Structures of human SRP72 complexes provide insights into SRP RNA remodeling and ribosome interaction
Nucleic Acids Research
Structures of human SRP72 complexes provide insights into SRP RNA remodeling and ribosome interaction
Matthias M. M. Becker 0
Karine Lapouge 0
Bernd Segnitz 0
Klemens Wild 0
Irmgard Sinning 0
0 Heidelberg University Biochemistry Center (BZH) , Im Neuenheimer Feld 328, D-69120 Heidelberg , Germany
Co-translational protein targeting and membrane protein insertion is a fundamental process and depends on the signal recognition particle (SRP). In mammals, SRP is composed of the SRP RNA crucial for SRP assembly and function and six proteins. The two largest proteins SRP68 and SRP72 form a heterodimer and bind to a regulatory site of the SRP RNA. Despite their essential roles in the SRP pathway, structural information has been available only for the SRP68 RNA-binding domain (RBD). Here we present the crystal structures of the SRP68 proteinbinding domain (PBD) in complex with SRP72-PBD and of the SRP72-RBD bound to the SRP S domain (SRP RNA, SRP19 and SRP68) detailing all interactions of SRP72 within SRP. The SRP72-PBD is a tetratricopeptide repeat, which binds an extended linear motif of SRP68 with high affinity. The SRP72-RBD is a flexible peptide crawling along the 5e- and 5floops of SRP RNA. A conserved tryptophan inserts into the 5e-loop forming a novel type of RNA kinkturn stabilized by a potassium ion, which we define as K+-turn. In addition, SRP72-RBD remodels the 5f-loop involved in ribosome binding and visualizes SRP RNA plasticity. Docking of the S domain structure into cryo-electron microscopy density maps reveals multiple contact sites between SRP68/72 and the ribosome, and explains the role of SRP72 in the SRP pathway.
The universally conserved signal recognition particle (SRP)
mediates co-translational targeting of proteins to
membranes by recognizing hydrophobic N-terminal signal
sequences of its client proteins emerging from ribosomes
(1). In eukaryotes, SRP––ribosome nascent chain
complexes (RNCs) are targeted to the endoplasmic reticulum
by guanosine-5 -triphosphate- (GTP-) dependent
interaction with the membrane-bound SRP receptor (SR ) and
the RNC is transferred to a vacant translocation channel
(Sec 61 complex) (2,3). The heterodimeric targeting
complex formed by the SRP GTPases SRP54 and SR
constitutes the core of the SRP system and regulates the entire
process (4–6). After the RNC is handed over to the
translocation channel and translation resumes, the targeting
complex relocates on SRP RNA and the SRP GTPases are
stimulated by a regulatory ‘distal site’ of the RNA. This is so far
only shown for Escherichia coli (7,8) despite the
conservation of the targeting complex and its binding sites on the
SRP RNA. Upon GTP-hydrolysis, SRP dissociates from
the SR and the SRP targeting cycle is closed.
Human SRP is a ribonucleoprotein complex comprising
the highly base-paired SRP RNA (also referred to as 7SL
RNA) of 300 nucleotides and six proteins (SRP9, SRP14,
SRP19, SRP54, SRP68 and SRP72) (Figure 1) (9). It is
divided into the Alu domain (SRP9/14 and 5 /3 ends of SRP
RNA) responsible for elongation retardation (10) and the S
domain, which recognizes the signal sequence of the
ribosome nascent chain and binds to the SR. Both S domain
functions are performed by the highly conserved SRP54
protein while SRP19 is a scaffolding protein clamping
helices 6 and 8 of SRP RNA (11,12). SRP19 binding to the
apical tetra-nucleotide loops (tetraloops) of helices 6 and 8
prepares the flexible internal ‘asymmetric loop’ within helix
8 for SRP54 binding. The two largest -solenoidal proteins
SRP68 and SRP72 (60% of SRP protein mass) belong to the
S domain and form a stable heterodimer essential for SRP
function (13). Reconstituted SRP with alkylated SRP68/72
or without the heterodimer is not functional in
translocation assays (14). The SRP68/72 heterodimer is assembled
into the pre-SRP particle in the nucleus and its presence is
necessary for export to the cytoplasm. Knockout of the
heterodimer in yeast leads to an accumulation of pre-SRP in
the nucleus (15) and gene silencing of SRP68/72 with RNAi
in Trypanosoma brucei causes cell death due to the toxicity
of nuclear SRP-accumulation (16). SRP72 is cleaved
during apoptosis by caspases and a 6 kDa C-terminal
fragment that is phosphorylated on serine residues is released
(17). Proteolytic cleavage of this peptide is connected with
the development of autoantibodies against SRP72
triggering the formation of autoimmune diseases, like systemic
lupus erythematosus (17,18). Mutations in the SRP72 gene
are causative for familial aplastic anemia and an increased
risk of acute myeloid leukemia (19).
The subunits of the SRP68/72 heterodimer (Figure 1)
bind individually to SRP RNA (20–22). The N-terminal
SRP68 RNA-binding domain (RBD) locates to a central
RNA three-way junction between RNA helices 5, 6 and
8 (22). SRP68 binding to the RNA kinks the S domain
RNA and remodels the nearby 5f-loop (23). Kinking is
important for ribosome interaction at the previously
described ‘C4-contact’ (24), while 5f-loop remodeling might
activate the targeting complex as described in E. coli (7,8).
Here, a conserved guanine base complements the
interface of the targeting complex and activates GTP
hydrolysis. SRP72 binding to SRP RNA was pinpointed to the
adjacent 5e-loop (21). The C-terminal SRP72-RBD,
preceding the caspase cleavage and phosphorylation sites of
SRP72 (17), comprises a lysine-rich cluster and a linear
sequence motif (SRP72-Pfam) harboring a conserved
tryptophan residue (21) (Supplementary Figure S1). Heterodimer
formation with SRP68 occurs via the N-terminal
proteinbinding domain (SRP72-PBD), which is predicted to
consist of four tetratricopeptide repeats (TPRs) (25). TPRs
consist of 34 amino acids, which form two antiparallel helices
with a conserved residue pattern. TPR domains contain 3
to 16 repeats and form right handed -solenoid structures
(26). Usually, small peptides bind to the concave side of the
TPR domain in a linear mode ranging from an extended coil
to -helical conformation (27). The TPRs of SRP72-PBD
bind to a highly conserved C-terminal peptide of SRP68
(SRP68-PBD). However, despite the importance of SRP72
for SRP biogenesis and function and despite the progress
with recent high-resolution cryo-EM structures of
SRPRNC complexes (28,29), it remained the last mammalian
SRP protein without any structural information available.
To close this gap, we determined the structures of the
human SRP68/72-PBD complex and SRP72-RBD bound to
SRP RNA in context of a quaternary S domain complex,
providing important insights into SRP RNA plasticity and
revealing all relevant interactions of SRP72 within SRP.
MATERIALS AND METHODS
DNA manipulations and plasmids construction
All constructs used in this study are listed in Supplementary
Table S1. Total human mRNA was extracted from HEK
cells (SV Total RNA Isolation System, Promega),
reversedtranscribed into cDNA (Transcription First Strand cDNA
Synthesis Kit with dT primers, Roche), and used for cloning
in this study. The DNA sequence encoding human
SRP72RBD (512–662) fused to a C-terminal His6-tag was cloned
into the pET24d vector (Novagen) digested NcoI/BamHI.
DNA encoding for human SRP72-PBD (8–166) was cloned
into the N-terminal His6-Sumo fusion vector pCA528 (30)
using the BsaI/BamHI restriction sites. Untagged human
SRP68-PBD (546–614) was cloned into the NcoI/BamHI
restriction sites of pET16b (Novagen) and into pETtrx1a
(31) containing an N-terminal thioredoxin. Short human
SRP72-RBD peptides P1 (560–605), P2 (557–605) and P3
(549–605) were cloned into the NcoI/BamHI restriction
sites of the pETtrx1a vector.
For yeast two-hybrid analyses, human SRP72-PBD
(1–159) was fused to an N-terminal GAL4 binding
(BD) and activation (AD) domain using the
plasmids pG4ADN111/pG4BDN22 (32), respectively.
Human SRP68-PBD (585–607) was fused to a
Cterminal GAL4-BD and GAL4-AD using the plasmids
pG4BDC22/pG4ADC111 (32). Point mutations were
introduced according to the QuikChange Lightning kit
(Agilent) site-directed mutagenesis protocol.
The 145 nucleotides long (105–249) human SRP RNA
gene was amplified from human genomic DNA, fused to
a 3 -hammerhead ribozyme and cloned under the
control of the T7 RNA polymerase promotor into pSP64
vector (Promega) digested EcoRI/HindIII. Numbering
and sequences correspond to UniProtKB o76094 (human
SRP72), UniProtKB q9uhb9 (human SRP68) and
GenBank accession no. X04248.1 (human SRP RNA).
Protein/RNA production and purification
The human SRP72-RBD constructs were transformed into
BL21(DE3) E. coli cells (Novagen) and proteins were
expressed by auto-induction for 16 h at 25◦C (33).
Sumofused human SRP72-PBD and non-tagged SRP68-PBD
were co-expressed by auto-induction for 16 h at 25◦C.
Seleno-methionine labeled SRP72-RBD as well as
SRP72PBD (L31M/L65M) were expressed in BL21 (DE3) E.
coli cells grown in M9 medium supplemented with
selenomethionine as described in (34). Cells were grown at 37◦C
to an OD600 of 0.6 and induced with of 0.5 mM IPTG for
16 h at 22◦C.
Purification of SRP72-RBD, SRP19 and SRP68-RBD
were performed as follows: the cell pellet was resuspended in
lysis buffer containing 40 mM Tris/HCl pH 8.0, 1 M NaCl,
5 mM MgCl2, 5 mM KCl, 20 mM imidazole, 10% (v/v)
glycerol, and 0.02% (v/v) 1-thioglycerol. Cells were lysed
using an M-110L Microfluidizer (Microfluidics), the cleared
lysate was loaded onto a Ni-NTA column (GE Healthcare),
the column was washed with the lysis buffer and the protein
was eluted by an imidazole gradient (20–400 mM). The
eluate was further purified on a size exclusion chromatography
(SEC) column Superdex 75 26/60 (GE Healthcare)
equilibrated with a buffer containing 20 mM Hepes pH 7.5, 500
mM NaCl, 5 mM MgCl2, 5 mM KCl, 5% (v/v) glycerol and
0.02% (v/v) 1-thioglycerol.
Purification of SRP72-PBD, SRP68-PBD (both proteins
individually or in complex) and SRP72-RBD P1, P2 and
P3 were performed as described above using buffers
containing 200 mM NaCl. In order to remove the fusion
constructs, SRP72-PBD and SRP72-RBD P1, P2 and P3 were
treated with Ulp1 Sumo- and Tev-protease, respectively,
after the first Ni-NTA elution. The proteins were dialyzed
against a buffer containing 20 mM Tris/HCl pH 8.0, 200
mM NaCl, 5 mM MgCl2, 5 mM KCl, 10% (v/v) glycerol
and 0.02% (v/v) 1-thioglycerol for 16 h at 4◦C. The
proteases and cleaved fusion tags were bound to a reverse
NiNTA column and the flow through with the containing
proteins was purified via SEC. All proteins were concentrated
using Amicon Centrifugal filters (Millipore), frozen in
liquid nitrogen and stored at -80◦C.
In vitro run-off transcription of the 145 nucleotides long
SRP RNA construct was performed according to a
modified protocol described previously (35). In vitro
transcription was performed at 37◦C for 3 h. The RNA was purified
by urea-polyacrylamide gel electrophoresis and extracted
by crush-and-soak using 0.3 M sodium acetate followed by
isopropanol precipitation and desalting (PD10, GE
Reconstitution of the human quaternary S domain
SRP RNA was refolded using a snap cool protocol. The
RNA was heated to 90◦C for 1 min, cooled immediately
on ice for 1 min, diluted with 6× folding buffer
containing 120 mM Tris/HCl pH 8.0, 1.2 M NaCl, 60 mM KCl,
60 mM MgCl2, 52% (v/v) glycerol and incubated for 10
min at 37◦C. SRP19, SRP68-RBD and SRP72-RBD were
added to the RNA, the salt concentration was adjusted to
200 mM and the complex was incubated at 25◦C for 10
min. The reconstituted S domain was purified by SEC
using a Superdex S200 16/60 column (GE Healthcare)
equilibrated with 20 mM HEPES/NaOH pH 7.5, 150 mM NaCl,
5 mM MgCl2, and 5 mM KCl. SRP19, SRP68-RBD and
SRP72-RBD were dialyzed two times against a buffer
containing 20 mM HEPES/NaOH pH 7.5, 500 mM NaCl, 5
mM MgCl2, 5 mM CsCl, 5% (v/v) glycerol and 0.02% (v/v)
1-thioglycerol for 2 h at 4◦C to replace potassium for cesium
ions. Folding buffer and SEC buffer for S domain
reconstitution were prepared with CsCl using the respective KCl
concentrations. S domain reconstitution was performed as
Crystallization and data collection
Crystals of the SRP68/72-PBD complex (native and
selenomethionine labeled SRP68/72-PBD L31M/L65M) were
grown by automated crytallization in sitting drops
containing 0.2 M K2SO4 and 20% (w/v) PEG3350 at 18◦C. The
concentration of the complex was adjusted to 12 mg/ml
and drops were set in a 1:1 ratio of protein solution to
crystallization buffer. Crystals of the quaternary S
domain (native, seleno-methionine labeled SRP72-RBD
either L562M, I598M, or cesium containing S domain) grew
at 4◦C in hanging drops manually set containing 17–20%
(w/v) PEG3350 and 0.2 M KF. The complex had a
concentration of 10 mg/ml and drops were set in 1:1 ratio.
Crystals were cryoprotected in 20% (v/v) glycerol added
to the mother liquor and were frozen in liquid nitrogen.
Data were collected at the European Synchrotron
Radiation Facility (ESRF, Grenoble). Single-wavelength
anomalous dispersion (SAD) data were collected for the
selenomethionine labeled samples or cesium containing crystals
at anomalous peak wavelengths.
Structure determination and refinement
Data processing was performed with XDS (36) and the
CCP4i-implemented program AIMLESS (37) for all
collected datasets. The structure was solved using SAD data
at 3.0 A˚ resolution using the Phenix suite (38,39). The
structure was refined against the native dataset using
Phenix.refine (40) and model building was done in COOT
(41). The structure was solved by molecular replacement
with the program Phenix.phaser (42) using the human
ternary S domain complex (4p3e (23)) as a search model.
The structure was refined with Phenix and built in iterative
cycles in COOT with the help of the anomalous signal for
methionine of SRP72-RBD L562M or I598M. Surface
potentials were calculated by APBS (43) in PyMol (44).
Superposition of structures was performed in COOT. Figures of
the structures and electron density maps were created using
PyMol and Chimera (45), respectively.
Isothermal titration calorimetry (ITC)
ITC experiments were performed using a MicroCal
PEAQ-ITC (Malvern) in a buffer containing 20 mM
HEPES/NaOH pH 7.5, 200 mM NaCl, 10 mM MgCl2, 10
mM KCl and 10% (v/v) glycerol (for RNA binding) or 5%
(v/v) glycerol for the protein interaction. The titrations were
conducted in 19 injection of 2 l aliquots at 25◦C, a
reference power of 10 cal/s and a stirring speed of 750 rpm.
Typical concentrations of the titrants were between 100–200
M (RNA-binding domains) and 150–200 M
(SRP68PBD), the concentrations in the cell were between 7–17 M
(S domain) and 20–30 M (SRP72-PBD). The
measurements were analyzed using the MicroCal PEAQ-ITC
Analysis Software. ITC experiments were performed at least in
Multi-angle light scattering (MALS)
A total of 150 l of the SRP68/72-PBD complex (2.5
mg/ml) was injected onto a Superdex 75 10/300 column
(GE Healthcare) coupled to a MALS system (Dawn Heleos
II 8+ and Optilab T-rEX, Wyatt Technology). Data was
analyzed using Astra 6 software (Wyatt Technology).
A total of 500 l of 40 M His6-SRP68-PBD were loaded
on spin-columns with 100 l Ni-NTA beads equilibrated
with the SRP68/72-PBD SEC buffer (buffer A) and
incubated for 5 min at 4◦C on a slow stirring wheel. The beads
were washed once with 500 l buffer A and once with 100
l buffer A containing 50 mM imidazole (buffer B). A
total of 500 l of 40 M SRP72-PBD were loaded on the
Ni-NTA beads and incubated for 5 min at 4◦C. The beads
were washed twice with 100 l buffer B before elution of the
complex with 100 l buffer A containing 500 mM
imidazole (buffer C). As control, SRP72-PBD was loaded
without His6-SRP68-PBD to 100 l Ni-NTA beads and
incubated for 5 min at 4◦C. The column was washed with 500 l
buffer A, twice with 100 l buffer B and once with 100 l
buffer C. All flow through samples were analyzed on a 15%
sodium dodecyl sulphate-polyacrylamide gel
Yeast two-hybrid assays
The Saccharomyces cerevisiae strain PJ69–4A was
cotransformed with a bait and prey plasmid. Double
transformants were selected on SDC medium lacking leucine and
tryptophan. For each transformation, four colonies were
arrayed in 96-well microtiter plates. The colonies were diluted
to an OD600 of 3.0 in water followed by 10-fold serial
dilutions and spotted onto SDC-Leu-Trp, SDC-Leu-Trp-His
and SDC-Leu-Trp-Ade plates. Growth was determined
after 3 days incubation at 30◦C. Transactivation controls were
systematically performed for each construct with the
opposite vector without insert.
Structure of the SRP68/72-PBD complex
In order to characterize the SRP68/72 interaction, the
SRP72-PBD was co-expressed and co-purified with
SRP68PBD. Stoichiometric complex formation was confirmed by
pull-down assays and analytical SEC coupled with
multiangle light scattering (Supplementary Figure S2). The
complex was crystallized in space group C2 with one molecule
per asymmetric unit. A native dataset was collected to
1.6 A˚ resolution. However, the structure could not be
solved by molecular replacement. Therefore, the double
mutant SRP72-PBD (L31M/L65M) was prepared for
selenomethionine based de novo single-wavelength anomalous
dispersion (SAD) phasing. The final model was refined to an
Rwork (Rfree) of 14.4% (18.6%) and Ramachandran statistics
show 100% of the residues in allowed regions (Table 1 and
Supplementary Figure S3).
SRP72-PBD (residues S10 to S159) is composed of
nine anti-parallel -helices arranged in a right-handed
solenoid (Figure 2A). SRP72-PBD comprises three
classical TPRs (TPR1: 11–44, TPR2: 45–78, TPR4: 109–142).
Residues 79–108 form two anti-parallel -helices, which
also possess the characteristics of a TPR but are shorter
in length (Supplementary Figure S4). We define these
helices as TPR3*. The C-terminal helix 9 forms a cap and
probably corresponds to the first helix of a following TPR5.
SRP72-PBD accommodates the TPR-ligand SRP68-PBD
in a conserved groove on the concave side of the -solenoid,
as typical for TPR domains (Figure 2B). The tight
interaction was confirmed by isothermal titration calorimetry
(ITC; KD of 33 nM; Table 2 and Supplementary Figure
S5). SRP68-PBD (residues K587 to L607) adopts an extended
structure with a central short -helix (D592 to H597) and a
C-terminal helical turn (E604 to L607) predicted to continue
until K612. Superposition of SRP72-PBD with its nearest
structural homolog (PDB 3vty (46); r.m.s.d. of 2.8 A˚ for
148 C -atoms) indicates a closed -solenoid
(Supplementary Figure S4). Closure is induced by a tilt of TPR4 and
TPR5a toward the groove, which is enabled by the
shortening of TPR3*. The contact surface between SRP68 and
SRP72 is 1200 A˚ 2, which is 43% of the whole surface of
SRP68-PBD (47). The interface is larger than for common
TPR–ligand complexes with extended peptide
conformation like the Hop/Hsp70-Hsp90 multichaperone machine
(48) (peptide interfaces less than 10 residues and below 700
A˚ 2, KD in the M range). The difference in affinity reflects
the stability of the SRP68/72 complex in contrast to the
diverse and reversible Hop interactions.
The SRP68/72 interaction is rather hydrophobic and the
complex is stable in high salt conditions (1 M). Most
prominent, the aromatic rings of the phenylalanines F590 and F600
within SRP68-PBD are anchored in hydrophobic pockets
formed in between TPR4 and 5a, and TPR1 and 2,
respectively (Figure 2C). The SRP68-PBD helix forms the center
of the interface and is clamped in between TPR2, TPR3*
and TPR4. Hydrogen bonding to the SRP68-PBD
backbone are restricted to amide sidechains as typically found
for ‘amide ladders’ in other TPR or Armadillo-repeat
proteins (49). To test for the importance of individual residues
on the interaction we performed yeast-two-hybrid assays
(Figure 3). We show that the region P585-L607 of SRP68
is sufficient for SRP72 binding. Single alanine mutations
of the strictly conserved residues Y86 and D592 abolished
the binding completely, consistent with previous
interaction studies (25) and underlining the central role of the
intertwined hydrogen-bonding network between residues Y86,
R90, Q117 and D592 (Figure 2C and Supplementary Figure
S6). Similarly, the modification of the hydrophobic patch
quaternary S domain
Wilson B-factor ( A˚2)
*for 5% of all data.
P 21 21 21
P 21 21 21
by the single mutant F600A and the double alanine mutant
I56A/V598A resulted in a complete loss of binding.
Structure of the human S domain with SRP72-RBD
While interaction between the PBDs of SRP68 and SRP72
results in the formation of a stable heterodimer, both
proteins can bind to SRP RNA individually via their RBDs.
Previously, we showed that the SRP68-RBD remodels the
SRP RNA using a S domain RNA construct of 125
nucleotides (23). In order to characterize now the
RNAbinding of SRP72, we reconstituted a quaternary human
SRP sub-complex including the S domain RNA (using a
longer RNA variant of 145 nucleotides from 105 to 249),
SRP19, SRP68-RBD and SRP72-RBD. ITC measurements
were performed to determine binding affinities of
SRP68RBD and SRP72-RBD to SRP RNA (Table 2 and
Supplementary Figure S5). SRP19 was prebound in all
experiments to SRP RNA in order to clamp helices 6 and 8
together and thereby to provide the binding platform for
SRP68 and SRP72. SRP68-RBD binds strongly to SRP
RNA (KD of 2 nM) independent of the presence of
SRP72RBD. In contrast, the binding affinity for SRP72-RBD is
lower by two orders of magnitude with a twofold increase in
presence of SRP68 (KD values of 181 and 427 nM,
respectively). This indicates that RNA binding of SRP72 is
enhanced by interaction with SRP68 or that a SRP68-induced
conformational change in the SRP RNA facilitates
recruitment of SRP72. Interestingly, while SRP68-RBD binding
to SRP RNA is enthalpy-driven, SRP72-RBD binding is
entropy-driven suggesting folding upon binding.
To answer this question, we set out to determine the
structure of the human quaternary S domain including
SRP72RBD. Crystals could be obtained belonging to space group
P212121 with two molecules in the asymmetric unit. The
structure was solved by molecular replacement at 3.4 A˚
resolution (Figure 4A). The final model was refined to an Rwork
(Rfree) of 23.7% (28.0%) and Ramachandran statistics show
97.8% of the residues in allowed regions (Table 1 and
Supplementary Figure S3). SRP72-RBD and metal ion
assignments were validated by mutational analyses and
anomalous difference Fourier techniques (Table 1, Supplementary
Figure S7 and Supplementary Table S2). The structure
reveals the fold of the entire S domain RNA including the
5e-loop, that so far was only modeled (21,50). SRP19 and
SRP68-RBD structures as well as the way they interact with
SRP RNA are identical as in a ternary S domain complex
solved previously (23) (Supplementary Figure S8).
Especially the 20◦ kink at the RNA three-way junction as
induced by SRP68-RBD and the insertion of its arginine-rich
motif (ARM) into the major groove at the 5f-loop are
maintained. Comparison of the RNA-binding site for SRP54
(asymmetric loop of helix 8) shows that it adopts a different
structure than previously observed (23,51,52). The
difference is due to a concerted helix-bending induced by crystal
packing (Supplementary Figure S8) and reflects the
plasticity of this RNA site. However, asymmetric loop flexibility
has no influence on SRP72-RBD binding.
The SRP72-RBD (K557-Q603) appears as a linear
peptide that crawls along the ‘distal site’ of SRP RNA at the
5e- and 5f-loop (Figure 4B and C). At the N-terminus, the
lysine-rich motif involved in RNA-binding (21) is only
partially ordered (K557-559) probably due to the limited length
of our RNA construct (Supplementary Figure S9).
Binding studies showed that the lysine-rich motif is needed for
SRP72 binding to the SRP RNA (Supplementary Figure
S9). The SRP72-Pfam motif (572PDPERWLPMRER583) is
central to the interaction. The strictly conserved 572PDP574
motif is highly flexible and electron density is weak. Most
prominent, the strictly conserved tryptophan (W577) inserts
into the 5e-loop, which introduces a kink into SRP RNA
(Figure 4B and C). Together with the neighboring strictly
conserved arginine R576, the two residues form a
continuous stack with the nucleobases of C111 and C242 thus
stabilizing the 5e-loop geometry. Arginine R581 stabilizes
nucleotide A231 of the 5f-loop in a bulged-out conformation.
The 5f-loop is opened by the ARM of SRP68-RBD (23),
suggesting an allosteric stimulation of SRP72-RBD binding
(2-fold) as shown by our ITC data. Interestingly, while in the
S domain structure without SRP72-RBD two nucleotides
(A231 and G232) were found to be bulged-out, in the
structure with SRP72-RBD guanine G232 is base-pairing with
A119 underlining the previously observed plasticity of the
5f-loop (23). At the C-terminus following the Pfam motif,
a positively charged helix (C4-helix) aligns with the 5f-loop
region (Figure 4C) and together with nucleotide G113
mediates a crystal contact with SRP RNA (Supplementary
Figure S8). A strictly conserved 603QG604 motif locates to the
C-terminal end of the helix. The very C-terminus beyond
the C4-helix and the caspase cleavage site (D614) is
intrinsically disordered and not visible in the quaternary
complex. The C-terminus of SRP72-RBD is located close to
an extended SRP68-RBD loop (F100-K111) that forms a
hairpin inserting in the three-way junction of SRP RNA
connecting helices 5, 6 and 8 (Figure 4A and D). An
exposed phenylalanine (F108) within this loop stacks onto an
RNA U-turn as predicted (23).
The 5e-loop constitutes a potassium-binding RNA kink-turn
The 5e-loop has been described as a putative
‘kinkturn’ (K-turn) motif (21) that represents an extremely
widespread RNA-folding unit present in ribosomal RNAs
and riboswitches (53). Classical K-turns comprise a
threenucleotide bulge flanked by two highly conserved sheared
A◦G pairs on the 3 side (non-canonical NC-stem) and a
Watson-Crick helix (canonical C-stem) at the 5 side of the
loop (Figure 5A) (54). The 5e-loop does not contain the
A◦G pairs, and although resembling the canonical motif
with three unpaired nucleotides (A240, U241 and C242), it
significantly differs. Most strikingly, the 5e-loop is stabilized
by the presence of a central potassium ion (Figure 5B),
validated by cesium replacement (Supplementary Figure S7),
which is not present in classical K-turns. We therefore
define the 5e-loop geometry as ‘K+-turn’. The potassium
locates to a central G◦U wobble base pair starting the C-stem
(U110◦G243; -1b and -1n position in K-turn nomenclature
(54)) and is coordinated by four oxygens of G109, U110, U241
and G243. The potassium bridges the NC- to the C-stem as
in classical K-turns performed by the central adenines of
the A◦G pairs that are involved in A-minor motifs. C-stem
superposition of the K+-turn with a classical K-turn
indicates a diametric opposed kinking of the RNA (Figure 5B).
Although different in shape, most K-turns serve as protein
binding sites with varying structures (53). In order to test
the influence of the potassium ion for SRP72 binding, we
exchanged the central G◦U wobble for a standard G-C base
pair (U110C mutant) to impair the potassium coordination
sphere. ITC experiments revealed a twofold reduced
binding affinity compared to the wild-type RNA (KD of 427 nM,
Table 2 and Supplementary Figure S5).
Strikingly, a respective SRP RNA kink of similar extent
(∼50◦) is also present in bacteria and archaea (55,56)
despite the absence of SRP68/72 (Figure 5C). In an archaeal
structure, the three unpaired nucleotides are stacked into
the loop (56) as typical for other SRP RNA bulge-loops in
the absence of protein (23,51). The ligand sphere of
potassium including the G◦U wobble is present in archaea and
according to sequence comparisons, the K+-turn might be
a general feature of archaeal, eukaryotic, as well as
Grampositive bacterial SRP RNA (57). In Gram-negative
bacteria the 5e-loop corresponds to loop E, which has been
determined as ‘primary docking site’ for the targeting complex
formed by Ffh (SRP54 in archaea and eukaryotes) and its
receptor FtsY (SR in eukaryotes) (7) (Figure 5C). The
integrity of this ‘distal site’ has been found to be crucial for the
GTPase-activating function of SRP RNA on the SRP
GTPase heterodimer in E. coli (7,8). Our data showing a
conserved SRP RNA kink indicate the general conservation of
targeting complex activation by RNA. Such mechanism is
supported by the conservation of the respective RNA
interaction sites in the targeting complexes from all kingdoms of
The SRP72-RBD in the interface of SRP/RNC complexes
In order to analyze the function of SRP72-RBD for RNC
interactions, the quaternary S domain structure was
fitted into electron density maps of eukaryotic SRP-RNC
and SRP/SR-RNC complexes previously obtained by
cryoelectron microscopy (24,58) (Figure 6A). In these
structures, SRP binds across the ribosomal surface, spanning
from the polypeptide exit tunnel on the 60S subunit to the
elongation factor binding site in the ribosomal subunit
interface. The placement of our S domain structure suggests
an intriguing function for SRP68-RBD and SRP72-RBD
in guiding and modulating the SRP-RNC interaction at
the C4-contact. The -hairpin of SRP68-RBD in the SRP
RNA three-way junction is in close contact with the 28S
rRNA, an interaction that due to limited local resolution of
the cryo-EM structure escaped previous analyses.
SRP72RBD is not resolved in the cryo-EM structure, but threads
between SRP and the ribosome and contributes to the
interaction with its PDP motif and the C-terminal C4-helix.
The PDP motif is in proximity to an extended -hairpin of
the ribosomal protein RpL3 and apparently confers
rigidity to the interaction. The C4-helix contains multiple
positive charges (K590/591, K594/595) and is involved in a protein–
RNA contact in our S domain crystal (Figure 4C and
Supplementary Figure S8). On the ribosome, it reads out the
3 -terminal three-way junction of 28S rRNA (Figure 6B).
However, rigid-body docking of the SRP onto the ribosome
would result in a clash of the C4-helix with 28S rRNA
(Supplementary Figure S10) suggesting a flexible tethering of
the C4-helix within the S domain. Upon SR binding,
eukaryotic SRP rotates at the C4-contact in respect to the
ribosome (58). The rotation places the C4-helix within the
ribosomal three-way junction in a very similar way as in
our protein–RNA crystal contact. Therefore, we assume
this contact to mimick the physiological C4-contact. The
bulged-out nucleotide A231, which is stabilized by R581,
contributes to the C4-contact possibly forming a base pair with
the 28S rRNA (Figure 6C). SR dependent rotation of SRP
induces a switch of this RNA–RNA contact and results
in replacement of nucleotide A231 by G113. Thus, the
C4contact includes protein–protein, protein–RNA and RNA–
RNA interactions (Supplementary Figure S10). It is
maintained upon SR interaction, but adjusts during the SRP
cycle. Of note, all ribosomal contacts locate to eukaryotic
extensions not present in bacterial ribosomes, which do not
establish a stable C4-contact with SRP (59).
SRP72, the largest SRP protein, forms a stable heterodimer
with SRP68 and is essential for SRP function. It is the
only SRP protein, which is post-translationally modified by
MAPK phosphorylation and contains a C-terminal caspase
cleavage site. Caspase cleavage during apoptosis is discussed
as a regulatory step for SRP-dependent protein secretion.
Even though SRP72 is biochemically and pathologically
well described, its structure and its functional implications
have stayed enigmatic.
The presented crystal structures close this gap and show
in detail how the N-terminal TPR domain of SRP72
enfolds a conserved C-terminal peptide of SRP68 and how
SRP72 interacts with the potassium-bound 5e-loop of SRP
RNA. Strikingly, SRP72 binding to the 5e-loop influences
the adjacent 5f-loop, with a bulged-out nucleotide
providing a binding platform for the ribosomal C4-contact.
Docking of the S domain structure into cryo-EM densities of
entire SRP/SR–RNC complexes reveals the C4-contact to be
maintained during the SRP cycle and that SRP68, SRP72
and SRP RNA contribute to the ribosome contact. In
addition the kink-turn of the 5e-loop seems to be present in all
domains of life providing a binding platform for the
targeting complex. On the basis of this work we propose the
following scenario for the human SRP targeting cycle (Figure
7): Without SR, SRP72-RBD presents the 5f-loop (A231)
to ribosomal RNA and the flexibly linked C4-helix
tethers SRP onto the ribosome. Upon SR binding and
docking of the targeting complex to the 5e-loop, SRP rotates
and the 5f-loop becomes available for SRP GTPase
activation as shown for the bacterial system (7,8) potentially
involving two-bulged out nucleotides (A231 and G232) (23).
The bulged-out nucleotide (G113) of SRP RNA takes over
the RNA–RNA tertiary interaction and the SRP-ribosome
contact is maintained in contrast to Gram-negative bacteria
(59). This might explain why ribosomes are essential for the
GTPase activation in eukaryotes in contrast to prokaryotes
(60). Although the C4-helix of SRP72 stays in place, its
conserved C-terminus might aid in GTP-hydrolysis. This idea is
supported by the activation of an SRP GTPase dimer by a
glutamine residue located in an -helix of a protein
partner as observed for the third SRP GTPase FlhF (involved
in flagella biosynthesis) (61,62).
The caspase cleavage and phosphorylation sites within
SRP72 immediately follow the C4-helix and would come
in close proximity to the re-localized targeting complex.
Positively-charged surface patches on the targeting
complex (6) are readily available to bind to phosphorylated
serine residues within the SRP72 tail. As caspase cleavage
removes the C-terminal tail, SRP targeting would be clearly
impaired. This model matches with data on SRP72
cleavage in apoptosis, where protein secretion is indeed decreased
(17,63) and in disease by generating an SRP72 epitope for
autoimmune response finally resulting in myopathies or
In summary, although SRP is a conserved and ancient
machinery in all kingdoms of life, its complexity in structure
and regulation increased significantly during evolution. Our
data underline that all players need to be carefully
characterized on the atomic level to finally understand the
mechanism of the whole machinery.
Coordinates and structure factors are deposited in the
RCSB protein data bank (PDB) with the accession numbers
5M72 and 5M73.
Supplementary Data are available at NAR Online.
We acknowledge Friederike Schreiter and Jonathan Paulitz
for technical assistance. We thank Yvonne Hackmann for
human cDNA preparation; Gunter Stier for providing
the pETTrx1a vector and Bernd Bukau for providing the
pCA528 plasmid. We are grateful to Ed Hurt for
sharing plasmids pG4ADN111, pG4ADC111, pG4BDN22 and
pG4BDC22. We thank Ju¨ rgen Kopp and Claudia Siegmann
from the BZH/Cluster of Excellence:CellNetworks
crystallization platform. We acknowledge access to the beamlines
at the European Synchrotron Radiation Facility (ESRF) in
Grenoble and the support of the beamline scientists. I.S. is
an investigator of the Cluster of Excellence:CellNetworks.
Deutsche Forschungsgemeinschaft (DFG) [SFB638 to I.S.,
K.W., GRK1188 and the Leibniz programme to I.S.].
Funding for open access charge: DFG (Leibniz Programme).
Conflict of interest statement. None declared.
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