Structural Studies of Potassium Transport Protein KtrA Regulator of Conductance of K+ (RCK) C Domain in Complex with Cyclic Diadenosine Monophosphate (c-di-AMP)

Kim, Henna; Youn, Suk-Jun; Kim, Seong Ok; Ko, Jong Soo; Lee, Jie‐Oh; Choi, Byong‐Seok

doi:10.1074/jbc.m115.641340

Cited by 85 publications

(89 citation statements)

References 50 publications

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“…Nucleotide binding to RCK_N has been proposed to induce conformational changes in the KtrA ring that are transmitted to the KtrB module 62 . C-di-AMP, on the other hand, binds as a U-shaped monomer to the RCK_C dimers and could thus limit the conformational cycle of the active transporter, consistent with its inhibitory effects on potassium transport 18,63 .…”

Section: Membrane Transport and Ion Homeostasismentioning

confidence: 79%

Versatile modes of cellular regulation via cyclic dinucleotides

2017

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Since the discovery of c-di-GMP almost three decades ago, cyclic dinucleotides (CDNs) have emerged as widely used signaling molecules in most kingdoms of life. The family of second messengers now includes c-di-AMP and distinct versions of mixed cyclic GMP-AMP (cGAMP) compounds. Along with these nucleotides, a vast number of proteins for the production and turnover of these molecules have been described, as well as effectors that translate the signals into physiological responses. The latter include but are not limited to mechanisms for adaptation and survival in prokaryotes, persistence and virulence of bacterial pathogens, as well as immune responses to viral and bacterial invasion in eukaryotes. In this review we will focus on recent discoveries and emerging themes that illustrate the ubiquity and versatility of cyclic dinucleotide function at the transcriptional and post-translational levels and, in particular, on insights gained through mechanistic structure-function analyses.

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Section: Membrane Transport and Ion Homeostasismentioning

confidence: 79%

Versatile modes of cellular regulation via cyclic dinucleotides

2017

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“…8) (9,27). Since it has been reported that c-di-AMP negatively controls the influx of potassium ions into S. pneumoniae and S. aureus cells (16,17,48), one can imagine that the deregulation of potassium homeostasis increases the turgor pressure to lethal levels, which would result in cell lysis. This hypothesis is supported by the observation that the osmosensitive phenotype of an S. aureus ltaS mutant strain unable to synthesize lipoteichoic acid might be rescued by an increased intracellular c-di-AMP concentration (27).…”

Section: Discussionmentioning

confidence: 99%

“…Among these is the transcription factor DarR from Mycobacterium smegmatis, the DNAbinding activity of which is stimulated by c-di-AMP (15). c-di-AMP was also shown to bind to and to control the activities of the potassium transporters from Staphylococcus aureus and Streptococcus pneumoniae and to allosterically regulate the L. monocytogenes pyruvate carboxylase (16,17,18). Recently, members of a novel class of P II -like signal transduction proteins from B. subtilis, S. aureus, and L. monocytogenes were also shown to bind c-di-AMP (19,20,21,22).…”

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confidence: 99%

Phenotypes Associated with the Essential Diadenylate Cyclase CdaA and Its Potential Regulator CdaR in the Human Pathogen Listeria monocytogenes

et al. 2016

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Cyclic diadenylate monophosphate (c-di-AMP) is a second messenger utilized by diverse bacteria. In many species, including the Gram-positive human pathogen Listeria monocytogenes, c-di-AMP is essential for growth. Here we show that the single diadenylate cyclase of L. monocytogenes, CdaA, is an integral membrane protein that interacts with its potential regulatory protein, CdaR, via the transmembrane protein domain. The presence of the CdaR protein is not required for the membrane localization and abundance of CdaA. We have also found that CdaR negatively influences CdaA activity in L. monocytogenes and that the role of CdaR is most evident at a high growth temperature. Interestingly, a cdaR mutant strain is less susceptible to lysozyme. Moreover, CdaA contributes to cell division, and cells depleted of CdaA are prone to lysis. The observation that the growth defect of a CdaA depletion strain can be partially restored by increasing the osmolarity of the growth medium suggests that c-di-AMP is important for maintaining the integrity of the protective cell envelope. Overall, this work provides new insights into the relationship between CdaA and CdaR. IMPORTANCE Cyclic diadenylate monophosphate (c-di-AMP) is a recently identified second messenger that is utilized by the Gram-positive human pathogen Listeria monocytogenes.Here we show that the single diadenylate cyclase of L. monocytogenes, CdaA, is an integral membrane protein that interacts with CdaR, its potential regulatory protein. We show that CdaR is not required for membrane localization or abundance of the diadenylate cyclase, but modulates its activity. Moreover, CdaA seems to contribute to cell division. Overall, this work provides new insights into the relationship between CdaA and CdaR and their involvement in cell growth. Bacteria from diverse phyla produce the cyclic dinucleotide cyclic diadenylate monophosphate (c-di-AMP) that is synthesized and degraded by specific diadenylate cyclases and phosphodiesterases, respectively (1). The DNA integrity scanning protein DisA from Thermotoga maritima was the first diadenylate cyclase structurally and biochemically characterized (2), and its characterization led to the discovery of c-di-AMP. Many bacteria possess only a single, DisA-type, diadenylate cyclase (1), which is involved in the maintenance of DNA integrity (3, 4, 5). The cyclase activity of DisA is modulated by unusual DNA recombination intermediates (2), but it is presently unclear how c-di-AMP signals the cell that the chromosome integrity is affected.In addition to DisA, two diadenylate cyclases, CdaA and CdaS, are synthesized in the Gram-positive model bacterium Bacillus subtilis (6). While cdaA is expressed during vegetative growth, the cdaS gene is expressed during sporulation or germination of spores (7). The cdaS inactivation decreases the germination efficiency of spores, indicating a germination-specific function for this enzyme (8). Recently, it has been shown that c-di-AMP is essential for the growth of B. subtilis (6, 9, 10). c-d...

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“…Replacement of these bulky hydrophobic residues with alanines results in a substantial loss of c-di-AMP binding as measured by ITC, 20 indicating the important roles of these residues in stabilizing this complex. This is a similar scenario to that described here for the SaCpaA_RCK_CTD−c-di-AMP complex, in which the binding 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 affinity of SaCpaA_RCK_CTD for c-di-AMP is significantly reduced when the crucial Leu160 was altered to an alanine (Table I).…”

Section: Structural Comparison With Other "U-shape" C-di-amp Binding mentioning

confidence: 98%

“…3h) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 The SaKtrA_RCK_CTD−c-di-AMP complex structure was recently solved. 20 As this protein also contains a RCK domain that acts as a gate for K + transportation, it was thus interesting to compare how c-di-AMP binds to these two regulators. SaKtrA and SaCpaA have rather different primary sequences, with their RCK_CTD sequences sharing a similarity value of only 15 % after sequence alignment (Fig.…”

Section: Emerging Importance Of Cyclic-di-nucleotide Researchmentioning

confidence: 99%

Structural Insights into the Distinct Binding Mode of Cyclic Di-AMP with SaCpaA_RCK

et al. 2015

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Cyclic di-AMP (c-di-AMP) is a relatively new member of the family of bacterial cyclic dinucleotide second messengers. It has attracted significant attention in recent years because of the abundant roles it plays in a variety of Gram-positive bacteria. The structural features that allow diverse bacterial proteins to bind c-di-AMP are not fully understood. Here we report the biophysical and structural studies of c-di-AMP in complex with a bacterial cation-proton antiporter (CpaA) RCK (regulator of the conductance of K(+)) protein from Staphylococcus aureus (Sa). The crystal structure of the SaCpaA_RCK C-terminal domain (CTD) in complex with c-di-AMP was determined to a resolution of 1.81 Å. This structure revealed two well-liganded water molecules, each interacting with one of the adenine bases by a unique H2Olp-π interaction to stabilize the complex. Sequence blasting using the SaCpaA_RCK primary sequence against the bacterial genome database returned many CpaA analogues, and alignment of these sequences revealed that the active site residues are all well-conserved, indicating a universal c-di-AMP binding mode for CpaA_RCK. A proteoliposome activity assay using the full-length SaCpaA membrane protein indicated that c-di-AMP binding alters its antiporter activity by approximately 40%. A comparison of this structure to all other reported c-di-AMP-receptor complex structures revealed that c-di-AMP binds to receptors in either a "U-shape" or "V-shape" mode. The two adenine rings are stabilized in the inner interaction zone by a variety of CH-π, cation-π, backbone-π, or H2Olp-π interaction, but more commonly in the outer interaction zone by hydrophobic CH-π or π-π interaction. The structures determined to date provide an understanding of the mechanisms by which a single c-di-AMP can interact with a variety of receptor proteins, and how c-di-AMP binds receptor proteins in a special way different from that of c-di-GMP.

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Structural Studies of Potassium Transport Protein KtrA Regulator of Conductance of K+ (RCK) C Domain in Complex with Cyclic Diadenosine Monophosphate (c-di-AMP)

Cited by 85 publications

References 50 publications

Versatile modes of cellular regulation via cyclic dinucleotides

Versatile modes of cellular regulation via cyclic dinucleotides

Phenotypes Associated with the Essential Diadenylate Cyclase CdaA and Its Potential Regulator CdaR in the Human Pathogen Listeria monocytogenes

Structural Insights into the Distinct Binding Mode of Cyclic Di-AMP with SaCpaA_RCK

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