Spectroscopic and crystallographic studies of the mutant R416W give insight into the nucleotide binding traits of subunit B of the A₁A_o ATP synthase

Abstract

A strategically placed tryptophan in position of Arg416 was used as an optical probe to monitor adenosine triphosphate and adenosine-diphosphate binding to subunit B of the A(1)A(O) adenosine triphosphate (ATP) synthase from Methanosarcina mazei Gö1. Tryptophan fluorescence and fluorescence correlation spectroscopy gave binding constants indicating a preferred binding of ATP over ADP to the protein. The X-ray crystal structure of the R416W mutant protein in the presence of ATP was solved to 2.1 A resolution, showing the substituted Trp-residue inside the predicted adenine-binding pocket. The cocrystallized ATP molecule could be trapped in a so-called transition nucleotide-binding state. The high resolution structure shows the phosphate residues of the ATP near the P-loop region (S150-E158) and its adenine ring forms pi-pi interaction with Phe149. This transition binding position of ATP could be confirmed by tryptophan emission spectra using the subunit B mutant F149W. The trapped ATP position, similar to the one of the binding region of the antibiotic efrapeptin in F(1)F(O) ATP synthases, is discussed in light of a transition nucleotide-binding state of ATP while on its way to the final binding pocket. Finally, the inhibitory effect of efrapeptin C in ATPase activity of a reconstituted A(3)B(3)- and A(3)B(R416W)(3)-subcomplex, composed of subunit A and the B subunit mutant R416W, of the A(1)A(O) ATP synthase is shown.

The Transition-Like State and Pi Entrance into the Catalytic A Subunit of the Biological Engine A-ATP Synthase

“…[22][23][24] Significance of the second vanadate position Also novel in the A Vi structure presented is the presence of a second vanadate, positioned in the region exactly opposite the nucleotide-binding site. This region does not bind vanadate randomly, since the ATP molecule transiently associates here on its way to the final binding pocket in the subunit B structure 25 (Supplementary Fig. S2).…”

Structural Basis for a Unique ATP Synthase Core Complex from Nanoarcheaum equitans

“…These closed and open forms have also been observed in the bovine mitochondrial F 1 -ATP synthase (6) as well as in the V-type ATPases from T. thermophilus (PDB codes 3W3A, 3A5D, and 3A5C) (56). Furthermore, transitional states of ADP capture have been shown for the M. mazei subunit B, indicating this C-terminal entry route for the nucleotide (43,57,58) and the nucleotide binding-induced conformational change required for its catalytic mechanism (29,30). However, in our NeqAB complexes, we did not observe any significant conformational transitions as a result of nucleotide binding (Figs.…”

“…The SXSGLPHN motif is known to be the Walker A homologous region in the regulatory subunit B of the V/A-type ATP synthase family ( Fig. 2a) (43). The Arg-326 residue of NeqB was also found to be highly conserved across the homologs; this residue has been reported to be crucial for nucleotide hydrolysis (29,39).…”

Subunit F modulates ATP binding and migration in the nucleotide-binding subunit B of the A1AO ATP synthase of Methanosarcina mazei Gö1

“…The residues marked with the superscript ( * ) are highly conserved across the homologues of subunit B in the A-and F-ATP synthases as well as VATPases ( Fig. 7 described in Kumar et al 2009). …”