ATP-binding Modes and Functionally Important Interdomain Bonds of Sarcoplasmic Reticulum Ca super(2+)-ATPase Revealed by Mutation of Glycine 438, Glutamate 439, and Arginine 678

ATP binds to sarcoplasmic reticulum Ca super(2+)-ATPase both in a phosphorylating (catalytic) mode and in a nonphosphorylating (modulatory) mode, the latter leading to acceleration of phosphoenzyme turnover (Ca sub(2)E sub(1)P arrow right E sub(2)P and E sub(2)P arrow right E sub(2) reactions) and Ca super(2+) binding (E sub(2) arrow right Ca sub(2)E sub(1)). In some of the Ca super(2+)-ATPase crystal structures, Arg super(678) and Glu super(439) seem to be involved in the binding of nucleotide or an associated Mg super(2+) ion. We have replaced Arg super(678), Glu super(439), and Gly super(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 Arg super(678) in nucleotide binding and to the importance of interdomain bonds Glu super(439)-Ser super(186) and Arg super(678)-Asp super(203) in stabilizing the E sub(2)P and E sub(2) intermediates, respectively. Mutation of Arg super(678) had conspicuous effects on ATP/MgATP binding to the E sub(1) form and ADP binding to Ca sub(2)E sub(1)P, as well as ATP/MgATP binding in modulatory modes to E sub(2)P and E sub(2), whereas the effects on ATP/MgATP acceleration of the Ca sub(2)E sub(1)P arrow right E sub(2)P transition were small, suggesting that the nucleotide that accelerates Ca sub(2)E sub(1)P arrow right E sub(2)P binds differently from that modulating the E sub(2)P arrow right E sub(2) and E sub(2) arrow right Ca sub(2)E sub(1) reactions. Mutation of Glu super(439) hardly affected nucleotide binding to E sub(1), Ca sub(2)E sub(1)P, and E sub(2), but it led to disruption of the modulatory effect of ATP on E sub(2)P arrow right E sub(2) and acceleration of the latter reaction, indicating that ATP normally modulates E sub(2)P arrow right E sub(2) by interfering with the interaction between Glu super(439) and Ser super(186). Gly super(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 super(439) and Thr super(441).