Organization of cytoplasmic domains of sarcoplasmic reticulum Ca2+-ATPase in E1P and E1ATP states:: a limited proteolysis study

In order to characterize the domain organization of sarcoplasmic reticulum Ca2+-ATPase in different physiological states, limited proteolysis using three proteases (proteinase K (prtK), V8 and trypsin) was conducted systematically and quantitatively. The differences between E-2 and E2P were examined in our previous study and E2P was characterized by the complete resistance to all three proteases (except for trypsin attack at the very top of the molecule (T1 site)). The same strategies were employed in this study for E(1)ATP, E(1)PADP and E1P states. Because of the transient nature of these states, they were either stabilized by non-hydrolyzable analogues or made predominant by adjusting buffer conditions. Aluminum fluoride (without ADP) was found to stabilize E1P. All these states were characterized by strong (E(1)ATP) to complete (E(1)PADP and E1P) resistance to prtK and to V8 but only weak resistance to trypsin at the T2 site. Because prtK and V8 primarily attack the loops connecting the A domain to the transmembrane helices whereas the trypsin T2 site (Arg(198)) is located on the outermost loop in the A domain, these results lead us to propose that the A domain undergoes a large amount of rotation between Ell? and E2P. Combined with previous results, we demonstrated that four states can be clearly distinguished by the susceptibility to three proteases, which will be very useful for establishing the conditions for structural studies. (C) 2001 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.