roles.

Spider silk is self-assembled from silk proteins or spidroins. C-terminal domains (CTDs) of various types of spidroins are relatively conserved in amino acid sequences and are suggested to adopt similar structures and perform similar functional roles in spidroin storage and silk formation. Here, we solved the structure of the CTD from a capture-spiral silk protein (CTDFl) and characterized its stability and fibril formation in the presence and absence of a reducing agent at different pH values. CTDFl adopts a dimeric structure with 8 helices, but the CTDs of other types of spidroins exist in a domain-swapped dimeric structure with 10 helices. Despite the structural differences, CTDFl is pH-responsive in stability and fibril formation, similar to the CTDs from minor and major ampullate spidroins. Thus, the functional role of CTDs in silk fiber formation seems conserved. Comparing wild-type CTDFl and its mutants, we found that the pH-responsive behavior results from the protonation of H76, which is conserved from different spider species. In addition, the fibril formation rate of CTDFl correlates with its instability, suggesting that structural changes are involved in fibril formation.

Anti-Fungal Hevein-like Peptides Biosynthesized from Quinoa Cleavable Hololectins.

Chitin-binding hevein-like peptides (CB-HLPs) belong to a family of cysteine-rich peptides that play important roles in plant stress and defense mechanisms. CB-HLPs are ribosomally synthesized peptides that are known to be bioprocessed from the following two types of three-domain CB-HLP precursor architectures: cargo-carrying and non-cargo-carrying. Here, we report the identification and characterization of chenotides biosynthesized from the third type of precursors, which are cleavable hololectins of the quinoa (Chenopodium quinoa) family. Chenotides are 6-Cys-CB-HLPs of 29-31 amino acids, which have a third type of precursor architecture that encompasses a canonical chitin-binding domain that is involved in chitin binding and anti-fungal activities. Microbroth dilution assays and microscopic analyses showed that chenotides are effective against phyto-pathogenic fungi in the micromolar range. Structure determination revealed that chenotides are cystine knotted and highly compact, which could confer resistance against heat and proteolytic degradation. Importantly, chenotides are connected by a novel 18-residue Gly/Ala-rich linker that is a target for bioprocessing by cathepsin-like endopeptidases. Taken together, our findings reveal that chenotides are a new family of CB-HLPs from quinoa that are synthesized as a single multi-modular unit and bioprocessed to yield individual mature CB-HLPs. Importantly, such precursors constitute a new family of cleavable hololectins. This unusual feature could increase the biosynthetic efficiency of anti-fungal CB-HLPs, to provide an evolutionary advantage for plant survival and reproduction.

Critical role of minor eggcase silk component in promoting spidroin chain alignment and strong fiber formation.

Natural spider silk with extraordinary mechanical properties is typically spun from more than one type of spidroin. Although the main components of various spider silks have been widely studied, little is known about the molecular role of the minor silk components in spidroin self-assembly and fiber formation. Here, we show that the minor component of spider eggcase silk, TuSp2, not only accelerates self-assembly but remarkably promotes molecular chain alignment of spidroins upon physical shearing. NMR structure of the repetitive domain of TuSp2 reveals that its dimeric structure with unique charged surface serves as a platform to recruit different domains of the main eggcase component TuSp1. Artificial fiber spun from the complex between TuSp1 and TuSp2 minispidroins exhibits considerably higher strength and Young's modulus than its native counterpart. These results create a framework for rationally designing silk biomaterials based on distinct roles of silk components.

1H, 15N and 13C resonance assignments of a repetitive domain of tubuliform spidroin 2.

Spider silk is renowned for its excellent mechanical properties. Among six types of silk and one silk glue produced by different abdominal glands for various purposes, tubuliform (eggcase) silk is unique due to its high serine and low glycine content. Eggcase silk is spun from at least two spidroins, tubuliform spidroin 1 (TuSp1) and TuSp2. TuSp1 and TuSp2 were identified as the major and the minor components in tubuliform glands, respectively. TuSp2 consists of multiple repetitive (RP) domains with short terminal tails and shares very limited homology to all known spidroins. Here we report backbone and side chain resonance assignments of TuSp2-RP as a basis for structural and functional studies on eggcase silk formation.

Dengue virus protease activity modulated by dynamics of protease cofactor.

The viral protease domain (NS3pro) of dengue virus is essential for virus replication, and its cofactor NS2B is indispensable for the proteolytic function. Although several NS3pro-NS2B complex structures have been obtained, the dynamic property of the complex remains poorly understood. Using NMR relaxation techniques, here we found that NS3pro-NS2B exists in both closed and open conformations that are in dynamic equilibrium on a submillisecond timescale in aqueous solution. Our structural information indicates that the C-terminal region of NS2B is disordered in the minor open conformation but folded in the major closed conformation. Using mutagenesis, we showed that the closed-open conformational equilibrium can be shifted by changing NS2B stability. Moreover, we revealed that the proteolytic activity of NS3pro-NS2B correlates well with the population of the closed conformation. Our results suggest that the closed-open conformational equilibrium can be used by both nature and humanity to control the replication of dengue virus.

Self-assembly of tubuliform spidroins driven by hydrophobic interactions among terminal domains.

Spider silk has remarkable physical and biocompatible properties. Investigation of structure-function relationship and self-assembly process of spidroins is necessary for uncovering the mechanism of silk fiber formation. Nevertheless, how the terminal domains initiate self-assembly of soluble tubuliform spidroins to form solid eggcase silk is still not fully understood. Here we investigate the roles of both terminal domains of tubuliform spidroin 1 (TuSp1) in the silk fiber formation. We found that interactions among the terminal domains drive rapid TuSp1 self-assembly and fiber formation, which is insensitive to pH changes from 6.0 to 7.0. These interactions also contribute to the spidroin chain alignment in fiber formation upon shear-force exposure. Structural analysis and site-directed mutagenesis identified eight critical surface-exposed residues involved in hydrophobic interactions among terminal domains. Spidroins with single-point mutations of these residues fail to form intermediate micelle-like structures. The structural docking model indicates that multiple terminal domains of TuSp1 may interact with each other based on hydrophobic interactions and surface complementarity, which may lead to forming the surface of the micelle-like structure. Our results provide new insights into the structural mechanism of eggcase silk formation and the basis for designing and producing novel biomaterials derived from spider eggcase silk.

Fungal Wound Healing through Instantaneous Protoplasmic Gelation.

Multicellular organisms employ fluid transport networks to overcome the limit of diffusion and promote essential long-distance transport. Connectivity and pressurization render these networks especially vulnerable to wounding. To mitigate this risk, animals, plants, and multicellular fungi independently evolved elaborate clotting and plugging mechanisms. In the septate filamentous fungi, membrane-bound organelles plug septal pores in wounded hyphae. By contrast, vegetative hyphae in the early-diverging Mucoromycota are largely aseptate, and how their hyphae respond to wounding is unknown. Here, we show that wounding in the Mucorales leads to explosive protoplasmic discharge that is rapidly terminated by protoplasmic gelation. We identify Mucoromycota-specific Gellin proteins, whose loss of function leads to uncontrolled wound-induced protoplasmic bleeding. Gellins contain ten related ß-trefoil Gll domains, each of which possesses unique features that impart distinct gelation-related properties: some readily unfold and form high-order sheet-like structures when subjected to mechanical force from flow, while others possess hydrophobic motifs that enable membrane binding. In cell-free reconstitution, sheet-like structures formed by a partial Gellin incorporate membranous organelles. Together, these data define a mechanistic basis for regulated protoplasmic gelation, and provide new design principles for the development of artificial flow-responsive biomaterials.

Structural and functional analyses of the PPIase domain of Arabidopsis thaliana CYP71 reveal its catalytic activity toward histone H3.

Arabidopsis thaliana CYP71 (AtCYP71) is a chromatin-remodeling protein that promotes shoot apical meristem (SAM) differentiation. The N terminus of AtCYP71 contains a noncanonical WD domain, and the C terminus contains an enzymatic peptidyl-prolyl isomerase (PPIase) cyclophilin (CYP) domain. To date, there has been no characterization of CYP71, and its mode of action remains unknown. Here, we report the crystal structure of the CYP domain of AtCYP71 at 1.9 Å resolution. The structure shows key differences when compared to the canonical CYP fold of human CypA. To the best our knowledge, this is the first A. thaliana CYP structure with a conserved active site loop. Using nuclear magnetic resonance spectroscopy, we demonstrate that the CYP domain is active toward histone H3. Our findings suggest that the PPIase activity of the CYP domain is important for the function of AtCYP71 in chromatin remodeling during organogenesis.

Structural basis of DNA binding to human YB-1 cold shock domain regulated by phosphorylation.

Human Y-box binding protein 1 (YB-1) is a multifunctional protein and overexpressed in many types of cancer. It specifically recognizes DNA/RNA through a cold shock domain (CSD) and regulates nucleic acid metabolism. The C-terminal extension of CSD and the phosphorylation of S102 are indispensable for YB-1 function. Until now, the roles of the C-terminal extension and phosphorylation in gene transcription and translation are still largely unknown. Here, we solved the structure of human YB-1 CSD with a C-terminal extension sequence (CSDex). The structure reveals that the extension interacts with several residues in the conventional CSD and adopts a rigid structure instead of being disordered. Either deletion of this extension or phosphorylation of S102 destabilizes the protein and results in partial unfolding. Structural characterization of CSDex in complex with a ssDNA heptamer shows that all the seven nucleotides are involved in DNA-protein interactions and the C-terminal extension provides a unique DNA binding site. Our DNA-binding study indicates that CSDex can recognize more DNA sequences than previously thought and the phosphorylation reduces its binding to ssDNA dramatically. Our results suggest that gene transcription and translation can be regulated by changing the affinity of CSDex binding to DNA and RNA through phosphorylation, respectively.

Spitzenkörper assembly mechanisms reveal conserved features of fungal and metazoan polarity scaffolds.

The Spitzenkörper (SPK) constitutes a collection of secretory vesicles and polarity-related proteins intimately associated with polarized growth of fungal hyphae. Many SPK-localized proteins are known, but their assembly and dynamics remain poorly understood. Here, we identify protein-protein interaction cascades leading to assembly of two SPK scaffolds and recruitment of diverse effectors in Neurospora crassa. Both scaffolds are transported to the SPK by the myosin V motor (MYO-5), with the coiled-coil protein SPZ-1 acting as cargo adaptor. Neither scaffold appears to be required for accumulation of SPK secretory vesicles. One scaffold consists of Leashin-2 (LAH-2), which is required for SPK localization of the signalling kinase COT-1 and the glycolysis enzyme GPI-1. The other scaffold comprises a complex of Janus-1 (JNS-1) and the polarisome protein SPA-2. Via its Spa homology domain (SHD), SPA-2 recruits a calponin domain-containing F-actin effector (CCP-1). The SHD NMR structure reveals a conserved surface groove required for effector binding. Similarities between SPA-2/JNS-1 and the metazoan GIT/PIX complex identify foundational features of the cell polarity apparatus that predate the fungal-metazoan divergence.

Structural Basis of Oligomerization of N-Terminal Domain of Spider Aciniform Silk Protein.

Spider silk is self-assembled from water-soluble silk proteins through changes in the environment, including pH, salt concentrations, and shear force. The N-terminal domains of major and minor ampullate silk proteins have been found to play an important role in the assembly process through salt- and pH-dependent dimerization. Here, we identified the sequences of the N-terminal domains of aciniform silk protein (AcSpN) and major ampullate silk protein (MaSpN) from Nephila antipodiana (NA). Different from MaSpN, our biophysical characterization indicated that AcSpN assembles to form large oligomers, instead of a dimer, upon condition changes from neutral to acidic pH and/or from a high to low salt concentration. Our structural studies, by nuclear magnetic resonance spectroscopy and homology modelling, revealed that AcSpN and MaSpN monomers adopt similar overall structures, but have very different charge distributions contributing to the differential self-association features. The intermolecular interaction interfaces for AcSp oligomers were identified using hydrogen-deuterium exchange mass spectrometry and mutagenesis. On the basis of the monomeric structure and identified interfaces, the oligomeric structures of AcSpN were modelled. The structural information obtained will facilitate an understanding of silk fiber formation mechanisms for aciniform silk protein.

Ligand Entry into Fatty Acid Binding Protein via Local Unfolding Instead of Gap Widening.

Fatty acid binding proteins play an important role in the transportation of fatty acids. Despite intensive studies, how fatty acids enter the protein cavity for binding is still controversial. Here, a gap-closed variant of human intestinal fatty acid binding protein was generated by mutagenesis, in which the gap is locked by a disulfide bridge. According to its structure determined here by NMR, this variant has no obvious openings as the ligand entrance and the gap cannot be widened by internal dynamics. Nevertheless, it still takes up fatty acids and other ligands. NMR relaxation dispersion, chemical exchange saturation transfer, and hydrogen-deuterium exchange experiments show that the variant exists in a major native state, two minor native-like states, and two locally unfolded states in aqueous solution. Local unfolding of either ßB-ßD or helix 2 can generate an opening large enough for ligands to enter the protein cavity, but only the fast local unfolding of helix 2 is relevant to the ligand entry process.

Roseltide rT7 is a disulfide-rich, anionic, and cell-penetrating peptide that inhibits proteasomal degradation.

Disulfide-rich plant peptides with molecular masses of 2-6 kDa represent an expanding class of peptidyl-type natural products with diverse functions. They are structurally compact, hyperstable, and underexplored as cell-penetrating agents that inhibit intracellular functions. Here, we report the discovery of an anionic, 34-residue peptide, the disulfide-rich roseltide rT7 from Hibiscus sabdariffa (of the Malvaceae family) that penetrates cells and inhibits their proteasomal activities. Combined proteomics and NMR spectroscopy revealed that roseltide rT7 is a cystine-knotted, six-cysteine hevein-like cysteine-rich peptide. A pair-wise comparison indicated that roseltide rT7 is >100-fold more stable against protease degradation than its S-alkylated analog. Confocal microscopy studies and cell-based assays disclosed that after roseltide rT7 penetrates cells, it causes accumulation of ubiquitinated proteins, inhibits human 20S proteasomes, reduces tumor necrosis factor-induced IκBα degradation, and decreases expression levels of intercellular adhesion molecule-1. Structure-activity studies revealed that roseltide rT7 uses a canonical substrate-binding mechanism for proteasomal inhibition enabled by an IIML motif embedded in its proline-rich and exceptionally long intercysteine loop 4. Taken together, our results provide mechanistic insights into a novel disulfide-rich, anionic, and cell-penetrating peptide, representing a potential lead for further development as a proteasomal inhibitor in anti-cancer or anti-inflammatory therapies.

Prolyl isomerization of the CENP-A N-terminus regulates centromeric integrity in fission yeast.

Centromeric identity and chromosome segregation are determined by the precise centromeric targeting of CENP-A, the centromere-specific histone H3 variant. The significance of the amino-terminal domain (NTD) of CENP-A in this process remains unclear. Here, we assessed the functional significance of each residue within the NTD of CENP-A from Schizosaccharomyces pombe (SpCENP-A) and identified a proline-rich 'GRANT' (Genomic stability-Regulating site within CENP-A N-Terminus) motif that is important for CENP-A function. Through sequential mutagenesis, we show that GRANT proline residues are essential for coordinating SpCENP-A centromeric targeting. GRANT proline-15 (P15), in particular, undergoes cis-trans isomerization to regulate chromosome segregation fidelity, which appears to be carried out by two FK506-binding protein (FKBP) family prolyl cis-trans isomerases. Using proteomics analysis, we further identified the SpCENP-A-localizing chaperone Sim3 as a SpCENP-A NTD interacting protein that is dependent on GRANT proline residues. Ectopic expression of sim3+ complemented the chromosome segregation defect arising from the loss of these proline residues. Overall, cis-trans proline isomerization is a post-translational modification of the SpCENP-A NTD that confers precise propagation of centromeric integrity in fission yeast, presumably via targeting SpCENP-A to the centromere.

1H, 15N and 13C chemical shift assignments of the C-terminal domain of TRADD.

The tumor necrosis factor receptor-associated death domain protein, TRADD, is a multifunctional intracellular molecule participating in divergent signaling pathways, such as NF-κB and apoptosis. TRADD consists of two structurally distinct domains. Its N-terminal domain displays an α-ß plaits fold while its C-terminal domain belongs to the death domain (DD) superfamily. TRADD DD is a central component in the tumor necrosis factor receptor 1 signaling. It interacts with other DD-containing proteins through homotypic interactions. TRADD DD is also involved in p75NTR-mediated signalling in MCF-7 human breast cancer cells. Here we report backbone and sidechain 1H, 13C and 15N chemical shift assignments of TRADD DD in pure water as a basis for further structural and functional studies.

Structure of the C-terminal domain of TRADD reveals a novel fold in the death domain superfamily.

The TNFR1-associated death domain protein (TRADD) is an intracellular adaptor protein involved in various signaling pathways, such as antiapoptosis. Its C-terminal death domain (DD) is responsible for binding other DD-containing proteins including the p75 neurotrophin receptor (p75NTR). Here we present a solution structure of TRADD DD derived from high-resolution NMR spectroscopy. The TRADD DD comprises two super-secondary structures, an all-helix Greek key motif and a ß-hairpin motif flanked by two α helices, which make it unique among all known DD structures. The ß-hairpin motif is essential for TRADD DD to fold into a functional globular domain. The highly-charged surface suggests a critical role of electrostatic interactions in TRADD DD-mediated signaling. This novel structure represents a new class within the DD superfamily and provides a structural basis for studying homotypic DD interactions. NMR titration revealed a direct weak interaction between TRADD DD and p75NTR DD monomers. A binding site next to the p75NTR DD homodimerization interface indicates that TRADD DD recruitment to p75NTR requires separation of the p75NTR DD homodimer, explaining the mechanism of NGF-dependent activation of p75NTR-TRADD-mediated antiapoptotic pathway in breast cancer cell.

Structural Basis for pH-mediated Regulation of F-actin Severing by Gelsolin Domain 1.

Six-domain gelsolin regulates actin structural dynamics through its abilities to sever, cap and uncap F-actin. These activities are modulated by various cellular parameters like Ca2+ and pH. Until now, only the molecular activation mechanism of gelsolin by Ca2+ has been understood relatively well. The fragment comprising the first domain and six residues from the linker region into the second domain has been shown to be similar to the full-length protein in F-actin severing activity in the absence of Ca2+ at pH 5. To understand how this gelsolin fragment is activated for F-actin severing by lowering pH, we solved its NMR structures at both pH 7.3 and 5 in the absence of Ca2+ and measured the pKa values of acidic amino acid residues and histidine residues. The overall structure and dynamics of the fragment are not affected significantly by pH. Nevertheless, local structural changes caused by protonation of His29 and Asp109 result in the activation on lowering the pH, and protonation of His151 directly effects filament binding since it resides in the gelsolin/actin interface. Mutagenesis studies support that His29, Asp109 and His151 play important roles in the pH-dependent severing activity of the gelsolin fragment.