ATP utilization and RNA conformational rearrangement by DEAD-box proteins

Abstract

RNA helicase enzymes catalyze the in vivo folding and conformational re-arrangement of RNA. DEAD-box proteins (DBPs) make up the largest family of RNA helicases and are found across all phyla. DBPs are molecular motor proteins that utilize chemical energy in cycles of ATP binding, hydrolysis, and product release to perform mechanical work resulting in reorganization of cellular RNAs. DBPs contain a highly conserved motor domain helicase core. Auxiliary domains, enzymatic adaptations, and regulatory partner proteins contribute to the diversity of DBP function throughout RNA metabolism. In this review we focus on the current understanding of the DBP ATP utilization mechanism in rearranging and unwinding RNA structures. We discuss DBP structural properties, kinetic pathways, and thermodynamic features of nucleotide-dependent interactions with RNA. We highlight recent advances in the DBP field derived from biochemical and molecular biophysical investigations aimed at developing a quantitative mechanistic understanding of DBP molecular motor function.

Figure 1

DEAD-box proteins (DBPs) display a high degree of sequence conservation within the characteristic DBP sequence motifs. The DBP motor domain is made up of two RecA-like domains, and individual family members may have N-terminal extensions, C-terminal extensions, or both. Four example DBP sequences from a broad range of phyla are shown. Numbers indicate the number of amino acids between sequence motifs. The motifs are colored according to their biochemical function and structural location: ATP binding (orange), RNA binding (green), and coupling ATPase and RNA helicase functions (teal).

Figure 2

DEAD-box protein (DBP) structure and relative arrangement of ATP and RNA binding sites. (a) Structural arrangement of the characteristic sequence motifs mapped on the structure of Mss116 from Saccharomyces cerevisiae (27), colored the same as in Figure 1. The bound single-stranded RNA (ssRNA) is colored red, and the bound ATP analog (AMPpNp) is colored according to atom type: carbon (teal), oxygen (red), nitrogen (blue), phosphorus (gold). The two RecA-like domains are colored purple and gray, and the C-terminal extension is colored brown. (b) Aligned structures (calculated using only conserved motif residues) of Mss116 as in panel a, and DDX19 from Homo sapiens (23). Mss116 is colored teal and DDX19 is colored blue. The RNA and AMPpNp bound to Mss116 and DDX19 are colored red and gold, respectively.

Figure 3

Schematic depicting the RNA-activated ATPase cycle for a monomeric DEAD-box protein (DBP) helicase with a single RNA binding site. DBP exists in free (H) and RNA-bound forms with single-stranded RNA (sR) or double-stranded RNA (dR) and with or without bound adenine nucleotide ATP (T) or ADP (D) and inorganic phosphate (P_i). The displaced duplex fragment is indicated by sP. The k_i are rate constants for each of the ith ATPase cycle steps.

Figure 4

The ATP-dependent DEAD-box protein (DBP) double-stranded RNA unwinding cycle. The DBP is depicted as having two lobes that represent the two RecA-like domains of the helicase motor core. The RNA is colored purple and the bound nucleotide state is indicated with red letters. The different helicase and helicase-RNA complex states are named according to Figure 3. The unwinding cycle proceeds clockwise. Darkening in the DBP blue coloration indicates the weak-to-strong transition in RNA affinity during ATPase cycling.