H662 is the linchpin of ATP hydrolysis in the nucleotide-binding domain of the ABC transporter HlyB.
The ABC transporter HlyB is a central element of the HlyA secretion machinery, a paradigm of Type I secretion. Here, we describe the crystal structure of the HlyB-NBD (nucleotide-binding domain) with H662 replaced by Ala in complex with ATP/Mg2+. The dimer shows a composite architecture, in which two intact ATP molecules are bound at the interface of the Walker A motif and the C-loop, provided by the two monomers. ATPase measurements confirm that H662 is essential for activity. Based on these data, we propose a model in which E631 and H662, highly conserved among ABC transporters, form a catalytic dyad. Here, H662 acts as a 'linchpin', holding together all required parts of a complicated network of interactions between ATP, water molecules, Mg2+, and amino acids both in cis and trans, necessary for intermonomer communication. Based on biochemical experiments, we discuss the hypothesis that substrate-assisted catalysis, rather than general base catalysis might operate in ABC-ATPases.
Zaitseva
Jelena
J
Institute of Biochemistry, Biocenter, Johann-Wolfgang Goethe University Frankfurt, Frankfurt, Germany.
Jenewein
Stefan
S
Jumpertz
Thorsten
T
Holland
I Barry
IB
Schmitt
Lutz
L
D018528:ATP-Binding Cassette Transporters / Q000737:chemistry* / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000409:Alanine / Q000737:chemistry; D019943:Amino Acid Substitution; D001665:Binding Sites; D002384:Catalysis; D018360:Crystallography, X-Ray; D019281:Dimerization; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism; D018698:Glutamic Acid / Q000737:chemistry; D005973:Glutamine / Q000737:chemistry; D006460:Hemolysin Proteins / Q000737:chemistry* / Q000378:metabolism; D006639:Histidine / Q000737:chemistry / Q000502:physiology*; D006860:Hydrogen Bonding; D006868:Hydrolysis; D008958:Models, Molecular; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship
D018528:ATP-Binding Cassette Transporters; D029968:Escherichia coli Proteins; D006460:Hemolysin Proteins; D005973:Glutamine; D018698:Glutamic Acid; D006639:Histidine; D000255:Adenosine Triphosphate; C086257:hemolysin B; D000409:Alanine