All four putative selectivity filter glycine residues in KtrB are essential for high affinity and selective K+ uptake by the KtrAB system from Vibrio alginolyticus

The subunit KtrB of bacterial Na+-dependent K+-translocating KtrAB systems belongs to a superfamily of K+ transporters. These proteins contain four repeated domains, each composed of two transmembrane helices connected by a putative pore loop (p-loop). The four p-loops harbor a conserved glycine residue at a position equivalent to a glycine selectivity filter residue in K+ channels. We investigated whether these glycines also form a selectivity filter in KtrB. The single residues Gly70, Gly185, Gly290, and Gly402 from p-loops P(A) to P(D) of Vibrio alginolyticus KtrB were replaced with alanine, serine, or aspartate. The three alanine variants KtrB(A70), KtrB(A185), and KtrB(A290) maintained a substantial activity in KtrAB-mediated K+ uptake in Escherichia coli. This activity was associated with a decrease in the affinity for K+ by 2 orders of magnitude, with little effect on Vmax. Minor activities were also observed for three other variants: KtrB(A402), KtrB(S70), and KtrB(D185). With all of these variants, the property of Na+ dependence of K+ transport was preserved. Only the four serine variants mediated Na+ uptake, and these variants differed considerably in their K+/Na+ selectivity. Experiments on cloned ktrB in the pBAD18 vector showed that V. alginolyticus KtrB alone was still active in E. coli. It mediated Na+-independent, slow, high affinity, and mutation-specific K+ uptake as well as K+-independent Na+ uptake. These data demonstrate that KtrB contains a selectivity filter for K+ ions and that all four conserved p-loop glycine residues are part of this filter. They also indicate that the role of KtrA lies in conferring velocity and ion coupling to the Ktr complex.     

Na+-dependent K+ uptake Ktr system from the cyanobacterium Synechocystis sp. PCC 6803 and its role in the early phases of cell adaptation to hyperosmotic shock

Transmembrane ion transport processes play a key role in the adaptation of cells to hyperosmotic conditions. Previous work has shown that the disruption of a ktrB/ntpJ-like putative Na(+)/K(+) transporter gene in the cyanobacterium Synechocystis sp. PCC 6803 confers increased Na(+) sensitivity, and inhibits HCO(3)(-) uptake. Here, we report on the mechanistic basis of this effect. Heterologous expression experiments in Escherichia coli show that three Synechocystis genes are required for K(+) transport activity. They encode an NAD(+)-binding peripheral membrane protein (ktrA; sll0493), an integral membrane protein, belonging to a superfamily of K(+) transporters (ktrB; formerly ntpJ; slr1509), and a novel type of ktr gene product, not previously found in Ktr systems (ktrE; slr1508). In E. coli, Synechocystis KtrABE-mediated K(+) uptake occurred with a moderately high affinity (K(m) of about 60 microm), and depended on both Na(+) and a high membrane potential, but not on ATP. KtrABE neither mediated Na(+) uptake nor Na(+) efflux. In Synechocystis sp. PCC 6803, KtrB-mediated K(+) uptake required Na(+) and was inhibited by protonophore. A Delta ktrB strain was sensitive to long term hyperosmotic stress elicited by either NaCl or sorbitol. Hyperosmotic shock led initially to loss of net K(+) from the cells. The Delta ktrB cells shocked with sorbitol failed to reaccumulate K(+) up to its original level. These data indicate that in strain PCC 6803 K(+) uptake via KtrABE plays a crucial role in the early phase of cell turgor regulation after hyperosmotic shock.     

Change to alanine of one out of four selectivity filter glycines in KtrB causes a two orders of magnitude decrease in the affinities for both K+ and Na+ of the Na+ dependent K+ uptake system KtrAB from Vibrio alginolyticus.

KtrAB from Vibrio alginolyticus is a recently described new type of high affinity bacterial K+ uptake system. Its activity assayed in an Escherichia coli K+ uptake negative mutant depended on Na+ ions (Km of 40 microM). Subunit KtrB contains four putative P-loops. The selectivity filter from each P-loop contains a conserved glycine residue. Residue Gly-290 from the third P-loop selectivity filter in KtrB was exchanged for Ala, Ser or Asp. KtrB variants Ser-290 and Asp-290 were without activity. In contrast, KtrB variant Ala-290 was still active. This variant transported K+ with a two orders of magnitude decrease in apparent affinity for both K+ and Na+ with little effect on Vmax.     

Dissecting the Molecular Mechanism of Nucleotide-Dependent Activation of the KtrAB K+ Transporter

KtrAB belongs to the Trk/Ktr/HKT superfamily of monovalent cation (K⁺ and Na⁺) transport proteins that closely resemble K⁺ channels. These proteins underlie a plethora of cellular functions that are crucial for environmental adaptation in plants, fungi, archaea, and bacteria. The activation mechanism of the Trk/Ktr/HKT proteins remains unknown. It has been shown that ATP stimulates the activity of KtrAB while ADP does not. Here, we present X-ray structural information on the KtrAB complex with bound ADP. A comparison with the KtrAB-ATP structure reveals conformational changes in the ring and in the membrane protein. In combination with a biochemical and functional analysis, we uncover how ligand-dependent changes in the KtrA ring are propagated to the KtrB membrane protein and conclude that, despite their structural similarity, the activation mechanism of KtrAB is markedly different from the activation mechanism of K⁺ channels.     

Probing the structure of the dimeric KtrB membrane protein

The KtrAB ion transporter is a complex of two proteins, KtrB and KtrA. The integral membrane protein KtrB is expected to adopt the structural architecture typified by the pore domain of potassium channels. Here we show that homo-dimerization of KtrB proteins is most likely a general property of this family of transporters. Using cysteine mutants and bifunctional cross-linkers we define regions of the Bacillus subtilis KtrB molecule that are close to the molecular 2-fold axis and to the dimer interface. Fitting of the cross-linking data to a potassium channel-like model suggests structural similarities between potassium channels and KtrB proteins in the extracellular half of the molecule and differences in the cytoplasmic regions.     

KtrB, a member of the superfamily of K+ transporters

KtrB is the K(+)-translocating subunit of the K(+)-uptake system KtrAB from bacteria. It is a member of the superfamily of K(+)transporters (SKT proteins) with other sub-families occurring in archaea, bacteria, fungi, plants and trypanosomes. SKT proteins may have originated from small K(+) channels by at least two gene duplication and two gene fusion events. They contain four covalently linked M(1)PM(2) domains, in which M(1) and M(2) stand for transmembrane stretches, and P for a P-loop, which folds back from the external medium into the membrane. SKT proteins distinguish themselves in two important aspects from K(+) channels: first, with just one conserved glycine residue in their P-loops they contain a much simpler K(+)-selectivity filter sequence than K(+) channels with their conserved Thr-Val-Gly-Tyr-Gly sequence. Secondly, the middle part M(2C2) from the long transmembrane stretch M(2C) of KtrB from the bacterium Vibrio alginolyticus forms a gate inside the membrane, which prevents K(+) permeation to the cytoplasm. Beside the mechanism of K(+) transport via KtrB and other SKT proteins existing hypotheses of how the KtrA protein regulates the K(+)-transport activity of KtrB are discussed.     

The RCK domain of the KtrAB K+ transporter: multiple conformations of an octameric ring

The KtrAB ion transporter is a complex of the KtrB membrane protein and KtrA, an RCK domain. RCK domains regulate eukaryotic and prokaryotic membrane proteins involved in K(+) transport. Conflicting functional models have proposed two different oligomeric arrangements for RCK domains, tetramer versus octamer. Our results for the KtrAB RCK domain clearly show an octamer in solution and in the crystal. We determined the structure of this protein in three different octameric ring conformations that resemble the RCK-domain octamer observed in the MthK potassium channel but show striking differences in size and symmetry. We present experimental evidence for the association between one RCK octameric ring and two KtrB membrane proteins. These results provide insights into the quaternary organization of the KtrAB transporter and its mechanism of activation and show that the RCK-domain octameric ring model is generally applicable to other ion-transport systems.     

KtrAB, a New Type of Bacterial K+-Uptake System from Vibrio alginolyticus

Vibrio alginolyticus contained two adjacent genes, ktrA and ktrB, which encode a new type of bacterial K⁺-uptake system. KtrA and KtrB are peripheral and integral membrane proteins, respectively. Six of the nine sequenced bacterial genomes contain homologs to both ktrA and ktrB, suggesting that KtrAB is widespread.     

The Ktr potassium transport system in Staphylococcus aureus and its role in cell physiology,antimicrobial resistance and pathogenesis

Potassium (K⁺) plays a vital role in bacterial physiology, including regulation of cytoplasmic pH, turgor pressure and transmembrane electrical potential. Here, we examine the Staphylococcus aureus Ktr system uniquely comprised of two ion‐conducting proteins (KtrB and KtrD) and only one regulator (KtrA). Growth of Ktr system mutants was severely inhibited under K⁺ limitation, yet detectable after an extended lag phase, indicating the presence of a secondary K⁺ transporter. Disruption of both ktrA and the Kdp‐ATPase system, important for K⁺ uptake in other organisms, eliminated regrowth in 0.1 mM K⁺, demonstrating a compensatory role for Kdp to the Ktr system. Consistent with K⁺ transport mutations, S. aureus devoid of the Ktr system became sensitive to hyperosmotic conditions, exhibited a hyperpolarized plasma membrane, and increased susceptibility to aminoglycoside antibiotics and cationic antimicrobials. In contrast to other organisms, the S. aureus Ktr system was shown to be important for low‐K⁺ growth under alkaline conditions, but played only a minor role in neutral and acidic conditions. In a mouse competitive index model of bacteraemia, the ktrA mutant was significantly outcompeted by the parental strain. Combined, these results demonstrate a primary mechanism of K⁺ uptake in S. aureus and a role for this system in pathogenesis.     

The KtrA and KtrE Subunits Are Required for Na+-Dependent K+ Uptake by KtrB across the Plasma Membrane in Synechocystis sp. Strain PCC 6803

The Na⁺-dependent K⁺ uptake KtrABE system is essential for the adaptation of Synechocystis to salinity stress and high osmolality. While KtrB forms the K⁺-translocating pore, the role of the subunits KtrA and KtrE for Ktr function remains elusive. Here, we characterized the role of KtrA and KtrE in Ktr-mediated K⁺ uptake and in modulating Na⁺ dependency. Expression of KtrB alone in a K⁺ uptake-deficient Escherichia coli strain conferred low K⁺ uptake activity that was not stimulated by Na⁺. Coexpression of both KtrA and KtrE with KtrB increased the K⁺ transport activity in a Na⁺-dependent manner. KtrA and KtrE were found to be localized to the plasma membrane in Synechocystis. Site-directed mutagenesis was used to analyze the role of single charged residues in KtrB for Ktr function. Replacing negatively charged residues facing the extracellular space with residues of the opposite charge increased the apparent K_m for K⁺ in all cases. However, none of the mutations eliminated the Na⁺ dependency of Ktr-mediated K⁺ transport. Mutations of residues on the cytoplasmic side had larger effects on K⁺ uptake activity than those of residues on the extracellular side. Further analysis revealed that replacement of R262, which is well conserved among Ktr/Trk/HKT transporters in the third extracellular loop, by Glu abolished transport activity. The atomic-scale homology model indicated that R262 might interact with E247 and D261. Based on these data, interaction of KtrA and KtrE with KtrB increased the K⁺ uptake rate and conferred Na⁺ dependency.Cyanobacterium Synechocystis sp. strain PCC 6803 contains a number of different K⁺ uptake systems that may contribute to satisfying its requirement of K⁺ (3, 19, 36). Among these systems, Ktr has been shown to have a major role not only in K⁺ uptake but also in adaptation against high-osmolarity stress (3, 19). Inactivation of the ktr gene renders the cells hypersensitive to high concentrations of NaCl and the nonionic compound sorbitol. Ktr-mediated K⁺ uptake depends on the presence of Na⁺ in the medium, which is likely to be an adaptation to salinity stress. A requirement of Na⁺ for K⁺ transport activity has also been found in the homologous protein from Vibrio alginolyticus (21). This dependency on Na⁺ is a unique property of Ktr-type transporters and has not been found in other types of K⁺ transporters or channels (32). The structure and function of Ktr-type transporters have been studied in a number of organisms (3, 6, 7, 9, 11-14, 18-20, 30, 32-34). The Ktr system from Synechocystis consists of three subunits, KtrA, KtrB, and KtrE (19). The KtrE gene and the KtrB gene form a cistron, whereas the KtrA gene resides at a site distant from the KtrEB genes in the Synechocystis genome (19). KtrB, the K⁺-translocating subunit, is a member of the Ktr/Trk/HKT family of K⁺ transporters. These transporters have been proposed to have evolved from two membrane-spanning K⁺ channels (6, 7). According to the model, this type of transporter contains eight transmembrane domains, which consist of a 4-fold-repeated membrane-pore-membrane (M1-P-M2) motif (6, 7, 13, 18). An intramolecular electrostatic interaction of Synechocystis KtrB has been proposed to stabilize the protein in its active configuration (12). In addition, a conserved His in the external region in Synechocystis KtrB has been shown to be crucial for KtrB function (39). The region of the Vibrio Ktr protein responsible for gating of ion permeation has been identified (9). However, not much is known about the mechanism of Na⁺ binding to KtrB in Synechocystis.The KtrA subunit belongs to the family of KTR (K⁺-transport nucleotide binding)/RCK (regulating the conductance of K⁺ channels) proteins, which contain a Rossmann-fold sequence encoding β-α protein structure for NAD⁺/NADH binding (17). Accordingly KtrA has been proposed to regulate the K⁺ transport activity of KtrB by changing its binding from NAD⁺ to NADH through a ligand-mediated conformational switch mechanism (25). It has also been shown that ATP promotes complex formation between KtrA and KtrB and that KtrAB from V. alginolyticus when expressed in Escherichia coli cells requires both ATP and the membrane potential for its activity (17).KtrE is a unique subunit found only in Synechocystis; it is not involved in KtrB-mediated K⁺ transport in V. alginolyticus and Bacillus subtilis (11, 32). The termination codon of ktrE overlaps the initiation codon of ktrB in the same cistron, which has not been found in other bacterial ktrB-related genes. Coexpression of KtrA with KtrB alone does not complement the growth defect of an E. coli K⁺ uptake mutant. However, introduction of KtrE into the same mutant background in addition to KtrA and KtrB complements the mutation of the K⁺ uptake system (19). Interestingly, the KtrE protein has been shown to function as a digalactosyldiacylglycerol (DGDG) synthase (EC 2.4.6.241), an enzyme that produces DGDG from monogalactosyldiacylglycerol (MGDG). KtrE has therefore also been designated DgdA (1). Under nonstress conditions, DGDG is found in the thylakoid membranes, which helps stabilize the photosystem II complex in Synechocystis (29). Under phosphate-limited conditions, DGDG is synthesized instead of phospholipids in Synechocystis (1). However, KtrB functions as a major K⁺-conducting transport pore in the Synechocystis plasma membrane. The subcellular localization of KtrE has not been identified directly. Inactivation of ktrE (also called dgdA) in Synechocystis does not result in sensitivity to osmotic stress imposed by 300 mM sorbitol (1). This may be inconsistent with the requirement of KtrE for KtrB-mediated K⁺ uptake in the presence of KtrA in the E. coli expression system (19).Because of these uncertainties about the roles of the KtrA and KtrE subunits in K⁺ uptake by KtrB in Synechocystis and about the identity of the Na⁺ binding site in KtrB, we examined the subcellular localization and membrane association of KtrA and KtrE, the requirement of these subunits for KtrB-mediated K⁺ uptake, and the primary target for Na⁺ binding in KtrB.     

Potassium uptake in the unicellular cyanobacterium Synechocystis sp. strain PCC 6803 mainly depends on a Ktr-like system encoded by slr1509 (ntpJ)

The molecular basis of potassium uptake in cyanobacteria has not been elucidated. However, genes known from other bacteria to encode potassium transporters can be identified in the genome of Synechocystis sp. strain PCC 6803. Mutants defective in kdpA and ntpJ were generated and characterized to address the role of the Kdp and KtrAB systems in this strain. KtrAB is crucial for K(+) uptake, as the DeltantpJ mutant shows slowed growth, slowed potassium uptake kinetics, and increased salt sensitivity. The DeltakdpA mutant has the same phenotype as the wild type even at limiting potassium, but a DeltakdpADeltantpJ double mutant is not viable, indicating a role of Kdp for potassium uptake when the Ktr system is not functioning.     

Genetic requirements for potassium ion-dependent colony spreading in Bacillus subtilis

Undomesticated strains of Bacillus subtilis exhibit extensive colony spreading on certain soft agarose media: first the formation of dendritic clusters of cells, followed by spreading (pellicle-like) growth to cover the entire surface. These phases of colonization are dependent on the level of potassium ion (K(+)) but independent of flagella, as verified with a mutant with a hag gene replacement; this latter finding highlights the importance of sliding motility in colony spreading. Exploring the K(+) requirement, directed mutagenesis of the higher-affinity K(+) transporter KtrAB, but not the lower-affinity transporter KtrCD, was found to inhibit surface colonization unless sufficient KCl was added. To identify other genes involved in K(+)-dependent colony spreading, transposon insertion mutants in wild-type strain 3610 were screened. Disruption of genes for pyrimidine (pyrB) or purine (purD, purF, purH, purL, purM) biosynthetic pathways abolished the K(+)-dependent spreading phase. Consistent with a requirement for functional nucleic acid biosynthesis, disruption of purine synthesis with the folic acid antagonist sulfamethoxazole also inhibited spreading. Other transposon insertions disrupted acetoin biosynthesis (the alsS gene), acidifying the growth medium, glutamine synthetase (the glnA gene), and two surfactin biosynthetic genes (srfAA, srfAB). This work identified four classes of surface colonization mutants with defective (i) potassium transport, (ii) surfactin formation, (iii) growth rate or yield, or (iv) pH control. Overall, the ability of B. subtilis to colonize surfaces by spreading is highly dependent on balanced nucleotide biosynthesis and nutrient assimilation, which require sufficient K(+) ions, as well as growth conditions that promote sliding motility.     

Characterization of the molecular properties of KtrC,a second RCK domain that regulates a Ktr channel in Bacillus subtilis

RCK (regulating conductance of K⁺) domains are common regulatory domains that control the activity of eukaryotic and prokaryotic K⁺ channels and transporters. In bacteria these domains play roles in osmoregulation, regulation of turgor and membrane potential and in pH homeostasis. Whole-genome sequencing unveiled RCK gene redundancy, however the biological role of this redundancy is not well understood. In Bacillus subtilis, there are two closely related RCK domain proteins (KtrA and KtrC) that regulate the activity of the Ktr cation channels. KtrA has been well characterized but little is known about KtrC. We have characterized the structural and biochemical proprieties of KtrC and conclude that KtrC binds ATP and ADP, just like KtrA. However, in solution KtrC exist in a dynamic equilibrium between octamers and non-octameric species that is dependent on the bound ligand, with ATP destabilizing the octameric ring relative to ADP. Accordingly, KtrC-ADP crystal structures reveal closed octameric rings similar to those in KtrA, while KtrC-ATP adopts an open assembly with RCK domains forming a super-helix. In addition, both KtrC-ATP and -ADP octamers are stabilized by the signaling molecule cyclic-di-AMP, which binds to KtrC with high affinity. In contrast, c-di-AMP binds with 100-fold lower affinity to KtrA. Despite these differences we show with an E. coli complementation assay that KtrC and KtrA are interchangeable and able to form functional transporters with both KtrB and KtrD. The distinctive properties of KtrC, in particular ligand-dependent assembly/disassembly, suggest that this protein has a specific physiological role that is distinct from KtrA.     

Isolation, characterization, and expression of the Corynebacterium glutamicum betP gene, encoding the transport system for the compatible solute glycine betaine.

Corynebacterium glutamicum accumulates glycine betaine under conditions of high osmolarity. Previous work revealed the existence of a high-affinity glycine betaine permease which is osmotically regulated. In the present study, the corresponding gene was cloned. The betP gene, encoding the glycine betaine uptake carrier, was isolated by heterologous complementation of mutant strain Escherichia coli MKH13. From sequence analysis it is predicted to encode a protein of 595 amino acids. This protein shares 36% identity with the choline transport system BetT and 28% identity with the carnitine transport system CaiT of E. coli, as well as 38% identity with a protein with an unknown function from Haemophilus influenzae. Analysis of hydropathy indicated a common structure for all four transport proteins. After heterologous expression of betP in E. coli MKH13, the measured Km values for glycine betaine and the cotransported Na+ were similar to those found in C. glutamicum, whereas the modulation of activity by osmotic gradients was shifted to lower osmotic values.     

ATP binding to the KTN/RCK subunit KtrA from the K+ -uptake system KtrAB of Vibrio alginolyticus: its role in the formation of the KtrAB complex and its requirement in vivo

Subunit KtrA of the bacterial Na(+)-dependent K(+)-translocating KtrAB systems belongs to the KTN/RCK family of regulatory proteins and protein domains. They are located at the cytoplasmic side of the cell membrane. By binding ligands they regulate the activity of a number of K(+) transporters and K(+) channels. To investigate the function of KtrA from the bacterium Vibrio alginolyticus (VaKtrA), the protein was overproduced in His-tagged form (His(10)-VaKtrA) and isolated by affinity chromatography. VaKtrA contains a G-rich, ADP-moiety binding beta-alpha-beta-fold ("Rossman fold"). Photocross-linking and flow dialysis were used to determine the binding of [(32)P]ATP and [(32)P]NAD(+) to His(10)-VaKtrA. Binding of other nucleotides was estimated from the competition by these compounds of the binding of the (32)P-labeled nucleotides to the protein. [gamma-(32)P]ATP bound with high affinity to His(10)-VaKtrA (K(D) of 9 microm). All other nucleotides tested exhibited K(D) (K(i)) values of 30 microm or higher. Limited proteolysis with trypsin showed that ATP was the only nucleotide that changed the conformation of VaKtrA. ATP specifically promoted complex formation of VaKtrA with the His-tagged form of its K(+)-translocating partner, VaKtrB-His(6), as detected both in an overlay experiment and in an experiment in which VaKtrA was added to VaKtrB-His(6) bound to Ni(2+)-agarose. In intact cells of Escherichia coli both a high of membrane potential and a high cytoplasmic ATP concentration were required for VaKtrAB activity. C-terminal deletions in VaKtrA showed that for in vivo activity at least 169 N-terminal amino acid residues of its total of 220 are required and that its 40 C-terminal residues are dispensable.     

Expression and regulation of a Vibrio alginolyticus sucrose utilization system cloned in Escherichia coli.

A halotolerant collagenolytic Vibrio alginolyticus strain isolated from salted hides had intracellular sucrase activity and did not secret sucrase into the medium. The strain actively transported sucrose by a sucrose-inducible, Na+-independent process. A 10.4-kilobase DNA fragment of V. alginolyticus DNA was cloned into Escherichia coli. The recombinant E. coli(pVS100) could utilize sucrose as a sole carbon source. In contrast to V. alginolyticus, the recombinant E. coli produced both intra- and extracellular sucrase activities. Up to 20% of the total sucrase activity was in the supernatant. Sucrase synthesis in E. coli(pVS100) was inducible and was subject to glucose repression, which was relieved by cyclic AMP. Sucrose was actively transported by a sucrose-inducible, Na+-independent system in E. coli(pVS100). Sucrose uptake was inhibited by the addition of a proton conductor. The maximum velocity and apparent Km values of sucrose uptake for the V. alginolyticus strain and E. coli(pVS100) were 130 nmol/mg of protein per min and 50 microM and 6 nmol/mg of protein per min and 275 microM, respectively.     

Change to alanine of one out of four selectivity filter glycines in KtrB causes a two orders of magnitude decrease in the affinities for both K and Na of the Na dependent K uptake system KtrAB from Vibrio alginolyticus

KtrAB from Vibrio alginolyticus is a recently described new type of high affinity bacterial K⁺ uptake system. Its activity assayed in an Escherichia coli K⁺ uptake negative mutant depended on Na⁺ ions (K_m of 40 μM). Subunit KtrB contains four putative P-loops. The selectivity filter from each P-loop contains a conserved glycine residue. Residue Gly-290 from the third P-loop selectivity filter in KtrB was exchanged for Ala, Ser or Asp. KtrB variants Ser-290 and Asp-290 were without activity. In contrast, KtrB variant Ala-290 was still active. This variant transported K⁺ with a two orders of magnitude decrease in apparent affinity for both K⁺ and Na⁺ with little effect on V_max.     

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

Although it was only recently identified as a second messenger, c-di-AMP was found to have fundamental importance in numerous bacterial functions such as ion transport. The potassium transporter protein, KtrA, was identified as a c-di-AMP receptor. However, the co-crystallization of c-di-AMP with the protein has not been studied. Here, we determined the crystal structure of the KtrA RCK_C domain in complex with c-di-AMP. The c-di-AMP nucleotide, which adopts a U-shaped conformation, is bound at the dimer interface of RCK_C close to helices α3 and α4. c-di-AMP interacts with KtrA RCK_C mainly by forming hydrogen bonds and hydrophobic interactions. c-di-AMP binding induces the contraction of the dimer, bringing the two monomers of KtrA RCK_C into close proximity. The KtrA RCK_C was able to interact with only c-di-AMP, but not with c-di-GMP, 3′,3-cGAMP, ATP, and ADP. The structure of the KtrA RCK_C domain and c-di-AMP complex would expand our understanding about the mechanism of inactivation in Ktr transporters governed by c-di-AMP.