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Lieblingsbild
(2017)
Das Bild zeigt die Kryo-EM-Elektronendichtekarte des bakteriellen Kalium-Aufnahmesystems KtrAB mit ADP gebunden mit einer Auflösung von 6.6 Å. Das Bild ist mein Lieblingsbild, weil man bereits auf den ersten Blick eine dramatische Konformationsänderung im Vergleich zu einer früheren ATPgebundenen Struktur erkennen konnte, nämlich die Ausbildung langgestreckter α-Helices (hier gelb markiert), die die regulatorischen A-Untereinheiten (blau) mit den Kaliumionen-translozierenden B-Untereinheiten (grau) verbinden. ...
The capability of osmoadaptation is a prerequisite of organisms that live in an environment with changing salinities. Halobacillus halophilus is a moderately halophilic bacterium that grows between 0.4 and 3 M NaCl by accumulating both chloride and compatible solutes as osmolytes. Chloride is absolutely essential for growth and, moreover, was shown to modulate gene expression and activity of enzymes involved in osmoadaptation. The synthesis of different compatible solutes is strictly salinity- and growth phase-dependent. This unique hybrid strategy of H. halophilus will be reviewed here taking into account the recently published genome sequence. Based on identified genes we will speculate about possible scenarios of the synthesis of compatible solutes and the uptake of potassium ion which would complete our knowledge of the fine-tuned osmoregulation and intracellular osmolyte balance in H. halophilus.
Halobacillus halophilus, a moderately halophilic bacterium isolated from salt marshes, produces various compatible solutes to cope with osmotic stress. Glutamate and glutamine are dominant compatible solutes at mild salinities. Glutamine synthetase activity in cell suspensions of Halobacillus halophilus wild type was shown to be salt dependent and chloride modulated. A possible candidate to catalyze glutamine synthesis is glutamine synthetase A2, whose transcription is stimulated by chloride. To address the role of GlnA2 in the biosynthesis of the osmolytes glutamate and glutamine, a deletion mutant (ΔglnA2) was generated and characterized in detail. We compared the pool of compatible solutes and performed transcriptional analyses of the principal genes controlling the solute production in the wild type strain and the deletion mutant. These measurements did not confirm the hypothesized role of GlnA2 in the osmolyte production. Most likely the presence of another, yet to be identified enzyme has the main contribution in the measured activity in crude extracts and probably determines the total chloride-modulated profile. The role of GlnA2 remains to be elucidated.
Cryo-EM structures of KdpFABC suggest a K+ transport mechanism via two inter-subunit half-channels
(2018)
P-type ATPases ubiquitously pump cations across biological membranes to maintain vital ion gradients. Among those, the chimeric K+ uptake system KdpFABC is unique. While ATP hydrolysis is accomplished by the P-type ATPase subunit KdpB, K+ has been assumed to be transported by the channel-like subunit KdpA. A first crystal structure uncovered its overall topology, suggesting such a spatial separation of energizing and transporting units. Here, we report two cryo-EM structures of the 157 kDa, asymmetric KdpFABC complex at 3.7 Å and 4.0 Å resolution in an E1 and an E2 state, respectively. Unexpectedly, the structures suggest a translocation pathway through two half-channels along KdpA and KdpB, uniting the alternating-access mechanism of actively pumping P-type ATPases with the high affinity and selectivity of K+ channels. This way, KdpFABC would function as a true chimeric complex, synergizing the best features of otherwise separately evolved transport mechanisms.
Potassium homeostasis is vital for all organisms, but is challenging in single-celled organisms like bacteria and yeast and immobile organisms like plants that constantly need to adapt to changing external conditions. KUP transporters facilitate potassium uptake by the co-transport of protons. Here, we uncover the molecular basis for transport in this widely distributed family. We identify the potassium importer KimA from Bacillus subtilis as a member of the KUP family, demonstrate that it functions as a K+/H+ symporter and report a 3.7 Å cryo-EM structure of the KimA homodimer in an inward-occluded, trans-inhibited conformation. By introducing point mutations, we identify key residues for potassium and proton binding, which are conserved among other KUP proteins.
KdpFABC, a high-affinity K+ pump, combines the ion channel KdpA and the P-type ATPase KdpB to secure survival at K+ limitation. Here, we apply a combination of cryo-EM, biochemical assays, and MD simulations to illuminate the mechanisms underlying transport and the coupling to ATP hydrolysis. We show that ions are transported via an intersubunit tunnel through KdpA and KdpB. At the subunit interface, the tunnel is constricted by a phenylalanine, which, by polarized cation-π stacking, controls K+ entry into the canonical substrate binding site (CBS) of KdpB. Within the CBS, ATPase coupling is mediated by the charge distribution between an aspartate and a lysine. Interestingly, individual elements of the ion translocation mechanism of KdpFABC identified here are conserved among a wide variety of P-type ATPases from different families. This leads us to the hypothesis that KdpB might represent an early descendant of a common ancestor of cation pumps.
Cells maintain membrane fluidity by regulating lipid saturation, but the molecular mechanisms of this homeoviscous adaptation remain poorly understood. Here, we have reconstituted the core machinery for sensing and regulating lipid saturation in baker’s yeast to directly characterize its response to defined membrane environments. Using spectroscopic techniques and in vitro ubiquitylation, we uncover a unique sensitivity of the transcriptional regulator Mga2 to the abundance, position, and configuration of double bonds in lipid acyl chains and provide unprecedented insight into the molecular rules of membrane adaptivity. Our data challenge the prevailing hypothesis that membrane viscosity serves as the measured variable for regulating lipid saturation. Rather, we show that the signaling output of Mga2 correlates with the size of a single sensor residue in the transmembrane helix, which senses the lateral pressure and/or compressibility profile in a defined region of the membrane. Our findings suggest that membrane property sensors have evolved remarkable sensitivities to highly specific aspects of membrane structure and dynamics, thus paving the way toward the development of genetically encoded reporters for such membrane properties in the future.
K+ plays an essential role in a different cellular processes in bacteria, and is a central player in microbial adaptation towards a number of environmental challenges. Accordingly, K+ transporters are subject to tight regulation by a diverse set of mechanisms. Here, we discuss three regulatory strategies from three transport systems, as well as the general regulation of K+ homeostasis by the second messenger c-di-AMP.
Cells maintain membrane fluidity by regulating lipid saturation, but the molecular mechanisms of this homeoviscous adaptation remain poorly understood. We have reconstituted the core machinery for regulating lipid saturation in baker’s yeast to study its molecular mechanism. By combining molecular dynamics simulations with experiments, we uncover a remarkable sensitivity of the transcriptional regulator Mga2 to the abundance, position, and configuration of double bonds in lipid acyl chains, and provide insights into the molecular rules of membrane adaptation. Our data challenge the prevailing hypothesis that membrane fluidity serves as the measured variable for regulating lipid saturation. Rather, we show that Mga2 senses the molecular lipid-packing density in a defined region of the membrane. Our findings suggest that membrane property sensors have evolved remarkable sensitivities to highly specific aspects of membrane structure and dynamics, thus paving the way toward the development of genetically encoded reporters for such properties in the future.
Cyclic di-AMP is the only known essential second messenger in bacteria and archaea, regulating different proteins indispensable for numerous physiological processes. In particular, it controls various potassium and osmolyte transporters involved in osmoregulation. In Bacillus subtilis, the K+/H+ symporter KimA of the KUP family is inactivated by c-di-AMP. KimA sustains survival at potassium limitation at low external pH by mediating K+ ions uptake. However, at elevated intracellular K+ concentrations, further K+ accumulation would be toxic. In this study, we reveal the molecular basis of how c-di-AMP binding inhibits KimA. We report cryo-EM structures of KimA with bound c-di-AMP in detergent solution and reconstituted in amphipols. By combining structural data with functional assays and molecular dynamics simulations we reveal how c-di-AMP modulates transport. We show that an intracellular loop in the transmembrane domain interacts with c-di-AMP bound to the adjacent cytosolic domain. This reduces the mobility of transmembrane helices at the cytosolic side of the K+ binding site and therefore traps KimA in an inward-occluded conformation.