ATP-binding modes and functionally important interdomain bonds of sarcoplasmic reticulum Ca2+-ATPase revealed by mutation of glycine 438, glutamate 439, and arginine 678

ATP binds to sarcoplasmic reticulum Ca2+-ATPase both in a phosphorylating ( catalytic) mode and in a nonphosphorylating ( modulatory) mode, the latter leading to acceleration of phosphoenzyme turnover (Ca2E1P -> E2P and E2P -> E-2 reactions) and Ca2+ binding (E-2 -> Ca2E1). In some of the Ca2+-ATPase crystal structures, Arg(678) and Glu(439) seem to be involved in the binding of nucleotide or an associated Mg2+ ion. We have replaced Arg(678), Glu(439), and Gly(438) with alanine to examine their importance for the enzyme cycle and the modulatory effects of ATP and MgATP. The results point to the key role of Arg678 in nucleotide binding and to the importance of interdomain bonds Glu(439)-Ser(186) and Arg(678)-Asp(203) in stabilizing the E2P and E-2 intermediates, respectively. Mutation of Arg(678) had conspicuous effects on ATP/MgATP binding to the E-1 form and ADP binding to Ca2E1P, as well as ATP/MgATP binding in modulatory modes to E2P and E-2, whereas the effects on ATP/MgATPacceleration of the Ca2E1P -> E2P transition were small, suggesting that the nucleotide that accelerates Ca2E1P -> E2P binds differently from that modulating the E2P -> E-2 and E-2 -> Ca2E1 reactions. Mutation of Glu(439) hardly affected nucleotide binding to E-1, Ca2E1P, and E-2, but it led to disruption of the modulatory effect of ATP on E2P 3 E2 and acceleration of the latter reaction, indicating that ATP normally modulates E2P -> E-2 by interfering with the interaction between Glu(439) and Ser(186). Gly(438) seems to be important for this interaction as well as for nucleotide binding, probably because of its role in formation of the helix containing Glu(439) and Thr(441).