Crystal Structure of Conserved Domains 1 and 2 of the Human DEAD-box Helicase DDX3X in Complex with the Mononucleotide AMP

• DExD-box helicases are involved in all aspects of cellular RNA metabolism. Conserved domains 1 and 2 contain nine signature motifs that are responsible for nucleotide binding, RNA binding and ATP hydrolysis. The human DEAD-box helicase DDX3X has been associated with several different cellular processes, such as cell-growth control, mRNA transport and translation, and is suggested to be essential for the export of unspliced/partially spliced HIV mRNAs from the nucleus to the cytoplasm. Here, the crystal structure of conserved domains 1 and 2 of DDX3X, including a DDX3-specific insertion that is not generally found in human DExD-box helicases, is presented. The N-terminal domain 1 and the C-terminal domain 2 both display RecA-like folds comprising a central β-sheet flanked by α-helices. Interestingly, the DDX3X-specific insertion forms a helical element that extends a highly positively charged sequence in a loop, thus increasing the RNA-binding surface of the protein. Surprisingly, although DDX3X was crystallized in the presence of a large excess of ADP or the slowly hydrolyzable ATP analogue ATPγS the contaminant AMP was seen in the structure. A fluorescent-based stability assay showed that the thermal stability of DDX3X was increased by the mononucleotide AMP but not by ADP or ATPγS, suggesting that DDX3X is stabilized by AMP and elucidating why AMP was found in the nucleotide-binding pocket.

Ämnesord

NATURVETENSKAP  -- Biologi (hsv//swe)

NATURAL SCIENCES  -- Biological Sciences (hsv//eng)

Nyckelord

DEAD-box

helicase

HIV

nucleotide

RNA

adenosine diphosphate

adenosine phosphate

adenosine triphosphate

DEAD box protein

dead box protein ddx3x

mononucleotide amp

RNA binding protein

unclassified drug

amino acid sequence

amino terminal sequence

article

carboxy terminal sequence

crystal structure

crystallization

priority journal

Adenosine Monophosphate

Binding Sites

Conserved Sequence

Crystallography

X-Ray

DEAD-box RNA Helicases

Enzyme Stability

Humans

Hydrolysis

Models

Molecular

Molecular Sequence Data

Protein Structure

Tertiary

RNA-Binding Proteins

Sequence Homology

Amino Acid

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