conserved tyrosine
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2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Anna Lepesheva ◽  
Adriana Osickova ◽  
Jana Holubova ◽  
David Jurnecka ◽  
Sarka Knoblochova ◽  
...  

AbstractPore-forming repeats in toxins (RTX) are key virulence factors of many Gram-negative pathogens. We have recently shown that the aromatic side chain of the conserved tyrosine residue 940 within the acylated segment of the RTX adenylate cyclase toxin-hemolysin (CyaA, ACT or AC-Hly) plays a key role in target cell membrane interaction of the toxin. Therefore, we used a truncated CyaA-derived RTX719 construct to analyze the impact of Y940 substitutions on functional folding of the acylated segment of CyaA. Size exclusion chromatography combined with CD spectroscopy revealed that replacement of the aromatic side chain of Y940 by the side chains of alanine or proline residues disrupted the calcium-dependent folding of RTX719 and led to self-aggregation of the otherwise soluble and monomeric protein. Intriguingly, corresponding alanine substitutions of the conserved Y642, Y643 and Y639 residues in the homologous RtxA, HlyA and ApxIA hemolysins from Kingella kingae, Escherichia coli and Actinobacillus pleuropneumoniae, affected the membrane insertion, pore-forming (hemolytic) and cytotoxic capacities of these toxins only marginally. Activities of these toxins were impaired only upon replacement of the conserved tyrosines  by proline residues. It appears, hence, that the critical role of the aromatic side chain of the Y940 residue is highly specific for the functional folding of the acylated domain of CyaA and determines its capacity to penetrate target cell membrane.



2021 ◽  
Author(s):  
Van Thi Bich Le ◽  
Joseph Dang ◽  
Ee Qi Lim ◽  
Misty L. Kuhn


ACS Catalysis ◽  
2020 ◽  
Vol 10 (15) ◽  
pp. 8177-8186
Author(s):  
Saborni Biswas ◽  
Donald M. Kurtz ◽  
Samuel R. Montoya ◽  
Michael P. Hendrich ◽  
Emile L. Bominaar


Biochemistry ◽  
2020 ◽  
Vol 59 (25) ◽  
pp. 2328-2339
Author(s):  
Martin Klein ◽  
Erhan Deniz ◽  
Sabine Heit ◽  
Georg Wille ◽  
Werner Mäntele ◽  
...  


2020 ◽  
Vol 34 (S1) ◽  
pp. 1-1
Author(s):  
Ee Qi Lim ◽  
Van Le ◽  
Joseph Dang ◽  
Melissa Law ◽  
Misty L. Kuhn


Author(s):  
Jun Li ◽  
Wenyan Han ◽  
Kunwei Wu ◽  
Yuping Derek Li ◽  
Qun Liu ◽  
...  


2018 ◽  
Vol 115 (52) ◽  
pp. 13186-13191 ◽  
Author(s):  
Elizabeth O’Brien ◽  
Lauren E. Salay ◽  
Esther A. Epum ◽  
Katherine L. Friedman ◽  
Walter J. Chazin ◽  
...  

Eukaryotic DNA primases contain a [4Fe4S] cluster in the C-terminal domain of the p58 subunit (p58C) that affects substrate affinity but is not required for catalysis. We show that, in yeast primase, the cluster serves as a DNA-mediated redox switch governing DNA binding, just as in human primase. Despite a different structural arrangement of tyrosines to facilitate electron transfer between the DNA substrate and [4Fe4S] cluster, in yeast, mutation of tyrosines Y395 and Y397 alters the same electron transfer chemistry and redox switch. Mutation of conserved tyrosine 395 diminishes the extent of p58C participation in normal redox-switching reactions, whereas mutation of conserved tyrosine 397 causes oxidative cluster degradation to the [3Fe4S]+ species during p58C redox signaling. Switching between oxidized and reduced states in the presence of the Y397 mutations thus puts primase [4Fe4S] cluster integrity and function at risk. Consistent with these observations, we find that yeast tolerate mutations to Y395 in p58C, but the single-residue mutation Y397L in p58C is lethal. Our data thus show that a constellation of tyrosines for protein-DNA electron transfer mediates the redox switch in eukaryotic primases and is required for primase function in vivo.



2018 ◽  
Vol 1865 (9) ◽  
pp. 1173-1186 ◽  
Author(s):  
Susan I. Daraiseh ◽  
Ari Kassardjian ◽  
Karen E. Alexander ◽  
Raed Rizkallah ◽  
Myra M. Hurt


2018 ◽  
Vol 8 (1) ◽  
Author(s):  
Emmanuelle Schmitt ◽  
Gabrielle Bourgeois ◽  
Muriel Gondry ◽  
Alexey Aleksandrov


2018 ◽  
Vol 293 (21) ◽  
pp. 8161-8172 ◽  
Author(s):  
Heikki Takala ◽  
Heli K. Lehtivuori ◽  
Oskar Berntsson ◽  
Ashley Hughes ◽  
Rahul Nanekar ◽  
...  

Phytochromes are photoreceptors in plants, fungi, and various microorganisms and cycle between metastable red light–absorbing (Pr) and far-red light–absorbing (Pfr) states. Their light responses are thought to follow a conserved structural mechanism that is triggered by isomerization of the chromophore. Downstream structural changes involve refolding of the so-called tongue extension of the phytochrome-specific GAF-related (PHY) domain of the photoreceptor. The tongue is connected to the chromophore by conserved DIP and PRXSF motifs and a conserved tyrosine, but the role of these residues in signal transduction is not clear. Here, we examine the tongue interactions and their interplay with the chromophore by substituting the conserved tyrosine (Tyr263) in the phytochrome from the extremophile bacterium Deinococcus radiodurans with phenylalanine. Using optical and FTIR spectroscopy, X-ray solution scattering, and crystallography of chromophore-binding domain (CBD) and CBD–PHY fragments, we show that the absence of the Tyr263 hydroxyl destabilizes the β-sheet conformation of the tongue. This allowed the phytochrome to adopt an α-helical tongue conformation regardless of the chromophore state, hence distorting the activity state of the protein. Our crystal structures further revealed that water interactions are missing in the Y263F mutant, correlating with a decrease of the photoconversion yield and underpinning the functional role of Tyr263 in phytochrome conformational changes. We propose a model in which isomerization of the chromophore, refolding of the tongue, and globular conformational changes are represented as weakly coupled equilibria. The results also suggest that the phytochromes have several redundant signaling routes.



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