title: Spectroscopic and crystallographic studies of the mutant R416W give insight into the nucleotide binding traits of subunit B of the A1Ao 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.
authors.affiliation: Division of Structural and Computational Biology, Nanyang Technological University, School of Biological Sciences, 60 Nanyang Drive, Singapore 637551, Republic of Singapore.
authors.forename: Anil
authors.initials: A
authors.lastname: Kumar
authors.forename: Malathy Sony Subramanian
authors.initials: MS
authors.lastname: Manimekalai
authors.forename: Asha Manikkoth
authors.initials: AM
authors.lastname: Balakrishna
authors.forename: Cornelia
authors.initials: C
authors.lastname: Hunke
authors.forename: Sven
authors.initials: S
authors.lastname: Weigelt
authors.forename: Norbert
authors.initials: N
authors.lastname: Sewald
authors.forename: Gerhard
authors.initials: G
authors.lastname: Grüber
mesh_terms: D000244:Adenosine Diphosphate / Q000737:chemistry / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism*; D019943:Amino Acid Substitution; D019843:Archaeal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018360:Crystallography, X-Ray; D008958:Models, Molecular; D009711:Nucleotides / Q000737:chemistry / Q000378:metabolism; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000378:metabolism
chemical_list: D019843:Archaeal Proteins; D009711:Nucleotides; D021122:Protein Subunits; D000244:Adenosine Diphosphate; D014364:Tryptophan; D000255:Adenosine Triphosphate; C113203:A1A0 ATPase, Methanosarcina mazei; D006180:Proton-Translocating ATPases