Cyclic di-AMP traps proton-coupled K+ transporters of the KUP family in an inward-occluded conformation

Fuss, Michael F.; Wieferig, Jan-Philip; Corey, Robin A.; Hellmich, Yvonne; Tascón, Igor; Sousa, J.; Stansfeld, Phillip J.; Vonck, Janet; Hänelt, Inga

doi:10.1038/s41467-023-38944-1

Cited by 8 publications

(3 citation statements)

References 57 publications

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“…The second messenger cyclic-di-AMP has recently been shown to act as a backstop for the protein to prevent rampant accumulation of betaine, that is, when the volume has been restored but the ionic strength of the stress-adapted cells is still above the gating threshold. Importantly, cyclic-di-AMP also plays a key role in the control of potassium transport, the other key component of cell volume regulation in bacteria. − …”

Section: Structure Of Bacterial Cytoplasm and Physicochemical Homeost...mentioning

confidence: 99%

Macromolecular Crowding, Phase Separation, and Homeostasis in the Orchestration of Bacterial Cellular Functions

Monterroso,

Margolin,

Boersma

et al. 2024

Chem. Rev.

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Macromolecular crowding affects the activity of proteins and functional macromolecular complexes in all cells, including bacteria. Crowding, together with physicochemical parameters such as pH, ionic strength, and the energy status, influences the structure of the cytoplasm and thereby indirectly macromolecular function. Notably, crowding also promotes the formation of biomolecular condensates by phase separation, initially identified in eukaryotic cells but more recently discovered to play key functions in bacteria. Bacterial cells require a variety of mechanisms to maintain physicochemical homeostasis, in particular in environments with fluctuating conditions, and the formation of biomolecular condensates is emerging as one such mechanism. In this work, we connect physicochemical homeostasis and macromolecular crowding with the formation and function of biomolecular condensates in the bacterial cell and compare the supramolecular structures found in bacteria with those of eukaryotic cells. We focus on the effects of crowding and phase separation on the control of bacterial chromosome replication, segregation, and cell division, and we discuss the contribution of biomolecular condensates to bacterial cell fitness and adaptation to environmental stress. CONTENTS 1910 2.2.5. Fluidization of the Cytoplasm 1911 3. The Bacterial Nucleoid 1911 4. Crowding and Phase Separation in Membrane Environments 4.1. Remodeling of the Membrane by Crowding and Phase Separation 4.

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Section: Structure Of Bacterial Cytoplasm and Physicochemical Homeost...mentioning

confidence: 99%

Macromolecular Crowding, Phase Separation, and Homeostasis in the Orchestration of Bacterial Cellular Functions

Monterroso,

Margolin,

Boersma

et al. 2024

Chem. Rev.

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“…Together, these two regulatory mechanisms provide a rapid route to modulate transporter activity, with the cyclic-di-AMP acting as a ‘double brake’ to stop hyper accumulation of compatible solutes, which is in itself also detrimental to bacterial viability [ 70 ]. Interestingly, the KUP family of K + transporters can also be deactivated by binding of cyclic-di-AMP to its CTD, which in this case is a phosphopantetheine adenylyltransferase (PPAT) domain rather than a CBS domain [ 71 ].…”

Section: Small Molecule Regulation Of Transporter Activitymentioning

confidence: 99%

Flipping the switch: dynamic modulation of membrane transporter activity in bacteria

Elston,

Mulligan,

Thomas

2023

Microbiology

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The controlled entry and expulsion of small molecules across the bacterial cytoplasmic membrane is essential for efficient cell growth and cellular homeostasis. While much is known about the transcriptional regulation of genes encoding transporters, less is understood about how transporter activity is modulated once the protein is functional in the membrane, a potentially more rapid and dynamic level of control. In this review, we bring together literature from the bacterial transport community exemplifying the extensive and diverse mechanisms that have evolved to rapidly modulate transporter function, predominantly by switching activity off. This includes small molecule feedback, inhibition by interaction with small peptides, regulation through binding larger signal transduction proteins and, finally, the emerging area of controlled proteolysis. Many of these examples have been discovered in the context of metal transport, which has to finely balance active accumulation of elements that are essential for growth but can also quickly become toxic if intracellular homeostasis is not tightly controlled. Consistent with this, these transporters appear to be regulated at multiple levels. Finally, we find common regulatory themes, most often through the fusion of additional regulatory domains to transporters, which suggest the potential for even more widespread regulation of transporter activity in biology.

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“…To complement cryo-EM structures, molecular dynamics (MD) simulations are often performed. − These help in studying the dynamics of the protein and the solvent as well as the binding and unbinding of lipids, ligands, and ions. The approach of combining structural data with MD simulations is a powerful technique to understand protein structure and function.…”

Section: Introductionmentioning

confidence: 99%

Role of Protonation States in the Stability of Molecular Dynamics Simulations of High-Resolution Membrane Protein Structures

Lasham,

Djurabekova,

Zickermann

et al. 2024

J. Phys. Chem. B

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Classical molecular dynamics (MD) simulations provide unmatched spatial and time resolution of protein structure and function. However, the accuracy of MD simulations often depends on the quality of force field parameters and the time scale of sampling. Another limitation of conventional MD simulations is that the protonation states of titratable amino acid residues remain fixed during simulations, even though protonation state changes coupled to conformational dynamics are central to protein function. Due to the uncertainty in selecting protonation states, classical MD simulations are sometimes performed with all amino acids modeled in their standard charged states at pH 7. Here, we performed and analyzed classical MD simulations on high-resolution cryo-EM structures of two large membrane proteins that transfer protons by catalyzing protonation/deprotonation reactions. In simulations performed with titratable amino acids modeled in their standard protonation (charged) states, the structure diverges far from its starting conformation. In comparison, MD simulations performed with predetermined protonation states of amino acid residues reproduce the structural conformation, protein hydration, and protein−water and protein−protein interactions of the structure much better. The results support the notion that it is crucial to perform basic protonation state calculations, especially on structures where protonation changes play an important functional role, prior to the launch of any conventional MD simulations. Furthermore, the combined approach of fast protonation state prediction and MD simulations can provide valuable information about the charge states of amino acids in the cryo-EM sample. Even though accurate prediction of protonation states in proteinaceous environments currently remains a challenge, we introduce an approach of combining pK a prediction with cryo-EM density map analysis that helps in improving not only the protonation state predictions but also the atomic modeling of density data.

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Cyclic di-AMP traps proton-coupled K+ transporters of the KUP family in an inward-occluded conformation

Cited by 8 publications

References 57 publications

Macromolecular Crowding, Phase Separation, and Homeostasis in the Orchestration of Bacterial Cellular Functions

Macromolecular Crowding, Phase Separation, and Homeostasis in the Orchestration of Bacterial Cellular Functions

Flipping the switch: dynamic modulation of membrane transporter activity in bacteria

Role of Protonation States in the Stability of Molecular Dynamics Simulations of High-Resolution Membrane Protein Structures

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