Surface Accessibility and Conformational Changes in the N-terminal Domain of Type I Inositol Trisphosphate Receptors

Trehalose-6-phosphate (T6P) synthase (Tps1) catalyzes the formation of T6P from UDP-glucose (UDPG) (or GDPG, etc.) and glucose-6-phosphate (G6P), and structural basis of this process has not been well studied. MoTps1 (Magnaporthe oryzae Tps1) plays a critical role in carbon and nitrogen metabolism, but its structural information is unknown. Here we present the crystal structures of MoTps1 apo, binary (with UDPG) and ternary (with UDPG/G6P or UDP/T6P) complexes. MoTps1 consists of two modified Rossmann-fold domains and a catalytic center in-between. Unlike Escherichia coli OtsA (EcOtsA, the Tps1 of E. coli), MoTps1 exists as a mixture of monomer, dimer, and oligomer in solution. Inter-chain salt bridges, which are not fully conserved in EcOtsA, play primary roles in MoTps1 oligomerization. Binding of UDPG by MoTps1 C-terminal domain modifies the substrate pocket of MoTps1. In the MoTps1 ternary complex structure, UDP and T6P, the products of UDPG and G6P, are detected, and substantial conformational rearrangements of N-terminal domain, including structural reshuffling (β3–β4 loop to α0 helix) and movement of a ‘shift region' towards the catalytic centre, are observed. These conformational changes render MoTps1 to a ‘closed' state compared with its ‘open' state in apo or UDPG complex structures. By solving the EcOtsA apo structure, we confirmed that similar ligand binding induced conformational changes also exist in EcOtsA, although no structural reshuffling involved. Based on our research and previous studies, we present a model for the catalytic process of Tps1. Our research provides novel information on MoTps1, Tps1 family, and structure-based antifungal drug design.

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Interventional Thermal Injury of the Arterial Wall: Unfolding of von Willebrand Factor and Its Increased Binding to Collagen after 55° C Heating

Thrombosis and Haemostasis ◽

10.1055/s-0038-1650307 ◽

1996 ◽

Vol 75 (03) ◽

pp. 515-519 ◽

Cited By ~ 7

Author(s):

Mark J Post ◽

Anke N de Graaf-Bos ◽

George Posthuma ◽

Philip G de Groot ◽

Jan J Sixma ◽

...

Keyword(s):

Von Willebrand Factor ◽

Conformational Changes ◽

Arterial Wall ◽

Platelet Adhesion ◽

Type I ◽

Temperature Dependent ◽

Collagen Types ◽

Electron Microscopic ◽

Von Willebrand ◽

Willebrand Factor

Summary Purpose. Thermal angioplasty alters the thrombogenicity of the arterial wall. In previous studies, platelet adhesion was found to increase after heating human subendothelium to 55° C and decrease after heating to 90° C. In the present electron microscopic study, the mechanism of this temperature-dependent platelet adhesion to the heated arterial wall is elucidated by investigating temperature-dependent conformational changes of von Willebrand factor (vWF) and collagen types I and III and the binding of vWF to heated collagen. Methods. Purified vWF and/or collagen was applied to electron microscopic grids and heated by floating on a salt-solution of 37° C, 55° C or 90° C for 15 s. After incubation with a polyclonal antibody against vWF and incubation with protein A/gold, the grids were examined by electron microscopy. Results. At 37° C, vWF was coiled. At 55° C, vWF unfolded, whereas heating at 90° C caused a reduction in antigenicity. Collagen fibers heated to 37° C were 60.3 ± 3.1 nm wide. Heating to 55° C resulted in the unwinding of the fibers, increasing the width to 87.5 ± 8.2 nm (p < 0.01). Heating to 90° C resulted in denatured fibers with an enlarged width of 85.1 ± 6.1 nm (p < 0.05). Heating of collagen to 55° C resulted in an increased vWF binding as compared to collagen heated to 37° C or to 90° C. Incubation of collagen with vWF, prior to heating, resulted in a vWF binding after heating to 55° C that was similar to the 37° C binding and a decreased binding after 90° C. Conclusions. After 55° C heating, the von Willebrand factor molecule unfolds and collagen types I and III exhibit an increased adhesiveness for von Willebrand factor. Heating to 90° C denatures von Willebrand factor and collagen. The conformation changes of von Willebrand factor and its altered binding to collagen type I and III may explain the increased and decreased platelet adhesion to subendothelium after 55° C and 90° C heating, respectively.

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The Odd Faces of Oligomers: The Case of TRAF2-C, A Trimeric C-Terminal Domain of TNF Receptor-Associated Factor

International Journal of Molecular Sciences ◽

10.3390/ijms22115871 ◽

2021 ◽

Vol 22 (11) ◽

pp. 5871

Author(s):

Almerinda Di Venere ◽

Eleonora Nicolai ◽

Velia Minicozzi ◽

Anna Maria Caccuri ◽

Luisa Di Paola ◽

...

Keyword(s):

Conformational Changes ◽

Strong Dependence ◽

Conformational Dynamics ◽

Receptor Interaction ◽

Tnf Receptor ◽

Oligomeric State ◽

Dynamic Simulations ◽

Associated Factor ◽

Terminal Domain

TNF Receptor Associated Factor 2 (TRAF2) is a trimeric protein that belongs to the TNF receptor associated factor family (TRAFs). The TRAF2 oligomeric state is crucial for receptor binding and for its interaction with other proteins involved in the TNFR signaling. The monomer-trimer equilibrium of a C- terminal domain truncated form of TRAF2 (TRAF2-C), plays also a relevant role in binding the membrane, causing inward vesiculation. In this study, we have investigated the conformational dynamics of TRAF2-C through circular dichroism, fluorescence, and dynamic light scattering, performing temperature-dependent measurements. The data indicate that the protein retains its oligomeric state and most of its secondary structure, while displaying a significative increase in the heterogeneity of the tyrosines signal, increasing the temperature from ≈15 to ≈35 °C. The peculiar crowding of tyrosine residues (12 out of 18) at the three subunit interfaces and the strong dependence on the trimer concentration indicate that such conformational changes mainly involve the contact areas between each pair of monomers, affecting the oligomeric state. Molecular dynamic simulations in this temperature range suggest that the interfaces heterogeneity is an intrinsic property of the trimer that arises from the continuous, asymmetric approaching and distancing of its subunits. Such dynamics affect the results of molecular docking on the external protein surface using receptor peptides, indicating that the TRAF2-receptor interaction in the solution might not involve three subunits at the same time, as suggested by the static analysis obtainable from the crystal structure. These findings shed new light on the role that the TRAF2 oligomeric state might have in regulating the protein binding activity in vivo.

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The Carboxy-Terminal Domain of Glycoprotein N of Human Cytomegalovirus Is Required for Virion Morphogenesis

Journal of Virology ◽

10.1128/jvi.01463-06 ◽

2007 ◽

Vol 81 (10) ◽

pp. 5212-5224 ◽

Cited By ~ 32

Author(s):

Michael Mach ◽

Karolina Osinski ◽

Barbara Kropff ◽

Ursula Schloetzer-Schrehardt ◽

Magdalena Krzyzaniak ◽

...

Keyword(s):

Human Cytomegalovirus ◽

Critical Role ◽

Point Mutations ◽

Cysteine Residue ◽

Cysteine Residues ◽

Type I ◽

Terminal Domain ◽

Carboxy Terminal Domain ◽

Carboxy Terminal ◽

Assembly Compartment

ABSTRACT Glycoproteins M and N (gM and gN, respectively) are among the few proteins that are conserved across the herpesvirus family. The function of the complex is largely unknown. Whereas deletion from most alphaherpesviruses has marginal effects on the replication of the respective viruses, both proteins are essential for replication of human cytomegalovirus (HCMV). We have constructed a series of mutants in gN to study the function of this protein. gN of HCMV is a type I glycoprotein containing a short carboxy-terminal domain of 14 amino acids, including two cysteine residues directly adjacent to the predicted transmembrane anchor at positions 125 and 126. Deletion of the entire carboxy-terminal domain as well as substitution with the corresponding region from alpha herpesviruses or mutations of both cysteine residues resulted in a replication-incompetent virus. Recombinant viruses containing point mutations of either cysteine residue could be generated. These viruses were profoundly defective for replication. Complex formation of the mutant gNs with gM and transport of the complex to the viral assembly compartment appeared unaltered compared to the wild type. However, in infected cells, large numbers of capsids accumulated in the cytoplasm that failed to acquire an envelope. Transiently expressed gN was shown to be modified by palmitic acid at both cysteine residues. In summary, our data suggest that the carboxy-terminal domain of gN plays a critical role in secondary envelopment of HCMV and that palmitoylation of gN appears to be essential for function in secondary envelopment of HCMV and virus replication.

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Pretreatment with d-myo-Inositol Trisphosphate Reduces Infarct Size in Rabbit Hearts: Role of Inositol Trisphosphate Receptors and Gap Junctions in Triggering Protection

Journal of Pharmacology and Experimental Therapeutics ◽

10.1124/jpet.105.087742 ◽

2005 ◽

Vol 314 (3) ◽

pp. 1386-1392 ◽

Cited By ~ 18

Author(s):

Karin Przyklenk ◽

Michelle Maynard ◽

Chad E. Darling ◽

Peter Whittaker

Keyword(s):

Gap Junctions ◽

Infarct Size ◽

Inositol Trisphosphate ◽

Inositol Trisphosphate Receptors

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Characterisation and molecular dynamic simulations of J15 asparaginase from Photobacterium sp. strain J15.

Acta Biochimica Polonica ◽

10.18388/abp.2014_1840 ◽

2014 ◽

Vol 61 (4) ◽

Cited By ~ 10

Author(s):

Mohd Adilin Yaacob ◽

Wan Atiqah Najiah Wan Hasan ◽

Mohd Shukuri Mohamad Ali ◽

Raja Noor Zaliha Raja Abdul Rahman ◽

Abu Bakar Salleh ◽

...

Keyword(s):

Molecular Dynamic ◽

Conformational Changes ◽

Specific Activity ◽

3D Structure ◽

Coli Strain ◽

Exchange Chromatography ◽

Molecular Dynamic Simulations ◽

Type I ◽

Enzymatic Reactions ◽

Dynamic Simulations

Genome mining revealed a 1011 nucleotide-long fragment encoding a type I L-asparaginase (J15 asparaginase) from the halo-tolerant Photobacterium sp. strain J15. The gene was overexpressed in pET-32b (+) vector in E. coli strain Rosetta-gami B (DE3) pLysS and purified using two-step chromatographic methods: Ni(2+)-Sepharose affinity chromatography and Q-Sepharose anion exchange chromatography. The final specific activity and yield of the enzyme achieved from these steps were 20 U/mg and 49.2%, respectively. The functional dimeric form of J15-asparaginase was characterised with a molecular weight of ~70 kDa. The optimum temperature and pH were 25°C and pH 7.0, respectively. This protein was stable in the presence of 1 mM Ni(2+) and Mg(2+), but it was inhibited by Mn(2+), Fe(3+) and Zn(2+) at the same concentration. J15 asparaginase actively hydrolysed its native substrate, l-asparagine, but had low activity towards l-glutamine. The melting temperature of J15 asparaginase was ~51°C, which was determined using denatured protein analysis of CD spectra. The Km, Kcat, Kcat/Km of J15 asparaginase were 0.76 mM, 3.2 s(-1), and 4.21 s(-1) mM(-1), respectively. Conformational changes of the J15 asparaginase 3D structure at different temperatures (25°C, 45°C, and 65°C) were analysed using Molecular Dynamic simulations. From the analysis, residues Tyr₂₄ , His₂₂, Gly₂₃, Val₂₅ and Pro₂₆ may be directly involved in the 'open' and 'closed' lid-loop conformation, facilitating the conversion of substrates during enzymatic reactions. The properties of J15 asparaginase, which can work at physiological pH and has low glutaminase activity, suggest that this could be a good candidate for reducing toxic effects during cancer treatment.

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