scholarly journals Biophysical Characterization of the Tandem FHA Domain Regulatory Module from the Mycobacterium tuberculosis ABC Transporter Rv1747

Structure ◽  
2018 ◽  
Vol 26 (7) ◽  
pp. 972-986.e6 ◽  
Author(s):  
Florian Heinkel ◽  
Leo Shen ◽  
Melissa Richard-Greenblatt ◽  
Mark Okon ◽  
Jennifer M. Bui ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Abc Transporter ◽  
Fha Domain ◽  
Biophysical Characterization ◽  
Regulatory Module

Biophysical methods toolbox to study ABC exporter structure and function

2017 ◽  
Vol 398 (2) ◽  
pp. 229-235
Author(s):  
Thomas Marcellino ◽  
Vasundara Srinivasan
Keyword(s):  
Membrane Proteins ◽  
Abc Transporter ◽  
Integrated Approach ◽  
Structure And Function ◽  
Dynamic Membrane ◽  
Biophysical Characterization ◽  
Intermediate States ◽  
And Function

Abstract ABC exporters are highly dynamic membrane proteins that span a huge spectrum of different conformations. A detailed integrated approach of cellular, biochemical and biophysical characterization of these ‘open’, ‘closed’ and other intermediate states is central to understanding their function. Almost 40 years after the discovery of the first ABC transporter, thanks to the enormous development in methodologies, a picture is slowly emerging to visualize how these fascinating molecules transport their substrates. This mini review summarizes some of the biophysical tools that have made a major impact in understanding the function of the ABC exporters.

scholarly journals Phase separation and clustering of an ABC transporter in Mycobacterium tuberculosis

2019 ◽  
Vol 116 (33) ◽  
pp. 16326-16331 ◽  
Author(s):  
Florian Heinkel ◽  
Libin Abraham ◽  
Mary Ko ◽  
Joseph Chao ◽  
Horacio Bach ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Phase Separation ◽  
Lipid Bilayers ◽  
Abc Transporter ◽  
Single Molecule ◽  
Intracellular Signaling ◽  
Intrinsically Disordered ◽  
Regulatory Module ◽  
Condensate Formation ◽  
Associated Proteins

Phase separation drives numerous cellular processes, ranging from the formation of membrane-less organelles to the cooperative assembly of signaling proteins. Features such as multivalency and intrinsic disorder that enable condensate formation are found not only in cytosolic and nuclear proteins, but also in membrane-associated proteins. The ABC transporter Rv1747, which is important for Mycobacterium tuberculosis (Mtb) growth in infected hosts, has a cytoplasmic regulatory module consisting of 2 phosphothreonine-binding Forkhead-associated domains joined by an intrinsically disordered linker with multiple phospho-acceptor threonines. Here we demonstrate that the regulatory modules of Rv1747 and its homolog in Mycobacterium smegmatis form liquid-like condensates as a function of concentration and phosphorylation. The serine/threonine kinases and sole phosphatase of Mtb tune phosphorylation-enhanced phase separation and differentially colocalize with the resulting condensates. The Rv1747 regulatory module also phase-separates on supported lipid bilayers and forms dynamic foci when expressed heterologously in live yeast and M. smegmatis cells. Consistent with these observations, single-molecule localization microscopy reveals that the endogenous Mtb transporter forms higher-order clusters within the Mycobacterium membrane. Collectively, these data suggest a key role for phase separation in the function of these mycobacterial ABC transporters and their regulation via intracellular signaling.

scholarly journals Clinically relevant mutations of mycobacterial GatCAB inform regulation of translational fidelity

2021 ◽  
Author(s):  
Yang-Yang Li ◽  
Rong-Jun Cai ◽  
Jia-Ying Yang ◽  
Tamara L. Hendrickson ◽  
Ye Xiang ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Second Step ◽  
Loss Of Function ◽  
Wild Type ◽  
Translational Fidelity ◽  
Partial Loss ◽  
Biophysical Characterization ◽  
Minimal Impact ◽  
Mutant Strains

AbstractMost bacteria employ a two-step indirect tRNA aminoacylation pathway for the synthesis of aminoacylated tRNAGln and tRNAAsn. The heterotrimeric enzyme GatCAB performs a critical amidotransferase reaction in the second step of this pathway. We have previously demonstrated in mycobacteria that this two-step pathway is error-prone and translational errors contribute to adaptive phenotypes such as antibiotic tolerance. Furthermore, we identified clinical isolates of the globally important pathogen Mycobacterium tuberculosis with partial loss-of-function mutations in gatA, and demonstrated that these mutations result in high, specific rates of translational error and increased rifampicin tolerance. However, the mechanisms by which these clinically-derived mutations in gatA impact GatCAB function was unknown. Here, we describe biochemical and biophysical characterization of M. tuberculosis GatCAB, containing either wild-type gatA or one of two gatA mutants from clinical strains. We show that these mutations have minimal impact on enzymatic activity of GatCAB; however, they result in destabilization of the GatCAB complex as well as that of the ternary asparaginyl-transamidosome. Stabilizing complex formation with the solute trehalose increases specific translational fidelity of not only the mutant strains, but also of wild-type mycobacteria. Therefore, our data suggest that alteration of GatCAB stability may be a mechanism for modulation of translational fidelity.

Structural and biophysical characterization of Mycobacterium tuberculosis dodecin Rv1498A

2011 ◽  
Vol 175 (1) ◽  
pp. 31-38 ◽  
Author(s):  
Fengxia Liu ◽  
Junhui Xiong ◽  
Sundaramurthy Kumar ◽  
Chunyan Yang ◽  
Shengxiang Ge ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Biophysical Characterization

scholarly journals An ABC Transporter Containing a Forkhead-Associated Domain Interacts with a Serine-Threonine Protein Kinase and Is Required for Growth of Mycobacterium tuberculosis in Mice

2005 ◽  
Vol 73 (8) ◽  
pp. 4471-4477 ◽  
Author(s):  
Juliet M. Curry ◽  
Rachael Whalan ◽  
Debbie M. Hunt ◽  
Kalpesh Gohil ◽  
Molly Strom ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Protein Kinase ◽  
Abc Transporter ◽  
Bacterial Load ◽  
Causative Organism ◽  
Mouse Bone Marrow ◽  
Dependent Manner ◽  
Wild Type ◽  
Fha Domain

ABSTRACT Forkhead-associated (FHA) domains are modular phosphopeptide recognition motifs with a striking preference for phosphothreonine-containing epitopes. FHA domains have been best characterized in eukaryotic signaling pathways but have been identified in six proteins in Mycobacterium tuberculosis, the causative organism of tuberculosis. One of these, coded by gene Rv1747, is an ABC transporter and the only one to contain two such modules. A deletion mutant of Rv1747 is attenuated in a mouse intravenous injection model of tuberculosis where the bacterial load of the mutant is 10-fold lower than that of the wild type in both lungs and spleen. In addition, growth of the mutant in mouse bone marrow-derived macrophages and dendritic cells is significantly impaired. In contrast, growth of this mutant in vitro was indistinguishable from that of the wild type. The mutant phenotype was lost when the mutation was complemented by the wild-type allele, confirming that it was due to mutation of Rv1747. Using yeast two-hybrid analysis, we have shown that the Rv1747 protein interacts with the serine-threonine protein kinase PknF. This interaction appears to be phospho-dependent since it is abrogated in a kinase-dead mutant and by mutations in the presumed activation loop of PknF and in the first FHA domain of Rv1747. These results demonstrate that the protein coded by Rv1747 is required for normal virulent infection by M. tuberculosis in mice and, since it interacts with a serine-threonine protein kinase in a kinase-dependent manner, indicate that it forms part of an important phospho-dependent signaling pathway.

Structural and biophysical characterization of Rv3716c, a hypothetical protein from Mycobacterium tuberculosis

2018 ◽  
Vol 495 (1) ◽  
pp. 982-987
Author(s):  
A. Gopalan ◽  
G. Deka ◽  
M. Prabhavathi ◽  
H.S. Savithri ◽  
M.R.N. Murthy ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Hypothetical Protein ◽  
Biophysical Characterization
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