Rab1-AMPylation by Legionella DrrA is allosterically activated by Rab1

AbstractLegionella pneumophila infects eukaryotic cells by forming a replicative organelle – the Legionella containing vacuole. During this process, the bacterial protein DrrA/SidM is secreted and manipulates the activity and post-translational modification (PTM) states of the vesicular trafficking regulator Rab1. As a result, Rab1 is modified with an adenosine monophosphate (AMP), and this process is referred to as AMPylation. Here, we use a chemical approach to stabilise low-affinity Rab:DrrA complexes in a site-specific manner to gain insight into the molecular basis of the interaction between the Rab protein and the AMPylation domain of DrrA. The crystal structure of the Rab:DrrA complex reveals a previously unknown non-conventional Rab-binding site (NC-RBS). Biochemical characterisation demonstrates allosteric stimulation of the AMPylation activity of DrrA via Rab binding to the NC-RBS. We speculate that allosteric control of DrrA could in principle prevent random and potentially cytotoxic AMPylation in the host, thereby perhaps ensuring efficient infection by Legionella.

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Characterization of Enzymes from Legionella pneumophila Involved in Reversible Adenylylation of Rab1 Protein

Journal of Biological Chemistry ◽

10.1074/jbc.m112.396861 ◽

2012 ◽

Vol 287 (42) ◽

pp. 35036-35046 ◽

Cited By ~ 24

Author(s):

Matthias P. Müller ◽

Alexander V. Shkumatov ◽

Lena K. Oesterlin ◽

Stefan Schoebel ◽

Philip R. Goody ◽

...

Keyword(s):

Legionella Pneumophila ◽

Adenosine Monophosphate ◽

Complex Model ◽

Covalent Modification ◽

Vesicular Trafficking ◽

Host Cells ◽

X Ray Scattering ◽

Main Regulator ◽

Kinetics Of

After the pathogenic bacterium Legionella pneumophila is phagocytosed, it injects more than 250 different proteins into the cytoplasm of host cells to evade lysosomal digestion and to replicate inside the host cell. Among these secreted proteins is the protein DrrA/SidM, which has been shown to modify Rab1b, a main regulator of vesicular trafficking in eukaryotic cells, by transfer of adenosine monophosphate (AMP) to Tyr77. In addition, Legionella provides the protein SidD that hydrolytically reverses the covalent modification, suggesting a tight spatial and temporal control of Rab1 function by Legionella during infection. Small angle x-ray scattering experiments of DrrA allowed us to validate a tentative complex model built by combining available crystallographic data. We have established the effects of adenylylation on Rab1 interactions and properties in a quantitative way. In addition, we have characterized the kinetics of DrrA-catalyzed adenylylation as well as SidD-catalyzed deadenylylation toward Rab1 and have determined the nucleotide specificities of both enzymes. This study enhances our knowledge of proteins subverting Rab1 function at the Legionella-containing vacuole.

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Development of Fragility Curves for Piping and Slope Stability of River Levees

Water ◽

10.3390/w13050738 ◽

2021 ◽

Vol 13 (5) ◽

pp. 738

Author(s):

Nicola Rossi ◽

Mario Bačić ◽

Meho Saša Kovačević ◽

Lovorka Librić

Keyword(s):

Slope Stability ◽

Fragility Curves ◽

Safety Margin ◽

Water Levels ◽

Flood Event ◽

Soil Parameters ◽

Design Code ◽

A Site ◽

Insight Into

The design code Eurocode 7 relies on semi-probabilistic calculation procedures, through utilization of the soil parameters obtained by in situ and laboratory tests, or by the means of transformation models. To reach a prescribed safety margin, the inherent soil parameter variability is accounted for through the application of partial factors to either soil parameters directly or to the resistance. However, considering several sources of geotechnical uncertainty, including the inherent soil variability, measurement error and transformation uncertainty, full probabilistic analyses should be implemented to directly consider the site-specific variability. This paper presents the procedure of developing fragility curves for levee slope stability and piping as failure mechanisms that lead to larger breaches, where a direct influence of the flood event intensity on the probability of failure is calculated. A range of fragility curve sets is presented, considering the variability of levee material properties and varying durations of the flood event, thus providing crucial insight into the vulnerability of the levee exposed to rising water levels. The procedure is applied to the River Drava levee, a site which has shown a continuous trend of increased water levels in recent years.

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“IF YOU DON’T KNOW ME BY NOW…”

Du Bois Review Social Science Research on Race ◽

10.1017/s1742058x21000084 ◽

2021 ◽

pp. 1-28

Author(s):

Ellen M. Whitehead ◽

Allan Farrell ◽

Jenifer L. Bratter

Keyword(s):

Race And Ethnicity ◽

Mate Selection ◽

Racial Identities ◽

Hispanic Origin ◽

Socially Constructed ◽

Attitudinal Factors ◽

A Site ◽

Intimate Partnerships ◽

College Educated ◽

Insight Into

ABSTRACT The racial composition of couples is a salient indicator of race’s impact on mate selection, but how well do those in intimate partnerships know the racial identities of their partners? While prior research has revealed that an individual’s race may be perceived differently than how they identify, most of what is known comes from brief interactions, with less information on established relationships. This study examines whether discrepancies in the reports of a person’s race or ethnicity can be identified even within intimate relationships, as well as which relational, social, and attitudinal factors are predictive of divergent or concordant reports. We draw on the Fragile Families and Child Wellbeing Study (n=3467), a U.S.-based dataset that uniquely provides both the father’s self-reported race and Hispanic origin and the mother’s report of the father’s race and ethnicity. We compare reports of the father’s race/Hispanic origin from both parents to assess the extent of mismatch, and we distinguish between whether mothers view the father’s race as similar to or different from her own. We find roughly 14% of mothers provide a race and Hispanic origin that is inconsistent with the father’s report, with a large share reflecting differences in the self-identified and perceived race of fathers who are reported as Hispanic. Among mismatched reports, mothers are more likely to report a race/ethnicity for the father that matches her own, depressing the number reporting interracial unions. Perceptions of racial homogamy are especially likely when mothers view racial sameness as important to marriage. Further, mismatches are more common in the midst of weak relational ties (i.e. non-marital relationships) and are less common when both parents are college-educated. These findings reveal that intimate unions are a site where race is socially constructed and provide insight into how norms of endogamy manifest within formed relationships.

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Structure of a canonical RNase YoeB bound to the ribosome

Acta Crystallographica Section A Foundations and Advances ◽

10.1107/s2053273314097927 ◽

2014 ◽

Vol 70 (a1) ◽

pp. C207-C207

Author(s):

Yun Chen ◽

Shu Feng ◽

Katsuhiko Kamada ◽

Han Wang ◽

Kai Tang ◽

...

Keyword(s):

Specific Activity ◽

Base Catalysis ◽

Mass Spectrometry Data ◽

Catalytic Core ◽

General Base ◽

Rna Hydrolysis ◽

70S Ribosome ◽

A Site ◽

Insight Into

As a typical endoribonuclease, YoeB mediates cellular adaptation in diverse bacteria by degrading mRNAs on its activation. Although the catalytic core of YoeB is thought to be identical to well-studied nucleases, this enzyme specifically targets mRNA substrates that are associated with ribosomes in vivo. However, the molecular mechanism of mRNA recognition and cleavage by YoeB, and the requirement of ribosome for its optimal activity, largely remain elusive. Here, we report the structure of YoeB bound to 70S ribosome in pre-cleavage state, revealing that both the 30S and 50S subunits participate in YoeB binding. The mRNA is recognized by the catalytic core of YoeB, of which the general base/acid (Glu46/His83) are within hydrogen-bonding distance to their reaction atoms, demonstrating an active conformation of YoeB on ribosome. Also, the mRNA orientation involves the universally conserved A1493 and G530 of 16S rRNA. In addition, mass spectrometry data indicated that YoeB cleaves mRNA following the second position at the A-site codon, resulting in a final product with a 3'–phosphate at the newly formed 3' end. Our results demonstrate a classical acid-base catalysis for YoeB-mediated RNA hydrolysis and provide insight into how the ribosome is essential for its specific activity.

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Structural and functional studies of pyruvate carboxylase regulation by cyclic di-AMP in lactic acid bacteria

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1704756114 ◽

2017 ◽

Vol 114 (35) ◽

pp. E7226-E7235 ◽

Cited By ~ 20

Author(s):

Philip H. Choi ◽

Thu Minh Ngoc Vu ◽

Huong Thi Pham ◽

Joshua J. Woodward ◽

Mark S. Turner ◽

...

Keyword(s):

Binding Site ◽

Conformational Changes ◽

Pyruvate Carboxylase ◽

Adenosine Monophosphate ◽

Binding Mode ◽

Functional Studies ◽

Cellular Processes ◽

Wide Range ◽

A Site ◽

Amp Inhibition

Cyclic di-3′,5′-adenosine monophosphate (c-di-AMP) is a broadly conserved bacterial second messenger that has been implicated in a wide range of cellular processes. Our earlier studies showed that c-di-AMP regulates central metabolism inListeria monocytogenesby inhibiting its pyruvate carboxylase (LmPC), a biotin-dependent enzyme with biotin carboxylase (BC) and carboxyltransferase (CT) activities. We report here structural, biochemical, and functional studies on the inhibition ofLactococcus lactisPC (LlPC) by c-di-AMP. The compound is bound at the dimer interface of the CT domain, at a site equivalent to that in LmPC, although it has a distinct binding mode in the LlPC complex. This binding site is not well conserved among PCs, and only a subset of these bacterial enzymes are sensitive to c-di-AMP. Conformational changes in the CT dimer induced by c-di-AMP binding may be the molecular mechanism for its inhibitory activity. Mutations of residues in the binding site can abolish c-di-AMP inhibition. InL. lactis, LlPC is required for efficient milk acidification through its essential role in aspartate biosynthesis. The aspartate pool inL. lactisis negatively regulated by c-di-AMP, and high aspartate levels can be restored by expression of a c-di-AMP–insensitive LlPC. LlPC has high intrinsic catalytic activity and is not sensitive to acetyl-CoA activation, in contrast to other PC enzymes.

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C-terminal tail polyglycylation and polyglutamylation alter microtubule mechanical properties

10.1101/791194 ◽

2019 ◽

Author(s):

Kathryn P. Wall ◽

Harold Hart ◽

Thomas Lee ◽

Cynthia Page ◽

Taviare L. Hawkins ◽

...

Keyword(s):

Mechanical Properties ◽

Molecular Mechanisms ◽

High Stress ◽

Resonance Spectroscopy ◽

Post Translational Modification ◽

Cellular Functions ◽

Peptide Backbone ◽

Post Translational Modifications ◽

Intrinsic Stiffness ◽

Insight Into

ABSTRACTMicrotubules are biopolymers that perform diverse cellular functions. The regulation of microtubule behavior occurs in part through post-translational modification of both the α- and β- subunits of tubulin. One class of modifications is the heterogeneous addition of glycine and glutamate residues to the disordered C-terminal tails of tubulin. Due to their prevalence in stable, high stress cellular structures such as cilia, we sought to determine if these modifications alter the intrinsic stiffness of microtubules. Here we describe the purification and characterization of differentially-modified pools of tubulin from Tetrahymena thermophila. We found that glycylation on the α-C-terminal tail is a key determinant of microtubule stiffness, but does not affect the number of protofilaments incorporated into microtubules. We measured the dynamics of the tail peptide backbone using nuclear magnetic resonance spectroscopy. We found that the spin-spin relaxation rate (R2) showed a pronounced decreased as a function of distance from the tubulin surface for the α-tubulin tail, indicating that the α-tubulin tail interacts with the dimer surface. This suggests that the interactions of the α-C-terminal tail with the tubulin body contributes to the stiffness of the assembled microtubule, providing insight into the mechanism by which glycylation and glutamylation can alter microtubule mechanical properties.SIGNIFICANCEMicrotubules are regulated in part by post-translational modifications including the heterogeneous addition of glycine and glutamate residues to the C-terminal tails. By producing and characterizing differentially-modified tubulin, this work provides insight into the molecular mechanisms of how these modifications alter intrinsic microtubule properties such as flexibility. These results have broader implications for mechanisms of how ciliary structures are able to function under high stress.

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Spontaneous ribosomal translocation of mRNA and tRNAs into a chimeric hybrid state

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1901310116 ◽

2019 ◽

Vol 116 (16) ◽

pp. 7813-7818 ◽

Cited By ~ 18

Author(s):

Jie Zhou ◽

Laura Lancaster ◽

John Paul Donohue ◽

Harry F. Noller

Keyword(s):

Crystal Structure ◽

Elongation Factor ◽

Intermediate Complex ◽

Reading Frame ◽

Hybrid State ◽

Head Domain ◽

30S Subunit ◽

A Site ◽

Insight Into ◽

Factor G

The elongation factor G (EF-G)–catalyzed translocation of mRNA and tRNA through the ribosome is essential for vacating the ribosomal A site for the next incoming aminoacyl-tRNA, while precisely maintaining the translational reading frame. Here, the 3.2-Å crystal structure of a ribosome translocation intermediate complex containing mRNA and two tRNAs, formed in the absence of EF-G or GTP, provides insight into the respective roles of EF-G and the ribosome in translocation. Unexpectedly, the head domain of the 30S subunit is rotated by 21°, creating a ribosomal conformation closely resembling the two-tRNA chimeric hybrid state that was previously observed only in the presence of bound EF-G. The two tRNAs have moved spontaneously from their A/A and P/P binding states into ap/P and pe/E states, in which their anticodon loops are bound between the 30S body domain and its rotated head domain, while their acceptor ends have moved fully into the 50S P and E sites, respectively. Remarkably, the A-site tRNA translocates fully into the classical P-site position. Although the mRNA also undergoes movement, codon–anticodon interaction is disrupted in the absence of EF-G, resulting in slippage of the translational reading frame. We conclude that, although movement of both tRNAs and mRNA (along with rotation of the 30S head domain) can occur in the absence of EF-G and GTP, EF-G is essential for enforcing coupled movement of the tRNAs and their mRNA codons to maintain the reading frame.

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Further Probing of Cu2+-Dependent PNAzymes Acting as Artificial RNA Restriction Enzymes

Molecules ◽

10.3390/molecules24040672 ◽

2019 ◽

Vol 24 (4) ◽

pp. 672 ◽

Cited By ~ 3

Author(s):

Olivia Luige ◽

Merita Murtola ◽

Alice Ghidini ◽

Roger Strömberg

Keyword(s):

Peptide Nucleic Acid ◽

Cleavage Site ◽

Restriction Enzymes ◽

Rna Cleavage ◽

Artificial Enzymes ◽

Rna Sequences ◽

Specific Manner ◽

A Site ◽

Fold Excess ◽

Insight Into

Peptide nucleic acid (PNA)-neocuproine conjugates have been shown to efficiently catalyse the cleavage of RNA target sequences in the presence of Cu2+ ions in a site-specific manner. These artificial enzymes are designed to force the formation of a bulge in the RNA target, the sequence of which has been shown to be key to the catalytic activity. Here, we present a further investigation into the action of Cu2+-dependent PNAzymes with respect to the dependence on bulge composition in 3- and 4-nucleotide bulge systems. Cu2+-dependent PNAzymes were shown to have a clear preference for 4-nucleotide bulges, as the cleavage of 3-nucleotide bulge-forming RNA sequences was significantly slower, which is illustrated by a shift in the half-lives from approximately 30 min to 24 h. Nonetheless, the nucleotide preferences at different positions in the bulge displayed similar trends in both systems. Moreover, the cleavage site was probed by introducing critical chemical modifications to one of the cleavage site nucleotides of the fastest cleaved 4-nucleotide RNA bulge. Namely, the exclusion of the exocyclic amine of the central adenine and the replacement of the 2′-hydroxyl nucleophile with 2′-H or 2′-OMe substituents in the RNA severely diminished the rate of RNA cleavage by the Cu2+-dependent PNAzyme, giving insight into the mechanism of cleavage. Moreover, the shorter recognition arm of the RNA/PNAzyme complex was modified by extending the PNAzyme by two additional nucleobases. The new PNAzyme was able to efficiently promote the cleavage of RNA when fully hybridised to a longer RNA target and even outperform the previous fastest PNAzyme. The improvement was demonstrated in cleavage studies with stoichiometric amounts of either PNAzyme present, and the extended PNAzyme was also shown to give turnover with a 10-fold excess of the RNA target.

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Lightweight WLCG Sites

EPJ Web of Conferences ◽

10.1051/epjconf/201921407019 ◽

2019 ◽

Vol 214 ◽

pp. 07019 ◽

Cited By ~ 1

Author(s):

Mayank Sharma ◽

Maarten Litmaath ◽

Eraldo Silva Junior ◽

Renato Santana

Keyword(s):

Grid Services ◽

Management Tools ◽

Batch Systems ◽

Common Interface ◽

Container Orchestration ◽

A Site ◽

Automation Tools ◽

Orchestration Tools ◽

Insight Into ◽

New Framework

This article describes a new framework, called SIMPLE, for settingup and maintaining classic WLCG sites with minimal operational efforts and insights needed into the WLCG middleware. The framework provides a single common interface to install and configure any of its supported grid services, such as Compute Elements, Batch Systems, Worker Nodes and miscellaneous middleware packages. It leverages modern container orchestration tools like Kubernetes, Docker Swarm, and confiuration management tools like Puppet, Ansible, to automate deployment of the WLCG services on behalf of a site admin. The framework is modular and extensible by design. Therefore, it is easy to add support for more grid services as well as infrastructure automation tools to accommodate diverse scenarios at different sites. We provide insight into the design of the framework and our efforts towards development, release and deployment of its first implementation featuring CREAM E, TORQUE Batch System and TORQUE based Worker Nodes.

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Mapping of Allosteric Modulator Effects in the Active Site of Factor IXa.

Blood ◽

10.1182/blood.v110.11.2695.2695 ◽

2007 ◽

Vol 110 (11) ◽

pp. 2695-2695

Author(s):

Karla Villegas ◽

Kimberly Baker-Deadmond ◽

Pierre F. Neuenschwander

Keyword(s):

Ethylene Glycol ◽

Active Site ◽

Allosteric Modulation ◽

Factor Ix ◽

Ionic Calcium ◽

Gla Domain ◽

A Site ◽

Hydrolysis Of ◽

Insight Into ◽

Active Site Region

Abstract Activated factor IX (fIXa) is a vitamin K-dependent blood coagulation serine protease involved in propagation of the coagulant response through activation of fX. Maximal enzymatic and procoagulant activity of fIXa requires the presence of several cofactors; one of which is ionic calcium, which is known to bind to a site in the protease domain of fIXa as well as several sites within the light chain Gla domain region. One of the roles of calcium appears to be allosteric modulation of the fIXa active site as evidenced by an increase in enzymatic activity towards small peptidyl substrates. We and others have additionally found that certain small hygroscopic molecules can also enhance fIXa amidolytic activity. The molecular details involved in either of these effects are not well understood. Previous studies by us have shown that a pentapeptide substrate (AGRSL; the reactive site sequence of antithrombin) is hydrolyzed by fIXa in the absence of cofactors or modulators. This hydrolysis is enhanced in the presence of ionic calcium, ethylene glycol or low molecular weight heparin suggesting effects of these molecules on the immediate active site vicinity of fIXa. In order to gain insight into the potential allosteric modulation that each of these effectors may affect in fIXa, we examined the hydrolysis of four peptide libraries based on the AGRSL pentapeptide sequence, in the presence and absence of various fIXa modulators. The four libraries synthesized were XGRSL, AXRSL, AGRXL and AGRSX; where X denotes any of the possible 20 amino acids. Each of these libraries were screened for hydrolysis by fIXa under various conditions with substrates and products being identified en masse using MALDI-TOF mass spectrometry. The results suggest that ionic calcium enhances fIXa reactivity in part by modulation of the S2 subsite in fIXa. In contrast, ethylene glycol enhances fIXa activity via modulation of the S3 subsite and heparin was found to effect the overall active site region.

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