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DEAD-box proteins are the largest family of nucleic acid helicases and are crucial to RNA metabolism throughout all domains of life1,2. They contain a conserved ‘helicase core’ of two RecA-like domains (domains 1 and 2; D1 and D2, respectively), which uses ATP to catalyze the unwinding of short RNA duplexes by nonprocessive, local strand separation3. This mode of action differs from that of translocating helicases and allows DEAD-box proteins to remodel large RNAs and RNA-protein complexes without globally disrupting RNA structure4. However, the structural basis for this distinctive mode of RNA-unwinding remains unclear. Here, structural, biochemical, and genetic analyses of the yeast DEAD-box protein Mss116p indicate that the helicase core domains have modular functions that enable a novel mechanism for RNA duplex recognition and unwinding. By investigating D1 and D2 individually and together, we find that D1 acts as an ATP-binding domain and D2 functions as an RNA-duplex recognition domain. D2 contains a nucleic acid-binding pocket that is formed by conserved DEAD-box protein sequence motifs and accommodates A-form but not B-form duplexes, providing a basis for RNA substrate specificity. Upon a conformational change in which the two core domains join to form a ‘closed-state’ with an ATPase active site, conserved motifs in D1 promote the unwinding of duplex substrates bound to D2 by excluding one RNA strand and bending the other. Our results provide a comprehensive structural model for how DEAD-box proteins recognize and unwind RNA duplexes. This model explains key features of DEAD-box protein function and affords new perspective on how the evolutionarily related cores of other RNA and DNA helicases diverged to use different mechanisms.

Topological knots are found in a considerable number of protein structures, but it is not clear how they knot and fold within the cellular environment. We investigated the behavior of knotted protein molecules as they are first synthesized by the ribosome using a cell-free translation system. We found that newly translated knotted proteins can spontaneously self-tie and do not require the assistance of molecular chaperones to fold correctly to their trefoil-knotted structures. This process is slow but efficient, and we found no evidence of misfolded species. A kinetic analysis indicates that the knotting process is rate limiting, occurs post-translationally, and is specifically and significantly (P < 0.001) accelerated by the GroEL-GroES chaperonin complex. This demonstrates a new active mechanism for this molecular chaperone and suggests that chaperonin-catalyzed knotting probably dominates in vivo. These results explain how knotted protein structures have withstood evolutionary pressures despite their topological complexity.

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Structures that contain a knot formed by the path of the polypeptide backbone represent some of the most complex topologies observed in proteins. How or why these topological knots arise remains unclear. By developing a method to experimentally trap and detect knots in nonnative polypeptide chains, we find that two knotted methyltransferases, YibK and YbeA, can exist in a trefoil-knot conformation even in their chemically unfolded states. The unique denatured-state topology of these molecules explains their ability to efficiently fold to their native knotted structures in vitro and offers insights into the potential role of knots in proteins. Furthermore, the high prevalence of the denatured-state knots identified here suggests that they are either difficult to untie or that threading of any untied molecules is rapid and spontaneous. The occurrence of such knotted topologies in unfolded polypeptide chains raises the possibility that they could play an important, and as yet unexplored, role in folding and misfolding processes in vivo.

In the last decade, a new class of proteins has emerged that contain a topological knot in their backbone. Although these structures are rare, they nevertheless challenge our understanding of protein folding. In this review, we provide a short overview of topologically knotted proteins with an emphasis on newly discovered structures. We discuss the current knowledge in the field, including recent developments in both experimental and computational studies that have shed light on how these intricate structures fold.

The mitochondrial DEAD-box proteins Mss116p of Saccharomyces cerevisiae and CYT-19 of Neurospora crassa are ATP-dependent helicases that function as general RNA chaperones. The helicase core of each protein precedes a C-terminal extension and a basic tail, whose structural role is unclear. Here we used small-angle X-ray scattering to obtain solution structures of the full-length proteins and a series of deletion mutants. We find that the two core domains have a preferred relative orientation in the open state without substrates, and we visualize the transition to a compact closed state upon binding RNA and adenosine nucleotide. An analysis of complexes with large chimeric oligonucleotides shows that the basic tails of both proteins are attached flexibly, enabling them to bind rigid duplex DNA segments extending from the core in different directions. Our results indicate that the basic tails of DEAD-box proteins contribute to RNA-chaperone activity by binding nonspecifically to large RNA substrates and flexibly tethering the core for the unwinding of neighboring duplexes.EAD-box proteins comprise the largest family of helicases and play critical roles in all aspects of RNA metabolism, including RNA splicing, translation, ribosome assembly, RNA degradation, and RNA transport (1, 2). Although their functions vary broadly, all DEAD-box proteins have a conserved helicase core consisting of two RecA-like domains (denoted domains 1 and 2) separated by a flexible linker and operate by a mechanism involving conformational changes within this core (3-6). These conformational changes couple cycles of ATP binding and hydrolysis to RNA binding and release by the core, enabling DEADbox proteins to promote local RNA unwinding and remodeling of structured RNAs and RNA-protein complexes (3-5).Structural and biochemical studies have given important insights into how DEAD-box proteins unwind RNA (2-4). The two helicase core domains are separated from each other in an open state in the absence of substrates, but they interact to form a compact, closed state upon binding of ATP and RNA. In this closed state, the interface between the two core domains forms a catalytic site for ATP hydrolysis and an RNA-binding cleft, which can accommodate a short region of a duplex strand (7). The tight binding of the RNA strand within this cleft results in a bend that is incompatible with partner-strand base pairing, leading to local RNA unwinding (7-10). ATP hydrolysis and dissociation of the P i product are then necessary to release the bound single-stranded RNA (9,11,12).Whereas the helicase core is central to the biochemical activities of DEAD-box proteins, most also have substantial extensions or additional domains at their N-and/or C-termini (1, 3). These extensions vary widely in size, composition, and function, but many are thought to interact with RNA or protein components of complexes to direct the DEAD-box proteins to desired sites of action. A common type of extension is a basic C-terminal sequence (C-tail), which is typically predicted...