Domain 2 of the Saccharomyces cerevisiae
DEAD-Box Helicase Mss116p
Cristina Nunes '15 and Robert Turlington '16
Contents:
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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