Preferential phosphatidylinositol 5-phosphate binding contributes to a destabilization of the VHS domain structure of Tom1

AbstractTom1 transports endosomal ubiquitinated proteins that are targeted for degradation in the lysosomal pathway. Infection of eukaryotic cells by Shigella flexneri boosts oxygen consumption and promotes the synthesis of phosphatidylinositol-5-phosphate [PtdIns5P], which triggers Tom1 translocation to signaling endosomes. Removing Tom1 from its cargo trafficking function hinders protein degradation in the host and, simultaneously, enables bacterial survival. Tom1 preferentially binds PtdIns5P via its VHS domain, but the effects of a reducing environment as well as PtdIns5P on the domain structure and function are unknown. Thermal denaturation studies demonstrate that, under reducing conditions, the monomeric Tom1 VHS domain switches from a three-state to a two-state transition behavior. PtdIns5P reduced thermostability, interhelical contacts, and conformational compaction of Tom1 VHS, suggesting that the phosphoinositide destabilizes the protein domain. Destabilization of Tom1 VHS structure was also observed with other phospholipids. Isothermal calorimetry data analysis indicates that, unlike ubiquitin, Tom1 VHS endothermically binds to PtdIns5P through two noncooperative binding sites, with its acyl chains playing a relevant role in the interaction. Altogether, these findings provide mechanistic insights about the recognition of PtdIns5P by the VHS domain that may explain how Tom1, when in a different VHS domain conformational state, interacts with downstream effectors under S. flexneri infection.

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Trypanosome Capping Enzymes Display a Novel Two-Domain Structure

Molecular and Cellular Biology ◽

10.1128/mcb.18.8.4612 ◽

1998 ◽

Vol 18 (8) ◽

pp. 4612-4619 ◽

Cited By ~ 22

Author(s):

Erika Silva ◽

Elisabetta Ullu ◽

Ryuji Kobayashi ◽

Christian Tschudi

Keyword(s):

Domain Structure ◽

Consensus Sequence ◽

Rna Modification ◽

Phosphate Binding ◽

Amino Terminal ◽

Mrna Capping ◽

Capping Enzyme ◽

Eukaryotic Mrnas ◽

Carboxy Terminal ◽

Capping Enzymes

ABSTRACT The ubiquitous m7G cap of eukaryotic mRNAs and of precursors to the spliceosomal small nuclear RNAs (snRNAs) is the result of an essential RNA modification acquired during transcript elongation. In trypanosomes, the m7G cap is restricted to the spliced leader (SL) RNA and the precursors of U2, U3, and U4 snRNAs. mRNA capping in these organisms occurs posttranscriptionally bytrans splicing, which transfers the capped SL sequence to the 5′ ends of all mRNAs. The SL cap is the most elaborate cap structure known in nature and has been shown to consist of an m7G residue followed by four methylated nucleotides. UsingCrithidia fasciculata, we have characterized and purified the guanylyltransferase (capping enzyme), which transfers GMP from GTP to the diphosphate end of RNA. The corresponding gene codes for a protein of 697 amino acids, with the carboxy-terminal half of theC. fasciculata guanylyltransferase containing the six signature motifs previously identified in yeast capping enzymes. The amino-terminal half contains a domain that displays no resemblance to any other domain associated with capping enzymes. Intriguingly, this region harbors a consensus sequence for a phosphate-binding loop which is found in ATP- and GTP-binding proteins. This two-domain structure is also present in the Trypanosoma brucei capping enzyme, which shows 44% overall identity with the C. fasciculatacapping enzyme. Thus, this structure appears to be common to all trypanosomatid protozoa and defines a novel class of capping enzymes.

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Domains and domain nucleation in magnetron-sputtered CoCr thin films

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100151064 ◽

1993 ◽

Vol 51 ◽

pp. 1046-1047

Author(s):

B. G. Demczyk

Keyword(s):

Thin Films ◽

Domain Structure ◽

Magnetic Domain ◽

Domain Size ◽

Film Plane ◽

Magnetic Domain Structure ◽

Perpendicular Magnetization ◽

Columnar Grains ◽

Reversal Mechanism ◽

Lorentz Transmission Electron Microscopy

CoCr thin films have been of interest for a number of years due to their strong perpendicular anisotropy, favoring magnetization normal to the film plane. The microstructure and magnetic properties of CoCr films prepared by both rf and magnetron sputtering have been examined in detail. By comparison, however, relatively few systematic studies of the magnetic domain structure and its relation to the observed film microstructure have been reported. In addition, questions still remain as to the operative magnetization reversal mechanism in different film thickness regimes. In this work, the magnetic domain structure in magnetron sputtered Co-22 at.%Cr thin films of known microstructure were examined by Lorentz transmission electron microscopy. Additionally, domain nucleation studies were undertaken via in-situ heating experiments.It was found that the 50 nm thick films, which are comprised of columnar grains, display a “dot” type domain configuration (Figure 1d), characteristic of a perpendicular magnetization. The domain size was found to be on the order of a few structural columns in diameter.

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Ferroelectric Domain Structure of Pb(Zr.52Ti.48)03

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s1431927600000933 ◽

1980 ◽

Vol 38 ◽

pp. 214-215

Author(s):

E.K. Goo ◽

R.K. Mishra

Keyword(s):

Phase Transformation ◽

Domain Structure ◽

Tetragonal Phase ◽

Ferroelectric Domain ◽

Cubic Phase ◽

Ion Milling ◽

Thin Foils ◽

Ferroelectric Domain Structure ◽

Ferroelectric Domains

Ferroelectric domains are twins that are formed when PZT undergoes a phase transformation from a non-ferroelectric cubic phase to a ferroelectric tetragonal phase upon cooling below ∼375°C.,1 The tetragonal phase is spontaneously polarized in the direction of c-axis, making each twin a ferroelectric domain. Thin foils of polycrystalline Pb (Zr.52Ti.48)03 were made by ion milling and observed in the Philips EM301 with a double tilt stage.

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