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The Cbf5–Nop10 complex is a molecular bracket that
organizes box H/ACA RNPs
Tomoko Hamma1, Steve L Reichow2, Gabriele Varani2,3 & Adrian R Ferré-D’Amaré1
Box H/ACA ribonucleoprotein particles (RNPs) catalyze RNA pseudouridylation and direct processing of ribosomal RNA,
and are essential architectural components of vertebrate telomerases. H/ACA RNPs comprise four proteins and a multihelical
RNA. Two proteins, Cbf5 and Nop10, suffice for basal enzymatic activity in an archaeal in vitro system. We now report their
cocrystal structure at 1.95-Å resolution. We find that archaeal Cbf5 can assemble with yeast Nop10 and with human telomerase
RNA, consistent with the high sequence identity of the RNP components between archaea and eukarya. Thus, the Cbf5–Nop10
architecture is phylogenetically conserved. The structure shows how Nop10 buttresses the active site of Cbf5, and it reveals
two basic troughs that bidirectionally extend the active site cleft. Mutagenesis results implicate an adjacent basic patch in RNA
binding. This tripartite RNA-binding surface may function as a molecular bracket that organizes the multihelical H/ACA and
telomerase RNAs.
Box H/ACA and box C/D small nucleolar RNPs (snoRNPs) are responsible for the two most prevalent and evolutionarily conserved posttranscriptional modifications of cellular RNAs, pseudouridylation and
ribose 2′-O-methylation, respectively. RNPs of these classes are comprised of one of many guide snoRNAs, responsible for specifying the
site of modification by base pairing to substrate RNAs, and distinct
sets of proteins highly conserved across archaea and eukarya. Over 100
different snoRNAs, varying in length from 60 to 150 nucleotides, have
been documented in human. Despite the variability of the guide RNAs,
all H/ACA snoRNAs associate with the same four proteins, and all C/D
snoRNAs assemble with a different but conserved set of four proteins
to form stable snoRNPs (reviewed in ref. 1).
H/ACA snoRNAs consist minimally of a helix (P1) and a hairpin
(P2) flanking a bulge, with a single-stranded region including the conserved ACA sequence three nucleotides from the helix on the 3′ side
(Fig. 1a). Eukaryotic H/ACA RNAs usually contain two such structures
connected by a single-stranded segment with the related and conserved
hinge (ANANNA) sequence2,3. Archaeal H/ACA RNAs comprise one
to three helix-bulge-hairpin elements4,5. The pseudouridylation site is
precisely specified by two short helices formed by base pairing between
the internal bulge of the guide H/ACA RNAs and sequences flanking the
target nucleotide in the substrate RNAs.
The four-helix junction formed by association of the H/ACA and
substrate RNAs binds Cbf5 (NAP57 in rat and dyskerin in human), Gar1,
L7Ae (Nhp2 in eukarya) and Nop10. All four proteins are essential for
viability in yeast, and Cbf5, Nhp2 and Nop10 are required for in vivo stability of H/ACA RNAs6–10. L7Ae, the archaeal homolog of human Nhp2,
is an RNA-binding protein that recognizes a structural motif called the
K-turn4,11, which is present in numerous RNAs, including archaeal
H/ACA RNAs4,12. Gar1 and Nop10 are small, basic proteins of unknown
structure and function10,13 that directly interact with Cbf5 in vitro7,14–16
and in vivo17. Cbf5 and its homolog dyskerin are predicted to be pseudouridine (Ψ) synthases on the basis of their sequence similarity to
these enzymes6,18–20. Consistent with this prediction, point mutations21
or genetic depletion7 of Cbf5 results in reduced pseudouridylation of
rRNA in yeast. There have been no reports of the reconstitution of box
H/ACA RNPs from purified, recombinant eukaryotic proteins and RNA.
However, archaeal H/ACA RNPs have been reconstituted using recombinant proteins and shown to have pseudouridylation activity in vitro15,16.
These studies have also demonstrated that an archaeal Cbf5–Nop10–
RNA complex has basal pseudouridylation activity16, which is enhanced
by the addition of Gar1 or L7Ae15,16.
Although deletion of Cbf5 is lethal in yeast, overexpression of a
catalytically inert point mutant of the protein in the null yeast results
in heat- and cold-sensitive cells that lack Ψ in their rRNA21. This
implies that H/ACA RNPs have functions distinct from their pseudouridylation activity. To date, two additional biological functions of
H/ACA snoRNPs have been identified. First, U17 H/ACA snoRNA
(snR30 in Saccharomyces cerevisiae) specifies a cleavage site in the 35S
precursor to 18S rRNA rather than serving as a pseudouridylation
guide22,23. In yeast, snR30 is the only H/ACA RNA that is essential
for viability24, underscoring the importance of this RNP-directed
rRNA processing event. Second, vertebrate telomerase RNAs contain an H/ACA RNA–like structure near their 3′ end25 (Fig. 1b) that
does not seem to direct Ψ formation in any cellular RNA (reviewed
in ref. 1). Association of this domain with the four H/ACA snoRNP
1Division
of Basic Sciences, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North, Seattle, Washington 98109-1024, USA. 2Department of
Chemistry, University of Washington, Box 351700, Seattle, Washington 98195-1700, USA. 3Department of Biochemistry, University of Washington, Box 357350,
Seattle, Washington 98195-7350, USA. Correspondence should be addressed to A.R.F. ([email protected]) or G.V. ([email protected]).
Received 15 September; accepted 8 November; published online 15 November 2005; doi:10.1038/nsmb1036
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Figure 1 Sequences and secondary structures of a box H/ACA snoRNA
bound to its substrate RNA and of the box H/ACA snoRNA–like region of
human telomerase RNA. (a) Sequence and predicted secondary structure
of the pseudouridylation guide RNA afu-190 (from Archaeoglobus fulgidus)
bound to part of its substrate, 16S rRNA4,5. Archaeal L7Ae (green)
recognizes the K-turn structure at the tip of the hairpin4,12. Binding of
a box H/ACA RNA to its substrate RNA results in a structure resembling
a four-helix junction, with the pseudouridylation site (Ψ) at the center.
(b) Sequence and predicted secondary structure of the 3′ domain of
human telomerase RNA (residues 371–451) used in this study25,55.
Three mutations found in dyskeratosis congenita patients29 are shown in
red (∆ denotes deletion of all nucleotides on the 3´ side of the arrow).
Both RNAs start with two non-native G residues, added to facilitate
in vitro transcription.
proteins is nonetheless essential for in vivo assembly and stability of
the telomerase RNP25,26.
Mutations in dyskerin27 and in the H/ACA domain of human telomerase RNA28 are linked to the bone marrow–failure syndrome dyskeratosis congenita. Cells from dyskeratosis congenita patients have shorter
telomeres, reduced telomerase activity and decreased levels of telomerase
RNA, suggesting that dyskeratosis congenita is caused by telomerase dysfunction (reviewed in ref. 29). A mouse knock-in model of dyskeratosis
congenita has been constructed using a hypomorphic allele of dyskerin.
Its cells show reduced pseudouridylation of rRNA and impaired precursor-rRNA processing and translation, but no detectable telomerase
defect30. It thus seems that both the enzymatic and nonenzymatic functions of H/ACA RNPs may be involved in the development of dyskeratosis congenita.
To shed light on the architectural functions of Cbf5 and its human
homolog dyskerin, and as a starting point for understanding the function
of the essential and highly conserved H/ACA RNP core protein Nop10,
we have now determined the structure of an archaeal Cbf5–Nop10 complex at 1.95-Å resolution. Together with NMR analyses of free Nop10,
structure-guided mutagenesis and in vitro reconstitution experiments,
our structure indicates that this highly conserved heterodimeric protein complex organizes the multihelical structure of all H/ACA RNAs,
including telomerase RNA.
RESULTS
Structural and functional conservation of Nop10
Sequence analysis of Nop10 reveals a striking difference between eukaryotic and archaeal proteins in their N-terminal regions, whereas the
C-terminal half shows substantial sequence conservation across these
groups. Archaeal Nop10 proteins contain a predicted zinc-binding
CX2CX8CX2C consensus sequence (where X is any amino acid residue) that is absent in eukaryotes (Supplementary Fig. 1 online). NMR
was used to probe the tertiary structure of archaeal (Methanococcus
janaschii) and yeast (S. cerevisiae) Nop10 and to compare their secondary structures (Methods). The N-terminal region (residues 6–30) of
archaeal Nop10 (aNop10) adopts a very well-defined zinc-ribbon31
domain structure (Supplementary Fig. 2 online). Chemical shifts,
NOE interactions and the values of heteronuclear NOEs indicate the
presence of an α-helix in the C-terminal part of the protein (residues
45–49) that is connected to the N-terminal zinc ribbon by a disordered
linker. We could not detect any NOE interaction between the zincribbon domain and the C-terminal helix of aNop10. Isolated yeast
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Nop10 (yNop10) was found to form an N-terminal (residues 5–15)
β-hairpin that is structurally similar to the extreme N-terminus of
aNop10, as well as an incipient C-terminal helical region corresponding to the homologous region of aNop10 (Supplementary Fig. 1). These
structural features are found in the crystal structure of the archaeal
Cbf5–Nop10 complex (described below). Residues from these structured regions are involved in numerous intermolecular contacts, suggesting that binding to Cbf5 stabilizes the α-helical conformation of the
C-terminal segment of Nop10 and organizes the linker connecting the
N- and C-terminal secondary structures.
NMR analysis shows that virtually every amide chemical shift of
aNop10 is strongly perturbed upon complex formation with Cbf5 from
M. jannaschii (aCbf5) (Fig. 2a). Remarkably, nearly all amide resonances
of yNop10 are also strongly perturbed to generate a spectrum of very
high-quality and much-increased spectral dispersion upon mixing with
aCbf5 (Fig. 2b). These results demonstrate that yNop10 has a strong specific interaction with aCbf5. Because of the similarities in the secondary
structure of free yeast and archaeal Nop10, and the observed interactions of these structural elements of aNop10 when bound to aCbf5, we
speculate that eukaryotic Nop10 proteins bind Cbf5 in a manner similar
to what we observed for the archaeal complex.
Consistent with the high degree of conservation of box H/ACA
snoRNP protein-protein interactions implied by our NMR studies, we
find that recombinant aCbf5, aGar1, L7Ae and aNop10 can assemble
onto a human telomerase H/ACA-domain RNA (hTR; Fig. 1b) in vitro
(Fig. 2c, lanes 6–10). The proteins thereby reconstitute a particle that
seems similar to the complex they form with an archaeal H/ACA RNA
(Supplementary Fig. 3 online and refs. 15,16). This extends the previously reported association of the archaeal proteins with a eukaryal guide
RNA15 to a eukaryal H/ACA RNA domain with an architectural function. Moreover, we find that substitution of aNop10 with yNop10 results
in formation of RNPs with electrophoretic behaviors similar to those of
RNPs formed by the four archaeal proteins binding hTR (Fig. 2c, lanes
11–15) and by the archaeal proteins binding an archaeal H/ACA RNA
(Supplementary Fig. 3).
Structure of the Cbf5–Nop10 complex
aCbf5 was biochemically unstable and only modestly soluble on its own,
but it formed a soluble complex with aNop10. The crystal structure of
the complex was determined through multiwavelength diffraction using
selenomethionyl (SeMet) aNop10, and it has been refined to an Rfree factor of 22.9% (Methods). The main domain of aCbf5 is comprised of a
six-stranded β-sheet, decorated on one face by several loops and α-helices
that flank the active site cleft (Fig. 3a,b). A pseudouridine synthase and
archaeosine transglycosylase (PUA) domain19 and several additional
helices protrude from one of the corners of the main domain, extending
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Figure 2 Functional conservation of Nop10
and the Cbf5–Nop10 complex between archaea
and eukarya. (a) 15N HSQC spectral overlay of
15N-labeled aNop10 free (blue) and bound to
unlabeled aCbf5 (pink). Peak broadening in
the complex spectrum is at least partly due to
aggregation of the sample during concentration.
(b) 15N HSQC spectral overlay of 15N-labeled
yNop10 free (green) and bound to unlabeled
aCbf5 (pink). (c) Assembly of archaeal H/ACA
RNP proteins and yeast Nop10 with the
32P-labeled H/ACA–like domain of human
telomerase RNA (hTR; Fig. 1b) analyzed by
nondenaturing gel electrophoresis. Cartoons
depict the composition of the complexes. Note
that aGar1, L7Ae, yNop10 or aNop10 alone
do not directly bind hTR but enter the RNP in
complex with aCbf5. Under these low–ionic
strength conditions, aGar1 binding to the
complex also produces aggregates with reduced
mobility (asterisk) and some that fail to enter the
gel (Well). This may also account for the smeary
nature of the free-RNA bands in lanes 9, 10, 14
and 15.
its comparatively flat face. The N-terminal zinc ribbon and C-terminal
α-helix detected in free aNop10 by NMR are present in the complex,
separated by the extended linker. However, both the C-terminal helix
and the linker sequence are well ordered in the complex, unlike what
is observed for the free protein. The linker spans ~20 Å. aNop10 binds
the main domain of aCbf5 on the side opposite the PUA domain. The
N- and C-terminal domains of aNop10 extend away from the back of
aCbf5, producing a ~20-Å-wide trough that opens out from the active
site cleft. The PUA domain flanks a similarly sized trough on the opposite
end of the active site. As expected from the perturbation of most aNop10
resonances seen upon complex formation with aCbf5 (Fig. 2a), the
aCbf5-aNop10 molecular interface (Fig. 3c) is extensive (over 2,300 Å2
of solvent-accessible surface area), burying ~46% of the total surface of
aNop10. A DALI32 search of the current structural database found no
other proteins that are structurally similar to full-length aNop10.
As implied33 by the ~30% sequence identity between aCbf5 and the
Escherichia coli Ψ synthase TruB20, the two proteins adopt very similar
overall structures (Fig. 4a,b). However, TruB (which introduces Ψ55
into all elongator transfer RNAs) functions without guide RNAs or
accessory proteins. Three substantial structural differences between
aCbf5 and TruB reflect the participation of aCbf5 (but not TruB) in
an elaborate RNP. First, TruB has two characteristic polypeptide segments that are directly involved in TΨC-loop recognition20. These are
absent from aCbf5 and its orthologs. In place of one of these segments,
aCbf5 has a shorter loop that is disordered in our structure (Figs. 3 and
4). One of the TΨC loop–recognizing segments of TruB is also disordered34,35 or in a completely different conformation36 in the absence
of substrate RNA. Second, the PUA domain (which binds the acceptor stem of tRNA in TruB37) is considerably larger in aCbf5 and its
orthologs (Fig. 4a,b). aCbf5 extends the PUA
domain further by enveloping it with N- and
C-terminal extensions of its main domain
(Fig. 3a,b). Sequences of these extensions are
phylogenetically conserved (Supplementary
Figs. 4 and 5 online). A large number of mutations associated with dyskeratosis congenita in
humans map to the PUA domain of dyskerin
and to the enveloping N-terminal extension of the main domain. Third,
the C-terminal helix of aNop10 packs against helix α5 of aCbf5, which
adopts a different orientation from that of the equivalent helix of TruB
(which does not participate in protein-protein interactions; Fig. 4a).
The active sites of aCbf5 and TruB superimpose closely. Three residues
that are present in all Ψ synthase active sites37,38 (an aspartate, a basic
residue and a tyrosine or phenylalanine; Asp81, Arg180 and Tyr109,
respectively, in aCbf5) occupy conserved positions (Fig. 4c). Arg180, a
basic residue that corresponds to one making a buried salt bridge with
the catalytic aspartate in the TruB–RNA complex, adopts a different
orientation in our RNA-free aCbf5–aNop10 structure. However, it is
very likely that the catalytic mechanism of the box H/ACA snoRNP is
identical to those of stand-alone Ψ synthases.
The interface between aCbf5 and aNop10 contains many highly conserved residues, especially in the linker segment (Supplementary Figs. 1
and 4), along with a dense network of hydrogen bonds, van der Waals
interactions and salt bridges (Fig. 3c). An absolutely conserved proline
of Nop10 (Pro33) interacts with two equally conserved prolines of aCbf5
(Pro53 and Pro56) through van der Waals interactions (Fig. 3c,d). The
latter prolines reside in a sequence element conserved throughout Ψ
synthases (Motif I)18 that is important for protein stability39. Mutation
of one of the equivalent prolines in TruB results in severe destabilization of the enzyme. The interaction with aNop10 positions the absolutely conserved Lys52 of Motif I of aCbf5 for hydrogen bonding to the
carbonyl oxygen of the catalytic Asp81 (Fig. 3d). Thus, it seems that
Motif I of aCbf5 bridges Nop10 and the enzyme’s active site, providing
a rationale for the lack of catalytic activity of aCbf5 in the absence of
aNop10 (refs. 15,16). Consistent with the importance of this interaction,
mutation of a Cbf5 Motif I proline results in growth defects in yeast21.
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Figure 3 Structure of the Cbf5–Nop10 complex. (a) Overall view. In Cbf5,
asterisks mark the active site cleft; dots represent a disordered polypeptide
loop; the structurally important Motif I18 is green, Motif II and other active
site residues are yellow and the PUA domain19 is gray. In Nop10, the zinc
ribbon, linker and C-terminal helix segments are red, pink and purple,
respectively (aNop10 color-coding is as in Supplementary Fig. 1; aCbf5 colorcoding is as in Supplementary Figs. 4 and 5). (b) View rotated 90°. (c) The
aCbf5–aNop10 interface. (d) aNop10 stabilizes the active site of aCbf5 by
buttressing residues in Motif I (green) of the enzyme. Water molecules are
shown as red spheres, hydrogen bonds as arrows. The catalytic aspartate
(Asp81) of aCbf5 is in the lower left corner.
Mutation of two conserved basic residues from the central positive
patch of aCbf5 (R94Q, K97Q) strongly diminishes binding to the guide
RNA (Fig. 5e). Although solvent-exposed aromatic side chains are often
involved in RNA binding, mutation of Trp42 of aNop10 and of the adjacent Tyr45 of aNop10 only moderately decreased RNA binding by the
aCbf5–aNop10 complex (Fig. 5e). The conserved Trp42 residue projects
into the trough from near the C-terminal helix of aNop10 (Figs. 3 and
5). We hypothesize that these residues are more important for binding
the duplexes formed between the guide and substrate RNAs than for
binding guide RNA alone.
Deletion of the loop formed by aCbf5 residues 139–149 did not markedly decrease RNA binding (Fig. 5e). However, this loop may still have
a role in the function of the full RNP, given its location, which is analogous to that of a substrate RNA–binding element of TruB. Although
RNA-binding surfaces of Cbf5–Nop10
The surface electrostatic potential of the aCbf5–aNop10 complex
(Fig. 5a,b) is characterized by a prominently positively charged girdle
extending to either side of the active site cleft and into troughs formed
by aNop10 on one side and the PUA domain on the other. In addition, a strongly positive surface patch is present near the interface of
the main and the PUA domains of aCbf5. These positively charged surfaces coincide with regions of high sequence conservation (Fig. 5c,d)
and seem ideally placed to bind several of the helices that comprise
the guide RNAs and their complexes with substrate RNAs. The basic troughs have dimensions appropriate for accommodating RNA
double helices (Fig. 5c and Supplementary
Fig. 6 online), and the patch is in a position
equivalent to the acceptor stem–binding surface of TruB (Fig. 4a). Furthermore, the most
common disease-associated human dyskerin
mutation29, A353V (Supplementary Figs. 4
and 5), maps to the trough surface of the PUA
domain. Although there is no evidence that
the side chain of Ala353 directly interacts with
RNA, the prevalence of this mutation among
dyskeratosis congenita patients underscores the
functional importance of the trough. As Cbf5
is essential in yeast, mutations that completely Figure 4 Structural comparison of the Cbf5–Nop10 complex and TruB. (a) Superposition of the structure
20
disrupt dyskerin function might be expected to of TruB (white and magenta) bound to a TΨC stem-loop of tRNA (black) (PDB entry 1K8W) on the
aCbf5–aNop10 complex (colored as in Figure 3). The r.m.s. deviation between aCbf5 and TruB (219 Cα
be embryonic lethal in human and would not
pairs) is 1.95 Å. The PUA domain and characteristic TΨC loop–binding segments of TruB are shown in
be isolated from patients.
magenta. The Cbf5 PUA domain is structurally very similar to the PUA domains of the RNA-modifying
We tested the involvement of the central enzyme arcTGT41 and of the nuclear protein Nip7 (ref. 56), involved in 60S-ribosome biogenesis in yeast
positive patch and the Nop10 trough in guide- (Supplementary Fig. 5). (b) View rotated 90°. (c) Superposition of conserved active site residues of aCbf5
RNA binding by site-directed mutagenesis. and TruB. The isomerized nucleotide present in the TruB–RNA complex20 is also shown.
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Figure 5 Conserved surfaces of the Cbf5–Nop10 complex interact with
H/ACA guide RNA. (a) Electrostatic potential, mapped onto the solventaccessible surface of the complex. Two prominently basic features are the
girdle extending to either side of the active site (asterisk) and a patch at the
interface of the main and PUA domains of aCbf5. (b) View rotated 90°. Two
acidic patches represent potential binding sites for the strongly basic H/ACA
RNP protein Gar1 (refs. 7,14). Gar1 does not directly interact with RNA but
binds dyskerin–Nop10 and thus assembles into the RNP15. (c) Sequence
conservation of Cbf5 and Nop10 from archaea to eukarya (Supplementary
Figs. 1 and 4) mapped onto the molecular surface of aCbf5 and a worm
representation of aNop10 (used because a molecular surface or a solventaccessible surface representation of aNop10 would occlude the proteinprotein interface). Dark green, most highly conserved residues; white,
nonconserved residues. The aNop10 trough has dimensions appropriate
for accommodating A-form RNA (pink) (see Supplementary Fig. 6). (d) View
rotated 90°. (e) Results of electrophoretic mobility shift assays comparing
binding of wild-type and mutant aCbf5–aNop10 complexes (S) to an
archaeal box H/ACA RNA (F). Locations of mutations are indicated in c.
Numbers above the lanes denote protein complex concentrations (nM).
disordered in our structure, these residues are near the active site and
occupy a position equivalent to that of one of the TΨC loop–binding
polypeptides of TruB (Fig. 4a,b). As six of the deleted residues are basic,
the strong inhibition of guide-RNA binding resulting from mutations
of two basic residues in the central positive patch (Arg94 and Lys97 of
Cbf5) is not a non–sequence-specific electrostatic effect.
DISCUSSION
We present the first high-resolution view of two central components of
the box H/ACA RNP: Cbf5 and Nop10. NMR analyses and biochemical reconstitution suggest that the interface between the two proteins
is functionally conserved between archaea and eukarya, despite the
structural divergence between the N-terminal portions of archaeal and
eukaryal Nop10. The structure of the aCbf5–aNop10 complex suggests
two architectural roles for the association between the two proteins:
buttressing of the active site of Cbf5 by Nop10 binding and formation
of a basic trough that extends the active site cleft and binds one of the
helices that comprise the H/ACA guide RNA–substrate RNA complex.
Together with the structural conservation of H/ACA RNAs (Fig. 1) and
the ability of the archaeal proteins to associate with eukaryotic H/ACA
RNAs (Fig. 2c and ref. 15), our structural results imply that the molecular interface between the H/ACA guide RNAs and the Cbf5–Nop10
complex is highly similar in the eukaryal and archaeal RNPs.
Structural comparison of Ψ synthases belonging to the five different
known enzyme families has previously shown that a variety of unrelated
peripheral structural elements protrude from the conserved core in proteins from different families, and these are employed for recognition of
substrate RNAs37,38. Our structure shows that in addition to enveloping
its peripheral PUA domain with extensions of its polypeptide chain,
Cbf5 extends its structure by associating with Nop10. The importance
of this association is underscored by conservation of both sequence
(Supplementary Figs. 1 and 4) and function (Fig. 2 and Supplementary
Fig. 3) as well as by our experimental observation that complex formation is required for biochemical stability of aCbf5.
The conservation of Nop10 from archaea to human could simply
reflect an evolutionarily early choice. As Cbf5 and Nop10 associate with
numerous H/ACA RNAs encoded by unrelated genetic loci, the cost of
losing Nop10 and evolving a new peripheral domain in Cbf5 or dyskerin
may be too high. Alternatively, the specific biophysical properties of
Nop10 may be functionally important. The likely degradation of the
disordered isolated Nop10 and the poor stability of apo Cbf5 may ensure
stoichiometric amounts of these two RNP components. Often, when
a small polypeptide has evolved to bind a larger protein, the former is
intrinsically unstructured. By coupling folding of the small polypeptide
to complex formation, it is possible to achieve very large interfacial areas
and therefore specificity40. Our crystal structure shows how the central
region of Nop10, which becomes ordered upon complex formation,
buttresses the structurally important Motif I of Cbf5 (Fig. 3). Binding to
Motif I allows Nop10 to monitor the enzymatic dynamics of the active
site of Cbf5. Folding of Nop10 concomitant with its association with
Cbf5 may allow coupling of RNA binding by the Nop10 basic trough
with progress of the pseudouridylation reaction.
Our structure-guided mutagenesis implicates the conserved basic
patch at the interface of the main and PUA domains of Cbf5 in binding
H/ACA guide RNA. In vitro analysis of archaeal apo Cbf5 binding to
mutant H/ACA guide RNAs has demonstrated that alterations of the single-stranded ACA segment, helix P1 (Fig. 1a) or the pseudouridylation
pocket all adversely affect binding, whereas alterations of helix P2 or its
terminal loop have little effect15. In the snoRNP, the pseudouridylation
site must be directly apposed to the catalytic aspartate of Cbf5 or dyskerin. This constraint, and the results of protein and RNA mutagenesis,
lead us to speculate that it is helix P1 of the H/ACA guide RNA that
binds the basic patch of Cbf5, placing the ACA sequence close to the
PUA domain. Notably, the PUA domain of the unrelated RNA-modifying enzyme archaeosine tRNA-guanine transglycosylase (arcTGT) has
been shown to recognize the single-stranded acceptor (CCA) sequence
of a tRNA41.
The dyskerin–Nop10 complex organizes two types of RNPs with
functions essential for cell growth. Box H/ACA RNPs are required for
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formed using combined automated and manual methods in CYANA44. Torsionangle restraints were included for (φ,ψ) angles according to TALOS45. Hydrogen
bonding constraints were derived from amide D2O protection data. Additional
structural restraints for aNop10 enforced tetrahedral geometry between the four
conserved zinc-ligating Sγ atoms. Structure calculations for both Nop10 proteins
were completed with CYANA v2.0. Structural statistics from 20 of 100 lowestenergy structures are provided in Supplementary Table 1 (online). PROCHECK
analysis46 found 99.6% and 99.8% of residues for aNop10 and yNop10, respectively, in allowed regions of the Ramachandran plot. Figures were prepared using
GRASP47, MolMol48 and Ribbons49.
Table 1 Crystallographic statistics
aCbf5–aNop10
Data collectiona
Space group
C2
© 2005 Nature Publishing Group http://www.nature.com/nsmb
Cell dimensions
a, b, c (Å)
118.29, 63.19, 67.90
α, β, γ (°)
90, 102.62, 90
Wavelength (Å)
0.9798
0.9570
Resolution (Å)b
50.0–1.95 (2.02–1.95)
50.0–1.95 (2.02–1.95)
Rsym or Rmerge
(%)b
6.2 (41.7)
7.6 (47.4)
I / σI b
18.4 (1.3)
26.5 (2.0)
Completeness (%)b
89.6 (52.6)
94.6 (70.2)
Redundancyb
2.9 (2.1)
7.8 (5.4)
Refinement
Resolution (Å)
50.0–1.95
No. reflections
66,120
Rwork / Rfree
20.0/22.9
No. atoms
Protein
2,794
Ligand/ion
3
Water
175
B-factors
Protein
47.25
Ligand/ion
40.21
Water
52.38
R.m.s deviations
Bond lengths (Å)
0.0065
Bond angles (°)
1.32
aOne
crystal was used for all data measurements. bValues in parentheses are for the highestresolution shell.
ribosome biogenesis, and the H/ACA domain of vertebrate telomerase
is required for assembly and stability of this chromosomal end–maintenance complex. Thus, our structural analysis of the aCbf5–aNop10
complex opens the way to the development of novel structure-guided
interventions in the molecular machinery of cell proliferation.
METHODS
Protein and RNA preparation. Protein expression, purification and complex
reconstitution are described in Supplementary Methods (online). Archaeoglobus
fulgidus box H/ACA guide RNA (afu-190)4 (Fig. 1a) and the box H/ACA domain
of hTR25 (residues 371–451; Fig. 1b) were prepared by in vitro transcription and
32P-labeled at the 5′ end.
NMR spectroscopy. NMR experiments were recorded at 298 K on Bruker
DRX 500 and DMX 750-MHz spectrometers, on a Bruker 500-MHz spectrometer equipped with cryo-probe at the National Magnetic Resonance Facility
at Madison and on a Varian 800-MHz spectrometer at the Pacific Northwest
National Laboratory. Standard triple-resonance experiments (HNCO, HNCA,
HN(CO)CACB, HN(CO)CA, HBHA(CO)HN and HCCH-TOCSY)42 were used
to obtain nearly complete assignments for both polypeptides. Structural restraints
were obtained through heteronuclear 13C- and 15N-edited 3D experiments
and homonuclear NOESY experiments collected with mixing times of 120 ms
(aNop10) and 100 ms (yNop10). 15N HSQC and 15N transverse relaxation-optimized spectroscopy HSQC data were collected at 315 K on complexes of aCbf5
with 2H,15N-labeled aNop10 or yNop10 on the 750-MHz spectrometer. Spectra
were processed using NMRPipe43 and analyzed with Sparky (http://www.cgl.
ucsf.edu/home/sparky). NOE assignments and structure calculations were per-
1106
Biochemical analysis. For analysis of binding of wild-type or mutant aCbf5–
aNop10 to afu-190 as well as for in vitro reconstitution of the box H/ACA RNP
using archaeal proteins and hTR, RNAs (~5 nM) were mixed with the proteins
in a buffer containing 500 mM KCl, 20 mM HEPES-KOH (pH 7.5), 0.5 mM Tris(2-carboxyethyl)phosphine, 5% (v/v) glycerol, 1 mM MgCl2 and 0.14 g l–1 E. coli
tRNA. The complexes were incubated at 338 K for 10 min and then at 295 K for
50 min, resolved on 8% nondenaturing (0.5× TBE) polyacrylamide gels run at
311 K and visualized by phosphorimaging.
Crystallization and structure determination. The complexes between aCbf5–
aNop10 (native) and aCbf5-SeMet–aNop10 (derivative) were concentrated to
~3.6 mM in a solution containing 20 mM HEPES-KOH (pH 7.5), 0.5 mM Tris(2-carboxyethyl)phosphine, 5% (v/v) glycerol and 1 M KCl, and then supplemented with 0.8 mM octyl-β-D-glucopyranoside. Native crystals were grown at
295 K by vapor diffusion. Sitting drops were prepared by mixing equal volumes of
the protein complex and a solution containing 20% (v/v) 2-propanol, 20% (w/v)
PEG4000 and 100 mM sodium citrate (pH 5.8). The drops were equilibrated
over a reservoir containing 15% (v/v) 2-propanol, 15% (w/v) PEG4000, 75 mM
sodium citrate (pH 5.8) and 1 M KCl. Crystals grew within 7 d to typical dimensions of 200 × 100 × 100 µm3. Native crystals were crushed in their mother liquor
and used to seed equilibrated sitting drops of the derivative protein complex
prepared in the same way as the native sitting drops. Derivative crystals grew at
295 K in 3 d to typical dimensions of 100 × 80 × 40 µm3. Derivative crystals were
stabilized in a solution containing 1 M LiCl, 25% (w/v) PEG3350 and 75 mM
sodium citrate (pH 5.8) and flash-cooled in liquid nitrogen. Diffraction data
(Table 1) were collected from a single derivative crystal at two X-ray wavelengths
at 100 K at beamline 5.0.2 of the Advanced Light Source (Lawrence Berkeley
National Laboratory) using the inverse-beam method and reduced with the HKL
package50. The crystals contained one complex per asymmetric unit. Three selenium sites were located and initial phases calculated with SOLVE51. An anomalous-difference Fourier synthesis using these phases revealed the zinc site. Density
modification of phases calculated using the three selenium sites and one zinc site
resulted in an electron density map (Supplementary Fig. 7 online) into which
most of the protein residues and the bulk of water molecules could be built unambiguously. Before density modification, the mean overall (50- to 1.95-Å resolution) figure of merit was 0.29. The mean figure of merit was 0.57 for reflections
between 50- and 3.08-Å resolution and 0.15 for reflections between 3.08- and
1.95-Å resolution. After density modification, the mean overall (50- to 1.95-Å
resolution) figure of merit was 0.97. Rounds of energy minimization, simulated
annealing and restrained individual B-factor refinement against all diffraction
data to 1.95-Å resolution and experimental phase-probability distributions using
a maximum likelihood target52, interspersed with manual rebuilding53, produced
the current model, which has excellent stereochemistry and a cross-validated σA
mean coordinate precision of 0.22 Å (Table 1). The mean real-space R-factors
for aCbf5, aNop10, water and ions are 0.039, 0.079, 0.094 and 0.028, respectively.
MolProbity54 analysis shows that 96.6% of the amino acid residues lie in the
most favored regions of the Ramachandran plot, with the remaining residues
in additional allowed regions. There are no amino acid residues with disallowed
backbone conformations.
Accession codes. Protein Data Bank: Coordinates have been deposited with the
following accession codes: 2APO (aCbf5–aNop10 complex), 2AQC (aNop10) and
2AQA (yNop10). Chemical shift data has been deposited with the BioMagnetic
Research Bank.
Note: Supplementary information is available on the Nature Structural & Molecular
Biology website.
VOLUME 12 NUMBER 12 DECEMBER 2005 NATURE STRUCTURAL & MOLECULAR BIOLOGY
ARTICLES
© 2005 Nature Publishing Group http://www.nature.com/nsmb
ACKNOWLEDGMENTS
We thank C. Hoang for experimental contributions to initial stages of this project,
A. Roll-Mecak for advice on protein coexpression, J. Bolduc for in-house X-ray
support, the staff of Advanced Light Source beamline 5.0.2. for synchrotron data
collection support, N. Isern at Pacific Northwest National Laboratories and the
staff at the National Magnetic Resonance Facility at Madison for support with
NMR data collection, N. Leuliott (IBBMC, Université Paris-Sud) for providing
yNop10 expression vector and T. Edwards, K. Godin, D. Klein, J. Pitt, B. Shen, B.
Stoddard and H. Xiao for discussions. This work was supported by the US National
Institutes of Health (grants to A.R.F. and G.V. and Viral Oncology training grant
to T.H.). T.H. is a Leukemia & Lymphoma Society Special Fellow. A.R.F. is a
Distinguished Young Scholar in Medical Research of the W.M. Keck Foundation.
COMPETING INTERESTS STATEMENT
The authors declare that they have no competing financial interests.
Published online at http://www.nature.com/nsmb/
Reprints and permissions information is available online at http://npg.nature.com/
reprintsandpermissions/
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