2009-07, Weidemüller, Christian, Siu, Shirley W. I., Böckmann, Rainer A., Hauser, Karin

P-type ATPases actively transport cations across the membrane. The basic mechanisms of ion transport and energy transduction are supposed to be the same in all P-type ATPases. Both reaction mechanisms were studied computationally for the Ca2+-ATPase and Na+/K+-ATPase. The Ca2+ transport of the Ca2+-ATPase is associated with a proton countertransport from the SR to the cytoplasm. Binding of the protons is thought to take place at acidic residues in the binding sites. The protonation of acidic ligands were analyzed in different enzyme states of the Ca2+-ATPase by multiconformation continuum electrostatic calculations. Glu771, Asp800 and Glu908 are prime candidates for the proton countertransporting residues and are likely to receive and release their proton via the same path. The Glu309 instead might serve as a proton shuttle between Ca2+ binding site I and the cytoplasm. The reaction cycle of P-Type ATPases is physiologically initiated by the binding and hydrolysis of ATP but can also be induced experimentally by voltage jumps across the membrane. We simulated the applied electric field by an “ionic capacitor” and studied the impact on different enzyme states of the Ca2+-ATPase and the Na+/K+-ATPase by a combination of MCCE and MD. A selective activation of specific helices in response to the electric field is observed.

2008, Weidemüller, Christian, Hauser, Karin

P-type ATPases are membrane proteins acting as ion pumps that drive an active transport of cations across the membrane against a concentration gradient. The required energy for the ion transport is provided by binding and hydrolysis of ATP. A reaction mechanism of ion transport and energy transduction is assumed to be common for all P-type ATPases and generally described by the Post-Albers cycle. Transient currents and charge translocation of P-type ATPases were extensively investigated by electrical measurements that apply voltage jumps to initiate the reaction cycle. In this study, we simulate an applied voltage across the membrane by an electric field and perform electrostatic calculations in order to verify the experimentally-driven hypothesis that the energy transduction mechanism is regulated by specific structural elements. Side chain conformational and ionization changes induced by the electric field are evaluated for each transmembrane helix and the selectivity in response is qualitatively analyzed for the Ca2+-ATPase as well as for structural models of the Na+/K+-ATPase. Helix M5 responds with more conformer changes as compared to the other transmembrane helices what is even more emphasized when the stalk region is included. Thus our simulations support experimental results and indicate a crucial role for the highly conserved transmembrane helix M5 in the energy transduction mechanism of P-type ATPases.

2009-06, Weidemüller, Christian, Hauser, Karin

This paper summarizes our present electrostatic calculations on P-type ATPases and their contribution to understand the molecular details of the reaction mechanisms. One focus was set on analyzing the proton countertransport of the sarcoplasmic reticulum Ca2+-ATPase (SERCA1a). Protonation of acidic residues was calculated in dependence of pH for different enzyme states in the reaction cycle of the Ca2+-ATPase. We proposed that the acidic Ca2+ ligands Glu 771, Asp 800 and Glu 908 participate in the proton countertransport whereas Glu 309 is more likely to serve as a proton shuttle between binding site I and the cytoplasm. Complementary to infrared measurements, we assigned infrared bands to specific Ca2+ ligands that are hydrogen bonded. Ion pathways were proposed based on the calculations and structural data. Another focus was set on analyzing the energy transduction mechanism of P-type ATPases. In accordance to electrophysiological experiments, we simulated an electric field across the membrane. The impact of the electric field was studied by an accumulated number of residue conformational and ionization changes on specific transmembrane helices. Our calculations on the Ca2+-ATPase and the Na+/K+-ATPase indicated that the highly conserved transmembrane helix M5 is one structural element that is likely to act as energy transduction element in P-type ATPases. Perspectives and limitations of the electrostatic calculations for future computational studies are pointed out.

2008, Weidemüller, Christian, Hauser, Karin

The Na+/K+-ATPase pumps ions across the membrane which is necessary for maintaining the membrane potential. The energy for this active ion transport is provided by binding and hydrolysis of ATP and has to be transferred from the cytoplasmic nucleotide binding site to the transmembrane domain of ion transport. This transport cycle can also be induced experimentally by applying voltage jumps across the membrane. We simulated the applied electric field by an ionic capacitor and studied the impact on the Na+/K+-ATPase by a combination of multiconformation continuum electrostatics (MCCE) and molecular dynamics (MD). Our calculations show a selective activation of the helices M5, M6 and M8 by the electric field. Those helices are likely to act as energy transduction elements.