Intrinsically disordered N-terminal domain of the Helicoverpa armigera Ultraspiracle stabilizes the dimeric form via a scorpion-like structure

Nuclear receptors (NRs) are a family of ligand-dependent transcription factors activated by lipophilic compounds. NRs share a common structure comprising three domains: a variable N-terminal domain (NTD), a highly conserved globular DNA-binding domain and a ligand-binding domain. There are numerous papers describing the molecular details of the latter two globular domains. However, very little is known about the structure-function relationship of the NTD, especially as an intrinsically disordered fragment of NRs that may influence the molecular properties and, in turn, the function of globular domains. Here, we investigated whether and how an intrinsically disordered NTD consisting of 58 amino acid residues affects the functions of the globular domains of the Ultraspiracle protein from Helicoverpa armigera (HaUsp). The role of the NTD was examined for two well-known and easily testable NR functions, i.e., interactions with specific DNA sequences and dimerization. Electrophoretic mobility shift assays showed that the intrinsically disordered NTD influences the interaction of HaUsp with specific DNA sequences, apparently by destabilization of HaUsp-DNA complexes. On the other hand, multi-angle light scattering and sedimentation velocity analytical ultracentrifugation revealed that the NTD acts as a structural element that stabilizes HaUsp homodimers. Molecular models based on small-angle X-ray scattering indicate that the intrinsically disordered NTD may exert its effects on the tested HaUsp functions by forming an unexpected scorpion-like structure, in which the NTD bends towards the ligand-binding domain in each subunit of the HaUsp homodimer. This structure may be crucial for specific NTD-dependent regulation of the functions of globular domains in NR

Insight into the Binding and Hydrolytic Preferences of hNudt16 Based on Nucleotide Diphosphate Substrates

Nudt16 is a member of the NUDIX family of hydrolases that show specificity towards substrates consisting of a ucleoside diphosphate linked to another moiety X. Several substrates for hNudt16 and various possible biological functions have been reported. However, some of these reports contradict each other and studies comparing the substrate specificity of the hNudt16 protein are limited. Therefore, we quantitatively compared the affinity of hNudt16 towards a set of previously published substrates, as well as identified novel potential substrates. Here, we show that hNudt16 has the highest affinity towards IDP and GppG, with Kd below 100 nM. Other tested ligands exhibited a weaker affinity of several orders of magnitude. Among the investigated compounds, only IDP, GppG, m7GppG, AppA, dpCoA, and NADH were hydrolyzed by hNudt16 with a strong substrate preference for inosine or guanosine containing compounds. A new identified substrate for hNudt16, GppG, which binds the enzyme with an affinity comparable to that of IDP, suggests another potential regulatory role of this protein. Molecular docking of hNudt16-ligand binding inside the hNudt16 pocket revealed two binding modes for representative substrates. Nucleobase stabilization by Π stacking interactions with His24 has been associated with strong binding of hNudt16 substrates

Structural studies of selected proteins involved in defense response against pathogens in Hordeum vulgare

Wydział Fizyki: Zakład Fizyki MakromolekularnejRośliny nieustannie narażone są na ataki ze strony patogenów takich jak bakterie, wirusy czy grzyby. Jednym z elementów molekularnych mechanizmów obronnych rośliny są receptory R. Białka R rozpoznają patogenne cząsteczki wewnątrz komórki roślinnej i uruchamiają odpowiedź obronną. Do poprawnego pełnienia swojej funkcji białka R potrzebują udziału kompleksu białek HSP90-SGT1-RAR1. W niniejszej pracy zostały przedstawione wyniki badań nad strukturą poszczególnych białek kompleksu w roztworze za pomocą małokątowego promieniowania rentgenowskiego oraz spektroskopii dichroizmu kołowego. Analiza otrzymanych wyników pozwoliła na wymodelowanie niskorozdzielczej struktury badanych białek oraz wykazanie, że białka SGT1 i RAR1 mają dynamiczną konformację w roztworze. Jednocześnie wykazano, że dimeryzacja domeny TPR białka SGT1 z jęczmienia, rzodkiewnika pospolitego oraz drożdży zależy od siły jonowej, która nie ma wpływu na strukturę i stan oligomeryczny domeny TPR ludzkiego homologa. Zbadano również wpływ wiązania nukleotydów, temperatury i siły jonowej na dynamikę konformacyjną białka HSP90 z pszenicy. Zarówno siła jonowa i temperatura powodują przesunięcie równowagi w stronę konformacji otwartej. Wiązanie nukleotydów nie powoduje natomiast dramatycznych zmian konformacyjnych. Określono także, niskorozdzielczą strukturę kompleksu białek HSP90-SGT1ΔSGS z ADP. W kompleksie tym białko HSP90 ma otwartą konformację. Wiązanie białka HSP90 powoduje dysocjację dimeru białka SGT1. Jednocześnie monomer białka SGT1 wiąże się w sposób asymetryczny do białka HSP90 w stosunku stechiometrycznym 1:2.Plants are constantly exposed to contacts with a variety of pathogens like bacteria, viruses and fungi. One of the most important elements of this system on the molecular level are R proteins, that recognize pathogenic molecules and trigger the immune response. For their function R proteins require the protein complex that consists of three major proteins: HSP90, SGT1 and RAR1 proteins. This PhD thesis presents results of a study concerning the structure and dynamic in solution of the proteins of HSP90-SGT1-RAR1 complex studied using small angle x-ray scattering and circular dichroism. From the analysis of experimental data we obtained low resolution structure of the SGT1 and RAR1 proteins and proved that both proteins are flexible in solution. I also proved that the dimerisation of the TPR domain of SGT1 protein from barley, Arabidopsis thaliana and yeasts depends on ionic strength, that has no influence on the structure and oligomeric state of TPR domain from the human homolog. In this PhD thesis I also present results concerning the role of the nucleotide binding, temperature and ionic strength in the conformational equilibrium of the wheat HSP90 protein. Both, temperature and ionic strength shifts equilibrium to the more open conformation. Nucleotide binding has very little effect on HSP90 conformation. I was able to model the low resolution structure of the HSP90-SGT1ΔSGS complex with ADP. Within this complex HSP90 has open conformation. HSP90 binding causes dissociation of the SGT1 dimer. Simultaneously the monomer of the SGT1 protein binds to the HSP90 dimer in asymmetric manner that leads to the 1:2 stoichiometry

Oligomerization of Human Cystatin C—An Amyloidogenic Protein: An Analysis of Small Oligomeric Subspecies

Human cystatin C (HCC), an amyloidogenic protein, forms dimers and higher oligomers (trimers, tetramers and donut like large oligomers) via a domain-swapping mechanism. The aim of this study was the characterization of the HCC oligomeric states observed within the pH range from 2.2 to 10.0 and also in conditions promoting oligomerization. The HCC oligomeric forms obtained in different conditions were characterized using size exclusion chromatography, dynamic light scattering and small-angle X-ray scattering. The marked ability of HCC to form tetramers at low pH (2.3 or 3.0) and dimers at pH 4.0–5.0 was observed. HCC remains monomeric at pH levels above 6.0. Based on the SAXS data, the structure of the HCC tetramer was proposed. Changes in the environment (from acid to neutral) induced a breakdown of the HCC tetramers to dimers. The tetrameric forms of human cystatin C are formed by the association of the dimers without a domain-swapping mechanism. These observations were confirmed by their dissociation to dimers at pH 7.4

Regulation of Plant Microprocessor Function in Shaping microRNA Landscape

MicroRNAs are small molecules (∼21 nucleotides long) that are key regulators of gene expression. They originate from long stem–loop RNAs as a product of cleavage by a protein complex called Microprocessor. The core components of the plant Microprocessor are the RNase type III enzyme Dicer-Like 1 (DCL1), the zinc finger protein Serrate (SE), and the double-stranded RNA binding protein Hyponastic Leaves 1 (HYL1). Microprocessor assembly and its processing of microRNA precursors have been reported to occur in discrete nuclear bodies called Dicing bodies. The accessibility of and modifications to Microprocessor components affect microRNA levels and may have dramatic consequences in plant development. Currently, numerous lines of evidence indicate that plant Microprocessor activity is tightly regulated. The cellular localization of HYL1 is dependent on a specific KETCH1 importin, and the E3 ubiquitin ligase COP1 indirectly protects HYL1 from degradation in a light-dependent manner. Furthermore, proper localization of HYL1 in Dicing bodies is regulated by MOS2. On the other hand, the Dicing body localization of DCL1 is regulated by NOT2b, which also interacts with SE in the nucleus. Post-translational modifications are substantial factors that contribute to protein functional diversity and provide a fine-tuning system for the regulation of protein activity. The phosphorylation status of HYL1 is crucial for its activity/stability and is a result of the interplay between kinases (MPK3 and SnRK2) and phosphatases (CPL1 and PP4). Additionally, MPK3 and SnRK2 are known to phosphorylate SE. Several other proteins (e.g., TGH, CDF2, SIC, and RCF3) that interact with Microprocessor have been found to influence its RNA-binding and processing activities. In this minireview, recent findings on the various modes of Microprocessor activity regulation are discussed

The elution profile of the recombinant full-length barley SGT1 protein purified on a Superdex column.

<p>The elution profile of the recombinant full-length barley SGT1 protein purified on a Superdex column.</p

Shape determination of the full-length barley SGT1 monomer.

<p>Models of SGT1 domains superimposed on the averaged low-resolution model obtained using DAMMIN (A) and fit of the theoretical scattering curve of the model to MCR-ALS scattering data for the monomer (B). Models of the SGT1 domains superimposed on a low-resolution model obtained by GASBOR (C) and fit of theoretical scattering curve (red) of the model to MCR-ALS scattering data for the monomer (D).</p

SDS-PAGE analysis of extracts from all of the steps performed during purification of the full-length barley SGT1 protein.

<p>SDS-PAGE analysis of extracts from all of the steps performed during purification of the full-length barley SGT1 protein.</p

Figure 8

<p>Scattering profiles of the full-length barley SGT1 monomer (blue) and dimer (red) obtained using the MCR-ALS procedure (A). Normalized pair distance distribution functions p(r) and maximum diameter of particle computed using GNOM (B).</p

Results of <i>ab-initio</i> shape determination of the full-length barley SGT1 dimer.

<p>Models of SGT1 domains superimposed on the low-resolution model obtained by DAMMIN (A). The low-resolution models (red and blue) of SGT1 monomers superimposed on the low-resolution model of the SGT1 dimer obtained by DAMMIN (B). Fit of the theoretical scattering curve (red) to the MCR-ALS scattering data for the dimer (C).</p