| Popis: |
Ca2+-ATPases actively maintain electrochemical gradients for calcium and very low cytosolic calcium levels. The overall architecture of this class of ATPase is conserved among species, but differences in the kinetics and the stoichiometry of the ions transported have developed during evolution. Mechanisms of Ca2+-ATPases have been described in detail by crystal structures of the mammalian sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) stabilized by inhibitors at specific intermediate steps of the transport cycle, and this is assumed to extrapolate to other calcium pumps.To understand how adaptive mechanisms translate sequence variations among Ca2+-ATPases to specific functions, detailed structural information of a more diverse pool of Ca2+-ATPases is helpful. I have used X-ray crystallography to solve three crystal structures of the Ca2+-ATPase 1 from Listeria Monocytogenes (LMCA1) at 3-4Å, one of them being the first high-resolution structure of a bacterial Ca2+-ATPase. The structures provide details that can explain why LMCA1 both has an altered ion stoichiometry and dephosphorylate with a faster rate when compared to SERCA. These findings elucidate how prokaryotic and eukaryotic Ca2+-ATPases have evolved to work in a range of different environments across the domains of life that transport mechanisms must adapt to. When active, plasma membrane Ca2+-ATPases are driven by ATP hydrolysis to transport calcium across membranes against a nearly twenty thousand-fold concentration gradient (approx. 100 nM vs. 2 mM). To overcome this gradient, it is crucial that an essentially irreversible step is present in the cycle to avoid reflux. Earlier, a single-molecule FRET (smFRET) study of LMCA1 from our laboratory revealed an intermediate G4-[Ca]E2P state, which suggested that the irreversible step for LMCA1 is the release of calcium and possibly ADP. To better understand this, I have assessed multiple smFRET constructs that could reveal dynamics of both the ADP and calcium releasing step. The mutations affected the activity considerably, and none of the constructs assessed could be used for smFRE, but fortunately, I solved the intermediate G4-[Ca]E2P state at 3.5Å by cryogenic electronic microscopy (cryo-EM), and it reveals no bound ADP to this state. Moreover, missing density for key residues in the binding site suggests that it is dynamic and prepares for calcium release, which indicates that calcium release takes part in the irreversible step.Typically, these pumps are not continuously active and can turn off by autoregulatory mechanisms to conserve energy when not needed. From a collaboration with the group of Dimitrios Stamou we know that single-molecule LMCA1 switches between modes of active and resting periods (not yet published). Crystal structures of SERCA show that the nucleotide binding (N) domain is detached from the other cytosolic domains in the apo state, which is in sharp contrast to the more compact organization of the of cytosolic domains during active transportation. To reveal if the N domain takes part of the regulation mechanism, I have also assessed constructs for smFRET, but likewise for the irreversible transition no construct was suitable for measuring smFRET.To measure a FRET signal, fluorophores must conjugate to the protein via a thiol-maleimide reaction. A poor labeling efficiency was observed for all constructs, which motivated me to develop a new method to explore and optimize the labeling reaction, namely on-beads labeling. Several parameters were adjusted, but unfortunately none of them resulted in an increase in labeling efficiency. |
