The N-Terminal Domain of the Tomato Immune Protein Prf Contains Multiple Homotypic and Pto Kinase Interaction Sites

Small molecule inhibitors of clathrin-mediated endocytosis are highly desired for the dissection of membrane trafficking pathways in the lab and for potential use as anti-infectives in the clinic. One inhibition strategy is to prevent clathrin from contacting adaptor proteins so that clathrin-mediated endocytosis cannot occur. "Pitstop" compounds have been developed which block only one of the four functional interaction sites on the N-terminal domain of clathrin heavy chain. Despite this limitation, Pitstop 2 causes profound inhibition of clathrin-mediated endocytosis. In this study, we probed for non-specific activity of Pitstop 2 by examining its action in cells expressing clathrin heavy chain harbouring mutations in the N-terminal domain interaction sites. We conclude that the inhibition observed with this compound is due to non-specificity, i.e. it causes inhibition away from its proposed mode of action. We recommend that these compounds be used with caution in cells and that they should not be used to conclude anything of the function of clathrin’s N-terminal domain.

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Mapping Functional Interaction Sites of Human Prune C-Terminal Domain by NMR Spectroscopy in Human Cell Lysates

Chemistry - A European Journal ◽

10.1002/chem.201302168 ◽

2013 ◽

Vol 19 (37) ◽

pp. 12217-12220 ◽

Cited By ~ 7

Author(s):

Donatella Diana ◽

Giovanni Smaldone ◽

Pasquale De Antonellis ◽

Luciano Pirone ◽

MariaNeve Carotenuto ◽

...

Keyword(s):

Nmr Spectroscopy ◽

Human Cell ◽

Functional Interaction ◽

Cell Lysates ◽

Interaction Sites ◽

Terminal Domain

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A Temperature-Sensitive Lesion in the N-Terminal Domain of the Rotavirus Polymerase Affects Its Intracellular Localization and Enzymatic Activity

Journal of Virology ◽

10.1128/jvi.00062-17 ◽

2017 ◽

Vol 91 (7) ◽

Cited By ~ 5

Author(s):

Allison O. McKell ◽

Leslie E. W. LaConte ◽

Sarah M. McDonald

Keyword(s):

Enzymatic Activity ◽

Rna Synthesis ◽

Viral Proteins ◽

Viral Rna ◽

Host Cells ◽

Temperature Sensitive ◽

Mutant Form ◽

Interaction Sites ◽

Terminal Domain ◽

Infected Cells

ABSTRACT Temperature-sensitive (ts) mutants of simian rotavirus (RV) strain SA11 have been previously created to investigate the functions of viral proteins during replication. One mutant, SA11-tsC, has a mutation that maps to the gene encoding the VP1 polymerase and shows diminished growth and RNA synthesis at 39°C compared to that at 31°C. In the present study, we sequenced all 11 genes of SA11-tsC, confirming the presence of an L138P mutation in the VP1 N-terminal domain and identifying 52 additional mutations in four other viral proteins (VP4, VP7, NSP1, and NSP2). To investigate whether the L138P mutation induces a ts phenotype in VP1 outside the SA11-tsC genetic context, we employed ectopic expression systems. Specifically, we tested whether the L138P mutation affects the ability of VP1 to localize to viroplasms, which are the sites of RV RNA synthesis, by expressing the mutant form as a green fluorescent protein (GFP) fusion protein (VP1L138P-GFP) (i) in wild-type SA11-infected cells or (ii) in uninfected cells along with viroplasm-forming proteins NSP2 and NSP5. We found that VP1L138P-GFP localized to viroplasms and interacted with NSP2 and/or NSP5 at 31°C but not at 39°C. Next, we tested the enzymatic activity of a recombinant mutant polymerase (rVP1L138P) in vitro and found that it synthesized less RNA at 39°C than at 31°C, as well as less RNA than the control at all temperatures. Together, these results provide a mechanistic basis for the ts phenotype of SA11-tsC and raise important questions about the role of leucine 138 in supporting key protein interactions and the catalytic function of the VP1 polymerase. IMPORTANCE RVs cause diarrhea in the young of many animal species, including humans. Despite their medical and economic importance, gaps in knowledge exist about how these viruses replicate inside host cells. Previously, a mutant simian RV (SA11-tsC) that replicates worse at higher temperatures was identified. This virus has an amino acid mutation in VP1, which is the enzyme responsible for copying the viral RNA genome. The mutation is located in a poorly understood region of the polymerase called the N-terminal domain. In this study, we determined that the mutation reduces the ability of VP1 to properly localize within infected cells at high temperatures, as well as reduced the ability of the enzyme to copy viral RNA in a test tube. The results of this study explain the temperature sensitivity of SA11-tsC and shed new light on functional protein-protein interaction sites of VP1.

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Structure of clathrin cages and coats in ice

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s042482010014213x ◽

1986 ◽

Vol 44 ◽

pp. 94-97

Author(s):

G.P.A. Vigers ◽

R.A. Crowther ◽

B.M.F. Pearse

Keyword(s):

Electron Microscopy ◽

Heavy Chain ◽

Outer Part ◽

Coated Vesicles ◽

Eukaryotic Cells ◽

Coat Proteins ◽

Terminal Domain ◽

Clathrin Heavy Chain ◽

Membrane Components

Clathrin forms the polyhedral cage of coated vesicles, which mediate the transfer of selected membrane components within eukaryotic cells. Clathrin cages and coated vesicles have been extensively studied by electron microscopy of negatively stained preparations and shadowed specimens. From these studies the gross morphology of the outer part of the polyhedral coat has been established and some features of the packing of clathrin trimers into the coat have also been described. However these previous studies have not revealed any internal details about the position of the terminal domain of the clathrin heavy chain, the location of the 100kd-50kd accessory coat proteins or the interactions of the coat with the enclosed membrane.

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Crystal structures of Magnaporthe oryzae trehalose-6-phosphate synthase (MoTps1) suggest a model for catalytic process of Tps1

Biochemical Journal ◽

10.1042/bcj20190289 ◽

2019 ◽

Vol 476 (21) ◽

pp. 3227-3240 ◽

Cited By ~ 1

Author(s):

Shanshan Wang ◽

Yanxiang Zhao ◽

Long Yi ◽

Minghe Shen ◽

Chao Wang ◽

...

Keyword(s):

Crystal Structures ◽

Conformational Changes ◽

Magnaporthe Oryzae ◽

Structural Information ◽

Complex Structure ◽

Critical Role ◽

Catalytic Process ◽

Structural Basis ◽

Salt Bridges ◽

Terminal Domain

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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