title: Synergistic effects of ATP and RNA binding to human DEAD-box protein DDX1.
abstract: RNA helicases of the DEAD-box protein family form the largest group of helicases. The human DEAD-box protein 1 (DDX1) plays an important role in tRNA and mRNA processing, is involved in tumor progression and is also hijacked by several virus families such as HIV-1 for replication and nuclear export. Although important in many cellular processes, the mechanism of DDX1's enzymatic function is unknown. We have performed equilibrium titrations and transient kinetics to determine affinities for nucleotides and RNA. We find an exceptional tight binding of DDX1 to adenosine diphosphate (ADP), one of the strongest affinities observed for DEAD-box helicases. ADP binds tighter by three orders of magnitude when compared to adenosine triphosphate (ATP), arresting the enzyme in a potential dead-end ADP conformation under physiological conditions. We thus suggest that a nucleotide exchange factor leads to DDX1 recycling. Furthermore, we find a strong cooperativity in binding of RNA and ATP to DDX1 that is also reflected in ATP hydrolysis. We present a model in which either ATP or RNA binding alone can partially shift the equilibrium from an 'open' to a 'closed'-state; this shift appears to be not further pronounced substantially even in the presence of both RNA and ATP as the low rate of ATP hydrolysis does not change.
authors.affiliation: Department of Biomolecular Mechanisms, Max-Planck-Institute for Medical Research, Jahnstrasse 29, 69120 Heidelberg, Germany.
authors.forename: Julian N
authors.initials: JN
authors.lastname: Kellner
authors.affiliation: Department of Biomolecular Mechanisms, Max-Planck-Institute for Medical Research, Jahnstrasse 29, 69120 Heidelberg, Germany.
authors.forename: Jochen
authors.initials: J
authors.lastname: Reinstein
authors.affiliation: Department of Biomolecular Mechanisms, Max-Planck-Institute for Medical Research, Jahnstrasse 29, 69120 Heidelberg, Germany anton.meinhart@mpimf-heidelberg.mpg.de.
authors.forename: Anton
authors.initials: A
authors.lastname: Meinhart
mesh_terms: D000244:Adenosine Diphosphate / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000465:Algorithms; D001667:Binding, Competitive; D002942:Circular Dichroism; D053487:DEAD-box RNA Helicases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D011485:Protein Binding; D055503:Protein Multimerization; D017433:Protein Structure, Secondary; D012313:RNA / Q000235:genetics / Q000378:metabolism*
chemical_list: D000244:Adenosine Diphosphate; D012313:RNA; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; C086252:DDX1 protein, human; D053487:DEAD-box RNA Helicases