Domain 2 of the Saccharomyces cerevisiae DEAD-Box Helicase Mss116p

Cristina Nunes '15 and Robert Turlington '16


I. Introduction

The Saccharomyces cerevisiae protein Mss116p is a member of the DEAD-box family of helicase proteins that contributes to the folding and splicing of mitochondrial group I and II introns.2 Although these types of introns are capable of catalyzing their own splicing, they occasionally form stable inactive structures that must be resolved in order for RNA folding and splicing to occur.1 Mss116p helps to unfold these inactive structures and then rearrange them into an active form.  Mss116p, unlike many helicases, unwinds short sequences of dsRNA through a non-processive mechanism that involves local strand separation rather than translocation through a DNA or RNA duplex. As a result of its low processivity, Mss116p separates several consecutive complementary bases without disrupting the overall structure of the dsRNA.1 The lack of processivity in Mss116p results from the particular way in which it interacts with RNA.  Mss116p binds both dsRNA and an ATP molecule in the initial steps leading to strand separation.  It then separates the two strands through the hydrolysis of ATP and a resultant conformational change in the protein.  Importantly, Mss116p quickly dissociates from the RNA after catalyzing strand separation and must rebind the RNA again before performing further helicase activity.  Mss116p's low affinity for RNA after the hydrolysis of ATP and subsequent departure of ADP and Pi from the protein is ultimately what leads to its low processivity.4 

Mss116p is composed of two domains, each of which plays a fundamental role in dsRNA strand separation. One domain forms the structural basis for dsRNA recognition by Mss116p while the other functions as a conserved ATP binding domain required both for strand separation and ATP hydrolysis.2 Thus far, only domain 2 has been crystallized.  The crystal structure shown here contains four copies of this domain.  However, this tutorials will deal with only a .

II. General Structure of D2 of Mss116p

The active site of Mss116p is located at the interface between two core domains, D1 and D2. These two domains, attached through a peptide bond between residue 334 of the carboxy terminus of D1 and residue 335 of the amino terminus of D2, function independently in the initial steps leading to Mss116p helicase activity.1,2 Here we focus on the structure and function of , the dsRNA binding domain. The main body of D2 is made up of a seven-stranded surrounded by . D2 also includes a carboxy terminal extension consisting of and a two-stranded . The CTE of D2 plays an essential role in the initial binding of dsRNA by Mss116p in addition to stabilizing the helicase core through hydrophobic packing with the rest of D2. Mss116p substrate specificity is based on the geometry of the phosphate backbone of the dsRNA. The positively charged binding pocket of D2 binds selectively to . This selective binding of A-form dsRNA prevents Mss116p from binding to and unwinding dsDNA, which is most often found in the B-form.2

III. Interactions Between Strand 1 of the dsRNA and D2

All bonds between Mss116p and dsRNA involve non sequence-specific interactions between the protein and the sugar-phosphate backbone and bases of the dsRNA. The majority of these contacts are made between the conserved IV, IVA, V, and VA of D2 and of the dsRNA.1,2 Accordingly, studies have shown that the binding free energy of strand 1 with Mss116p is much lower than that of strand 2.5 In this crystal structure,  two guanine nucleotides on strand 1 of the dsRNA serve as a basis for . The guanidino group of Arg-415 of motif IVA forms ionic bonds with the two oxygen atoms of the 5' phosphate group of one guanine while the nitrogen of the protein backbone of Gly-436 of motif VA forms a hydrogen bond with the 5' oxygen atom of the other guanine. In addition to these interactions, D2 forms three other with the sugar-phosphate backbone of strand 1. Thr-433 of motif V and Gly-408 of motif IVA both form hydrogen bonds with the phosphate group of while the nitrogen of the protein backbone of Val-383 of motif IV forms a hydrogen bond with the oxygen of 's phosphate group. These residues and the hydrophobic interactions between D2 and strand 1 are responsible for forming the bonds that establish the basis of Mss116p's to dsRNA.2

IV. Interactions Between Strands 1 and 2 and the Carboxy-Terminal Extension (CTE)

The CTE of D2 plays an important role in initial dsRNA binding and helps to stabilize the helicase core of Mss116p.  Additionally, the CTE is responsible for forming a kink in the dsRNA through the positioning of .3 This kink is essential for efficient helicase activity. Like the interactions formed by the main body of D2, all interactions between the dsRNA and the CTE are non sequence-specific.  However, unlike the rest of D2, the CTE mainly interacts with of the dsRNA through hydrogen bonds with the 2'-OH groups of RNA nucleotides. In this crystal structure, Both Leu-580 and Arg-538 form hydrogen bonds with the 2'-OH of . Leu-580 forms this hydrogen bond via the oxygen atom of its protein backbone while Arg-538 forms two hydrogen bonds through its guanidino group. An additional interaction between strand 2 and the CTE is made through a hydrogen bond between the oxygen atom of the protein backbone of Ser-539 and the 2'-OH of . The CTE of Mss116p makes only one contact with strand one of the dsRNA. This interaction occurs through a hydrogen bond between the side-chain oxygen atom of Ser-535 and the 2'-OH group of . In total, D2 makes eleven with the dsRNA: six through the main body of D2 and five through the CTE.2

V. Interactions with dsRNA in the Closed Complex

Helicase activity of Mss116p is preceded by the independent binding of ATP and dsRNA to D1 and D2 respectively.  The binding of these two substrates results in increased interactions between the two domains and drives the formation of the closed-state complex and the development of a composite ATPase site between D1 and motif of D2.2  The formation of the closed-state complex must occur before Mss116p can hydrolyze ATP, a process required for strand separation followed by Mss116p's dissociation from the dsRNA after the release of ADP and Pi.4 Interestingly, while all interactions in the open complex between the conserved D2 motifs are maintained with the exception of that of of motif VA, all interactions between the CTE and the dsRNA are broken upon the formation of the . In one proposed model, dsRNA unwinding occurs as a result of a considerable conformational change in D1 during closed complex formation and subsequent ATP hydrolysis.2 As D1 undergoes this conformational change, it severs the hydrogen bonds between the two complementary RNA strands and strand 1 remains tightly bound to D2 as strand 2 is by D1.  It is also thought that D1 introduces another kink in strand 2 of the dsRNA in addition to that caused through the positioning of alpha helix 19 of the CTE. This additional kink impedes strand 2's ability to reanneal to strand 1 following strand separation.1,4 After the two complementary strands of the dsRNA have been separated, Ms116p releases its bound ADP and Pi and dissociates from strand 2, later rebinding another ATP molecule and dsRNA in order to catalyze further strand separation.

VI. References

1Del Campo, M.; Lambowitz, A. M. 2009. Structute of the yeast DEAD box protein MSS116p reveals two wedges that crimp RNA. Molecular Cell 35, 598-609.

2Mallam, A.L.; Del Campo, M.; Gilman, B.; Sidote, D. J.; Lambowitz, A. M. 2012. Structural basis for RNA-duplex recognition and unwinding by the DEAD-box helicase MSS116p. Nature 490: 121-124.

3Mohr, G.; Del Campo, M; Turner, K.G.; Gilman, B.; Wolf, R.Z.; Lambowitz, A. M. 2011. High-Throughput Genetic Identification of Functionally Important Regions of the Yeast DEAD-Box Protein Mss116p. Journal of Molecular Biology 413: 952-972.

4Sachsenmaier, N.; Waldsich, C. 2013. Mss116p a DEAD-box Protein Facilitates RNA Folding. Landes Bioscience 10, 71-82.

5Xue, Q.; Zhang, J.; Zheng, Q.; Cui, Y.; Chen, L.; Chu, W.; Zhang, H. 2013. Exploring the Molecular Basis of dsRNA Recognition by Mss116p Using Molecular Dynamics Simulations and Free-Energy Calculations. Langmuir 29, 11135-11144.

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