Acta Cryst. (2012). D68, 531–540doi:10.1107/S090744491200491X
Acta Crystallographica Section D
Novel b-structure of YLR301w from Saccharomyces
Kook-Han Kim, Hyung Jun Ahn,
Won-Kyu Lee, Cheolju Lee,
Myeong-Hee Yu and
Eunice EunKyeong Kim*
Biomedical Research Institute, Korea Institute of
Science and Technology, 39-1 Hawolkok-dong,
Sungbuk-gu, Seoul 136-791, Republic of Korea
Correspondence e-mail: firstname.lastname@example.org
# 2012 International Union of Crystallography
Printed in Singapore – all rights reserved
When the Z-type variant of human ?1-antitrypsin was
analysis identified YLR301w as one of the up-regulated
proteins. YLR301w is a 27.5 kDa protein with no sequence
homology to any known protein and has been reported to
interact with Sec72 and Hrr25. The crystal structure of
S. cerevisiae YLR301w has been determined at 2.3 A˚
resolution, revealing a novel ?-structure. It consists of an
N-terminal ten-stranded ?-barrel with two short ?-helices
connected by a 23-residue linker to a seven-stranded half-
barrel with two short helices at the C-terminus. The
N-terminal barrel has a highly conserved hydrophobic channel
that can bind hydrophobic molecules such as PEG. It forms
a homodimer both in the crystal and in solution. YLR301w
binds Sec72 with a Kdof 6.2 mM, but the biological significance
of this binding requires further investigation.
Received 26 September 2011
Accepted 4 February 2012
PDB Reference: YLR301w,
Proteins are the essential operators of cellular functions and
at least 30 000 different proteins are expected to play various
physiological roles in humans. However, the accumulation of
misfolded or unfolded proteins in the endoplasmic reticulum
(ER) causes ER stress, and chronic or unresolved ER stress
can lead to apoptosis. This stress-related apoptosis contributes
to pathophysiological processes involved in a number of
prevalent diseases such as diabetes, atherosclerosis, neuro-
degenerative diseases and renal diseases (Marciniak & Ron,
2006; Herczenik & Gebbink, 2008; Tabas & Ron, 2011).
One of the better characterized protein-folding diseases is
human ?1-antitrypsin deficiency, which is an autosomal co-
dominant genetic disorder caused by the defective production
of ?1-antitrypsin. ?1-Antitrypsin inhibits a wide variety of
proteases and protects tissues from the enzymes of inflam-
matory cells; e.g. in the absence of ?1-antitrypsin neutrophil
elastase is free to break down elastin, resulting in respiratory
complications such as emphysema or chronic obstructive
pulmonary disease in adults (Crystal, 1990; Stoller & Abous-
souan, 2005; Gooptu & Lomas, 2009). ?1-Antitrypsin is also
associated with cirrhosis in adults and children. The forms and
degree of deficiency depend on whether the patient has one or
two copies of the affected genes. To date, over 100 different
variants of ?1-antitrypsin have been described, including the
Z-type with a mutation of Glu342 to Lys (Lomas et al., 1992).
In the case of Z-type ?1-antitrypsin, protein folding is
extremely slow compared with the wild type owing to loop–
sheet polymerization and this ultimately results in protein
aggregation (Yu et al., 1995).
In order to better understand the cellular responses at the
protein level when such protein aggregation occurs, we over-
expressed Z-type ?1-antitrypsin in Saccharomyces cerevisiae,
as yeast has been widely used as a model system to understand
cellular processes (Gasch et al., 2000). In order to observe the
effect, we carried out proteomics analysis. Two-dimensional
gel electrophoresis and mass analysis of differentially
expressed proteins resulted in chaperones, antioxidant
proteins, mitochondrial proteins for ATP generation and
some others (data to be published elsewhere). Interestingly,
YLR301w, which has no sequence homology to any proteins
that have been characterized, is among the up-regulated
proteins. The YLR301w (UniProt code Q05905) gene is
located in chromosome 12 of S. cerevisiae and encodes a
protein of 244 amino acids (Mr= 27 501) which does not
appear to have an apparent signal sequence or transmem-
Genomic neighbourhood analysis searches suggest that
YLR301w is a protein of yeast origin of unknown function.
Searches in the Pfam database (Bateman et al., 2004) as well as
the Protein Data Bank (Berman et al., 2000) showed no shared
domains or motifs for YLR301w. However, YLR301w has
previously been identified as an interacting partner of Sec72,
which is associated with Sec62, Sec63 and Sec71, three integral
membrane proteins in the ER. The Sec63 complex is required
for translocation of pre-secretory proteins into the ER of
S. cerevisiae (Willer et al., 2003; Fang & Green, 1994; Rapo-
port, 2007; Zimmermann et al., 2006; Jermy et al., 2006). The
Sec72 null mutant accumulated a subset of secretory precur-
sors in vivo, but Sec72 is not essential for cell growth. In
addition, YLR301w was reported to increase by 1.7-fold at
the mRNA level when yeast was treated with rapamycin
(Bandhakavi et al., 2008) and YLR301w expression was down-
regulated in the presence of 300 mM citric acid pH 3.5
(Lawrence et al., 2004). In summary, YLR301w is a novel
protein that is located in the cytosol with no sequence
homology to any known proteins and that may play a role
in various environmental stresses. Protein structure is closely
linked to protein function and several recent structural
genomics efforts have resulted in the identification of function
based on structure (Chandonia & Brenner, 2006; Hrmova &
Fincher, 2009; Kim et al., 2010; Shin et al., 2007; Chin et al.,
2006). Therefore, we investigated the structure of YLR301w in
order to gain some insight into its function. Here, we report
a novel crystal structure of S. cerevisiae YLR301w at 2.3 A˚
2. Materials and methods
2.1. Protein expression and purification
The YLR301w (Swiss-Prot entry Q05905) and Sec72 (Swiss-
Prot entry P39742) genes were amplified by polymerase
chain reaction (PCR) from S. cerevisiae genomic DNA using
PfuTurbo polymerase (Stratagene). The PCR product of
YLR301w was cloned into plasmid pET28a, which encodes a
purification tag consisting of the amino acids Leu-Glu-His6
at the C-terminus of the protein. For Sec72–YLR301w inter-
action, YLR301w was cloned into a modified pGEX-2T vector
with an N-terminal GST tag and residues 39–193 of Sec72
were cloned into pET32a with a thioredoxin-His6tag added
at the N-terminus. All three constructs were expressed in
Escherichia coli BL21 (DE3) cells.
YLR301w was prepared and expressed from E. coli BL21
(DE3) as described previously (Ahn et al., 2007). Cells over-
expressing YLR301w were harvested by centrifugation and
resuspended in buffer consisting of 50 mM Tris–HCl pH 7.4,
100 mM NaCl, 2 mM ?-mercaptoethanol, 1 mM phenyl-
methylsulfonyl fluoride. The cells were disrupted by sonica-
tion; the crude lysate was centrifuged at 16 000g (Sorvall GSA
rotor) for 30 min at 277 K and the cell debris was discarded.
The supernatant was loaded onto a nickel-chelated HiTrap
chelating column (GE Healthcare) and eluted with a linear
gradient of 20–500 mM imidazole in 50 mM Tris–HCl pH 7.4,
100 mM NaCl, 2 mM ?-mercaptoethanol. Fractions containing
YLR301w were pooled based on SDS–PAGE analysis.
YLR301w was further purified by gel filtration using a HiLoad
26/60 Superdex 75 prep-grade column (GE Healthcare) and
was concentrated to 21 mg ml?1in 50 mM Tris–HCl pH 7.4,
100 mM NaCl, 2 mM ?-mercaptoethanol using a concentrator.
Since YLR301w only has a starting methionine, an additional
methionine was introduced at position Leu35 or Leu128 for
phasing. The Leu128Met mutant gave a clear single band
on SDS–PAGE, while the Leu35Met mutant did not. The
Leu128Met mutant was then cultured in E. coli BL21 B834
(DE3) cells in M9 culture medium which contained extra
amino acids including SeMet (Guerro et al., 2001). Purification
of the SeMet protein was carried out in the same manner as
for the native protein.
GST-YLR301w and Sec72 were overexpressed as described
above. YLR301w was purified using a GST affinity column
(GE Healthcare) followed by a HiLoad 26/60 Superdex 75
prep-grade column (GE Healthcare) and was concentrated to
6 mg ml?1in 50 mM Tris–HCl pH 7.4, 100 mM NaCl, 1 mM
DTT using a concentrator. Sec72 was purified utilizing a
nickel-chelating HiTrap column (GE Healthcare) and the
thioredoxin tag was removed by cleavage using thrombin. The
cleaved Sec72 was further purified by gel filtration using a
HiLoad 26/60 Superdex 75 prep-grade column (GE Health-
care) which was pre-equilibrated in 50 mM Tris–HCl pH 7.4,
300 mM NaCl, 1 mM DTT and concentrated to 10 mg ml?1.
Purified GST-YLR301w and Sec72 were subjected to gel
filtration using a Superdex 200 HR 10/30 column (GE
Healthcare) in PBS buffer (50 mM NaH2PO4, 150 mM NaCl
2.2. Crystallization and data collection
The crystallization condition for YLR301w protein was
initially obtained from sitting-drop vapour-diffusion experi-
ments using 96-well Intelli-Plates (Hampton Research) and a
Hydra II Plus One robot (Matrix Technology) at 295 K and
was optimized using hanging-drop vapour diffusion in 24-well
plates with a 1:1 mixture of protein solution and reservoir
Kim et al.
Acta Cryst. (2012). D68, 531–540
solution. Diffraction-quality crystals of YLR301w were
obtained by mixing a protein solution consisting of 21 mg ml?1
protein in 50 mM Tris–HCl pH 7.4, 100 mM NaCl, 2 mM
?-mercaptoethanol with a reservoir solution consisting of 33%
polyethylene glycol monomethyl ether 550 (PEG MME 550),
0.1 M HEPES pH 7.3. The crystals grew as hexagonal
bipyramids in 1–2 d. SeMet protein crystals were obtained
using an identical condition to that used for the native crystals.
The crystals were flash-cooled in liquid nitrogen and diffrac-
tion data were collected on beamline 4A of Pohang Light
Source, Pohang, Republic of Korea using an ADSC Quantum
210 CCD detector at 100 K. The native crystal diffracted to
2.3 A˚resolution, while the SeMet crystal diffracted to 3.0 A˚
resolution. Data were processed and scaled using the
programs DENZO and SCALEPACK from the HKL-2000
program suite (Otwinowski & Minor, 1997). The Matthews
coefficient for YLR301w was 3.29 A˚3Da?1and the estimated
solvent content was 62.6%; there were two YLR301w mole-
cules in an asymmetric unit. Data statistics are given in
2.3. Structure solution and refinement
Refinement of heavy-atom parameters and phase calcula-
tion was carried out using SOLVE (Terwilliger, 2004), which
found all four potential Se sites for the P6522 space group. The
identification of four Se sites by single-wavelength anomalous
dispersion (SAD) phasing led to a mean FOM of 0.75 to a
resolution of 3.5 A˚. The electron-density map was generated
through Fourier transformation. Maximum-likelihood density
modification and automated model building was carried out
using RESOLVE (Terwilliger, 2004). The resulting electron-
density map with a partial model revealed clear main-chain
density with substantial side-chain details. Manual building
was performed using Coot (Emsley & Cowtan, 2004) and
refinement was carried out using CNS (Bru ¨nger et al., 1998)
and PHENIX (Zwart et al., 2008). The final model was vali-
dated using PROCHECK (Laskowski et al., 1993). The DALI
server (Holm & Rosenstro ¨m, 2010) was used to search for
proteins with a similar fold. Solvent-accessible and interaction
areas were calculated by PISA (http://www.ebi.ac.uk/msd-srv/
prot_int/pistart.html). Figures were generated using PyMOL
2.4. Size-exclusion chromatography
In orderto determine the quaternary structure of YLR301w
in solution, the hydrodynamic volume was determined using
a Superdex 75 10/300 GL column (GE Healthcare) installed
on an FPLC (GE Healthcare) protein-purification system.
YLR301w and standard molecular-weight markers (Sigma)
were prepared separately in buffers consisting of 50 mM Tris–
HCl pH 7.4, 100 mM NaCl, 1 mM DTT. The proteins were
passed through the Superdex 75 10/300 GL column and the
elution profile was compared with the standard molecular-
weight marker profile to estimate the size of the proteins in
2.5. Circular-dichroism spectroscopy and dynamic light
The secondary structure of YLR301w was characterized
using a Jasco J-715 spectropolarimeter equipped with a
temperature-control unit. Standard far-UV circular-dichroism
(CD) spectra were collected at room temperature at a scan
speed of 20 nm min?1with a 0.1 nm step resolution using a
0.1 cm path-length cell with a 1 nm bandwidth and a 4 s
response time. Ten accumulations taken from 260 to 190 nm
were added and averaged, followed by subtraction of the
solvent CD signal. Dynamic light-scattering (DLS) measure-
ments were performed on a DynaPro molecular-sizing
instrument equipped with a microsampler (Protein Solutions).
The data were analyzed using the Dynamics v.4.0 and DynaLS
software as described previously (Moradian-Oldak et al.,
1994). Dynamics v.4.0 was used to calculate the Rh.
2.6. Enzyme-linked immunosorbent assay (ELISA)
An ELISA was carried out in order to estimate the
interaction between YLR301w and Sec72. Each well of a
nickel-coated 96-well plate (Pierce) was incubated with 1 mg
His-Sec72 protein in 100 ml PBS (50 mM NaH2PO4, 150 mM
NaCl pH 7.4) for 1 h at room temperature. The wells were
blocked with 5%(w/v) skimmed milk in PBST (0.1% Tween-20
in PBS) for 1 h at room temperature and different concen-
trations of GST-YLR301w protein were then added to the
wells and incubated at 1 h at room temperature. The wells
Acta Cryst. (2012). D68, 531–540 Kim et al.
Crystallographic data-collection and refinement statistics for YLR301w.
Values in parentheses are for the highest resolution shell.
Data set Se peakNative
X-ray wavelength (A˚)
Resolution range (A˚)
Unit-cell parameters (A˚)
a = 121.44, b = 121.44,
c = 171.45,
? = ? = 90, ? = 120
a = 122.32, b = 122.32,
c = 174.00,
? = ? = 90, ? = 120
Resolution range (A˚)
No. of protein atoms
No. of water molecules
No. of ligand molecules
Average B factor (A˚2)
R.m.s.d. bond length (A˚)
R.m.s.d. bond angles (?)
Ramachandran analysis (%)
not included in the calculation of the R value.
‡ Rfreewas calculated from a randomly selected 5% set of reflections
ijIiðhklÞ ? hIðhklÞij=P
iIiðhklÞ, where Ii(hkl) is the intensity of
the ith measurement of reflection hkl and hI(hkl)i is the mean value of I(hkl) for all i
were incubated with HRP-labelled GST-tag antibodies (1/1000
dilution in PBST) for 1 h at room temperature; the colour-
developing reaction was initiated by adding OPD solution
(Pierce) and the reaction was terminated by adding 2.5 N
H2SO4. The absorbance of each well was measured at 492 nm
using a TRIAD microplate reader (Dynex Technologies).
2.7. PDB code
The atomic coordinates and structure factors for YLR301w
from S. cerevisiae have been deposited in the RCSB Protein
Data Bank (PDB) with code 3rby.
3. Results and discussion
3.1. Structure of YLR301w
YLR301w from S. cerevisiae was overexpressed in E. coli
and purified. The CD spectra indicated significant ?-structure
content, i.e. 47% of the structure was ?-structure, as shown in
Fig. 1(a). Since YLR301w has only one methionine (that at
the N-terminus), we introduced an additional methionine for
phasing by mutating leucine residues randomly. Replacement
of Leu128 by Met gave soluble protein and the SAD method
gave a structure solution at 2.3 A˚resolution. All atoms of
YLR301w were well defined in the electron-density maps. The
final model included two protein molecules (each consisting
of residues 1–244 plus one N-terminal His residue), six PEG
molecules, one sulfate molecule and 146 water molecules. The
Ramachandran plot produced by PROCHECK (Laskowski
et al., 1993) showed that 96% of the residues were in the most
favoured regions and 4% were in additional allowed regions.
Statistics of the data collection and refinement are summar-
ized in Table 1.
The topology of YLR301w is shown in Fig. 1(b) and a
ribbon drawing of the molecule is shown in Fig. 1(c). As shown
in the figure, the structure is almost exclusively comprised of
?-strands and loops, with a few short helices. The structure
is composed of three parts: the N-terminal 131 or so residues
form a full antiparallel ?-barrel consisting of ten strands
(?1–?10) and two short helices, the C-terminal 80 residues
(residues 155–244) form a half-barrel consisting of seven
antiparallel strands (?11–?17) with two short helices and the
central region (residues 132–154) forms a long linker loop
between the two (Fig. 1b). The total ?-structural content
amounts to 45.7% and this is similar to the value estimated
based on the CD data. There are two YLR301w molecules in
the asymmetric unit and the two molecules are related by a
noncrystallographic twofold axis. The two molecules are
almost identical to each other, with an overall root-mean-
square deviation (r.m.s.d.) of 0.51 A˚over 245 C?atoms.
Kim et al.
Acta Cryst. (2012). D68, 531–540
Structure of YLR301w from S. cerevisiae. (a) Far-UV CD spectra of YLR301w. The spectra indicate about 47% ?-structure. (b) Topological diagram of
YLR301w. YLR301w is comprised of an N-terminal domain (residues 1–131), a linker (residues 132–154) and a C-terminal domain (residues 155–244),
which are coloured blue, green and orange, respectively. The N-terminal domain has a ten-stranded ?-structure with two short helices and the C-terminal
domain consists of a seven-stranded ?-structure with two short helices. Helices and strands are shown as cylinders and arrows, respectively. (c) Ribbon
diagram of YLR301w. The ten-stranded N-terminal domain forms a ?-barrel, while the seven-stranded C-terminal domain forms a half-barrel. The
domains and linker are coloured blue, orange and green as in (b).
Although YLR301w is annotated as an uncharacterized
protein with unknown function, a BLAST search using the
UniProt database (Wu et al., 2006) gave a number of hits:
putative uncharacterized proteins from Candida glabrata
(Q6FR71), Vanderwaltozyma polyspora (A7TL22), Zygo-
(Q6CUE9) and Lachancea thermotolerans (C5E2Q7) were
identified with sequence identities of 54, 52, 54, 51 and 47%,
respectively (see Fig. 2). Proteins with sequence identities in
the range 40–60% are generally considered to be homologous,
with potentially similar functions. Sequence alignment of
YLR301w with these proteins shows that the conserved resi-
dues are distributed in both the N-terminal and C-terminal
domains. As shown in Fig. 3, the conserved residues in the N-
terminal domain mostly face the interior of the barrel. The
residues in both the N-terminal and the C-terminal domain
that face each other, i.e. the residues at the interface, are also
relatively conserved. The residues lining the entrance of the
N-terminal barrel, i.e. Phe10, Trp52, Phe54, Phe75, Phe125 and
Trp129, are conserved among the YLR301w homologues.
Together, these observations suggest that these proteins most
likely to all have the same structural arrangement as
3.2. Interactions between the N-terminal and C-terminal
The N-terminal domain and the C-terminal domain are
connected by a 23-residue linker, as shown in Fig. 1. However,
the two domains make extensive interactions with each other,
as indicated by the buried surface area of 1208 A˚2. The
interactions involve both hydrophobic and polar residues. In
addition, residues from the linker loop make interactions with
the two barrels, with the interfaces being 420 and 480 A˚2,
respectively. The corresponding values are 1217, 421 and
472 A˚2for molecule B. The conserved residues at the
Acta Cryst. (2012). D68, 531–540 Kim et al.
Amino-acid sequence alignment. A BLAST search shows uncharacterized proteins from C. glabrata (Q6FR71), V. polyspora (A7TL22), Z. rouxii
(C5DWI2), K. lactis (Q6CUE9) and L. thermotolerans (C5E2Q7) as homologues of YLR301w. The residues in the red boxes are strictly conserved.
Secondary structures of YLR301w are depicted above the sequence; ?-helices and ?-strands are indicated by coils and arrows, respectively. Residues
within 3.5 A˚of the two PEG MME 550 molecules bound inside the barrel are highlighted by inverted blue triangles. The sequence alignment was
performed using ClustalW and the image was produced using ESPript 2.2.
N-terminal and C-terminal domain interface include both
hydrophobic and hydrophilic amino acids, i.e. Ser25, Ser27,
Leu35, Pro37, Pro49, Leu85 and Pro131 from the N-terminal
domain, and Ser155, Leu168, Gly173, Trp175, Gln177, Glu241
and Arg195 from the C-terminal domain (see Fig. 2). The
backbone atoms of Phe21, Leu23, Thr28, Asp29, Ser47 and
Gln182 make hydrogen bonds to the side-chain atoms of
Arg195, Gln177, Ser155, Glu241, Tyr210 and Tyr84, respec-
tively, while the side-chain atoms of Ser25, Ser27, Asp29
and His31 make hydrogen bonds to the side-chain atoms of
Gln177, Glu241, His243 and Glu241, respectively.
3.3. PEG binding to YLR301w
After all the protein atoms had been placed, extra electron
density still remained at the centre of the barrel in both
molecules A and B, as shown in Fig. 4(a). The electron density
was clearly separated from the protein atoms; it was
surrounded by the side-chain atoms of
mostly hydrophobic residues, i.e. Leu5,
Phe10, Trp52, Phe54, Val71, Phe75,
Phe77, Val95, Trp99, Val107, Val113 and
Phe125, and is therefore highly hydro-
phobic in nature (see Fig. 4a). Since the
crystallization buffer included PEG
MME 550 we modelled this molecule,
and two molecules of PEG MME 550
density as shown in Fig. 4(a). These
molecules make hydrophobic interac-
tions with the side-chain atoms of
Phe10, Tyr32, Trp52, Phe54, Phe75,
Phe77, Trp99 and Phe125. In addition,
hydrogen bonds are formed to residues
Ser24, Arg36, Glu109 and Glu127
through three water molecules. An
additional PEG molecule is located
between ?7 and the ?3–?4 loop in the
domain interface. In this case the PEG
Phe38, Pro49, Phe50 and Leu208. The
same interactions are observed in both
molecules of YLR301w.
It is interesting to note that the
aforementioned hydrophobic residues
lining the pocket of the full barrel are
for theobserved extra
Kim et al.
Acta Cryst. (2012). D68, 531–540
Internal cavity of YLR301w. (a) Bound PEG MME 550 at the centre of the ?-barrel. There are two PEG MME 550 molecules bound inside the ?-barrel.
The electron-density map (2Fo? Fc) for the PEG molecules is contoured at the 1? level and the residues involved in PEG binding are included. The
right-hand panel shows a side view. (b) Hydrophobicity of YLR301w. The molecular surface is coloured according to the hydrophobicity of the side
chain, with yellow representing hydrophobic residues and blue representing residues with polar side chains.
Conservation of YLR301w. Residues are colour-coded based on sequence conservation (see Fig. 2)
going from maroon to blue to white as the degree of conservation decreases. The conservation of (a)
the N-terminal ?-barrel domain and (b) the C-terminal half-barrel domain are shown as surface
models. The two domains are shown separately for clarity and the ribbon shows the relative
orientation of the molecule.
highly conserved, as indicated by blue triangles in Fig. 2. Both
sides of the N-terminal barrel are open to the solvent, i.e.
a narrow channel runs through the barrel. It is somewhat
narrower in the middle of the barrel and widens at both ends.
The entry to the hydrophobic cavity is defined by four loops:
Val12–Thr22, Phe38–Pro49, Asn76–Glu94 and Tyr114–Ser122.
This can be seen even more clearly in Fig. 4(b). The volume
of the channel calculated using the Pocket-Finder algorithm
(Burgoyne & Jackson, 2006) is about 910 A˚3; this is about 7%
of that of the entire protein. The change in the accessible
surface area owing to PEG MME 550 binding is about 435 A˚2.
The same was observed for the second YLR301w molecule
in the asymmetric unit; the corresponding values are 670 and
430 A˚2, respectively. Based on this, it is tempting to suggest
that YLR301w may bind other hydrophobic compounds that
are similar to PEG MME 550 and that YLR301w and its
homologues may function as carriers of some sort.
3.4. Comparison with other structures
In order to determine whether there are any structurally
homologous proteins despite the lack of sequence homology,
we searched the PDB using the DALI server (Holm &
Rosenstro ¨m, 2010). Initial attempts using either the monomer
or the dimer of YLR301w all failed, showing no hits. However,
when the N-terminal ?-barrel alone was used as a search
model hits were obtained. The hits with the highest Z scores
were the C-terminal haem-binding domain of human THAP
domain-containing protein 4 (cTHAP4; PDB entry 3ia8;
Bianchetti et al., 2011), cellular retinoic acid-binding protein
2 (PDB entry 3d95; Vaezeslami et al., 2008), hypothetical
protein MTUBF_01000852 (PDB entry 2fwv; Shepard et al.,
2007) and human brain fatty-acid-binding protein (B-FABP;
PDB entry 1fdq; Balendiran et al., 2000). There were several
additional hits as possible structural homologues, but they
were either hypothetical proteins or functionally annotated as
fatty acid-binding proteins. They all had a Z score of about 8,
with an r.m.s.d. ranging from 3.2 to 3.4 A˚. However, a search
using only the C-terminal half-barrel gave no hits. Therefore,
the N-terminal domain of YLR301w has a ?-barrel fold that
is similar to those of fatty acid-binding proteins and the half-
barrel structure in YLR301w is rather unique, suggesting that
the overall structure of YLR301w as a whole is quite unique
Similar to YLR301w, these fatty acid-binding proteins bind
ligands or chromophores inside the barrel. However, unlike
YLR301w they have a cavity rather than a channel, as shown
in Fig. 5(a). In the case of B-FABP the internal cavity is
estimated to be about 960 A˚3in volume. The structure of the
complex of B-FABP with oleic acid showed that the hydro-
carbon tail of oleic acid assumes a ‘U-shaped’ conformation,
while the hydrocarbon tail in the complex with docosa-
hexaenoic acid adopts a helical conformation. The binding of
these ligands has been reported to have a Kdin the range 28–
53 nM (Balendiran et al., 2000).
THAP4 belongs to a novel protein family termed thanatos-
associated proteins, which have a conserved N-terminal C2CH
zinc-finger DNA-binding motif. They are found in the
proteomes of all species except plants, yeast and bacteria; 12
human THAP domain-containing proteins have been identi-
fied. Although the exact function of the THAP proteins is not
well defined, they have been reported to play a role in many
cellular functions and have been implicated in a number of
human disease states, including heart disease and neurological
defects as well as cancers (Bianchetti et al., 2011). It is inter-
esting to note that THAP has also been found to be up-
regulated in response to heat shock (Gau et al., 2008). The
C-terminal domain of THAP4, which is evolutionarily
conserved, has a haem moiety which is located in the hydro-
phobic cavity; therefore, the structure is similar to that of
nitrobindin (r.m.s.d. of 0.68 A˚, Z score of 32.8; Bianchetti et al.,
2010), which is involved in nitric oxide transport. In this case,
the internal cavity volume excluding the haem is estimated to
be ?830 A˚3.
Acta Cryst. (2012). D68, 531–540Kim et al.
Structural comparison of the N-terminal ?-barrel. (a) The N-terminal ?-barrel of YLR301w from S. cerevisiae, fatty acid-binding protein from human
brain (PDB entry 1fdq; Balendiran et al., 2000) and the C-terminal domain of human THAP domain-containing protein 4 (PDB entry 3ia8; Bianchetti et
al., 2011) are shown in blue, green and magenta, respectively. The yellow sphere represents the channel or the cavity of the barrel calculated using the
Pocket-Finder algorithm. (b) Representation of structural comparisons of YLR301w, B-FABP and cTHAP4. The three ?-barrel structures are shown
superimposed and coloured as in (a). B-FABP and cTHAP show r.m.s.d.s of 2.3 A˚over 82 C?atoms and 2.8 A˚over 105 C?atoms, respectively.
Kim et al.
Acta Cryst. (2012). D68, 531–540
Molecular arrangement of the YLR301w dimer. (a) Dimeric arrangement. The two molecules in the asymmetric unit are related by a noncrystallographic
twofold axis; the overall arrangement of the dimer is a butterfly shape with dimensions of 47 ? 57 ? 81 A˚. (b) Dimer-interface residues of YLR301w.
Surface representation ofYLR301w; residues involved in thedimer interaction in molecule A are coloured and labelled. The ?-barrel domain iscoloured
blue and the linker-loop domain is coloured green. The right panel shows the electrostatic potential of the surface area of YLR301w (blue, positively
charged area; red, negatively charged area). The residues involved in the dimer interaction in molecule B are shown as stick models and the electrostatic
surface model was calculated by the program PyMOL. (c) A close-up view showing the residues involved in hydrogen bonds in the dimer interface.
These residues are shown as stick representations. (d) Analytical size-exclusion chromatography of YLR301w. Gel filtration was performed as described
in x2. Chromatographic absorbance traces at 280 nm and elution positions are shown for molecular standards and YLR301w in black and red,
respectively. The standard proteins used were bovine serum albumin (66 kDa), carbonic anhydrase (29 kDa) and cytochrome c (12 kDa).
Although all three proteins form a compact ten-stranded
?-barrel, there are several differences. Firstly, both ends of the
?-barrel are accessible in the case of YLR301w, while only
one end is accessible in the other two, as shown in Fig. 5(b).
Secondly, in the case of B-FABP the bound oleic acid is
located towards the rim of the ?-barrel, just beneath the
?-helical lid, and the haem moiety in cTHAP4 is also bound
towards the rim, while the two PEG molecules bound in
YLR301w are buried deeper in the ?-barrel. Thirdly, the
amino-acid composition of the ?-barrel is significantly more
hydrophobic in the case of YLR301w. There are seven Trp,
14 Phe and seven Tyr residues in the case of YLR301w, while
there are two Trp, eight Phe and two Tyr residues in B-FABP
and two Trp, eight Phe and three Tyr residues in cTHAP4.
Despite this, both ends of the ?-barrel in YLR301w display
3.5. Dimeric arrangement of YLR301w
As mentioned earlier, there are two YLR301w molecules
in the asymmetric unit and the two molecules are related by
a rotation of ?100?about the noncrystallographic axis. The
overall arrangement of the dimer is as a butterfly shape with
dimensions of 47 ? 57 ? 81 A˚, as shown in Fig. 6(a). The
interface is relatively flat, but the interaction between the two
change in accessible surface area, ?ASA, of about 1340 A˚2
(for molecule A; 1355 A˚2for molecule B). These values
correspond to about 11% of the surface area of a monomer.
The interactions of the residues from the two N-terminal
domain barrels and the linker loops are shown in Fig. 6(b).
They are coloured blue and green, respectively. As shown in
the middle panel, the interactions involve both hydrophobic
and hydrophilic residues, with the hydrophobic residues at the
centre and the polar residues on the peripheral surface. In
addition to the two salt bridges Lys40–Asp103 and Arg136–
Asp138, the backbone atoms of Pro14, Ser104, Leu139 and
Ile141 make hydrogen bonds to Arg126, Glu18, Ile141 and
Leu139 (see Fig. 6c). Further contacts are made by residues
Leu8, Leu9, Pro16, Trp102, Leu139 and Pro144 from each
monomer. Ser104, Glu18 and Trp102 are conserved (Fig. 2).
The methionine that was introduced at position 128 for
phasing is located at the dimer interface. The side chain is
harboured between Leu8 and Trp102. It is somewhat acces-
sible, in contrast to Leu35, which is located at the interface and
tightly packed by hydrophobic residues.
It is worth noting that B-FABP and cTHAP4 are reported
to form a dimer in their crystal structures. The orientation of
the dimer is also somewhat similar to that of YLR301w; the
two barrels in B-FABP and cTHAP4 are related by 150?and
170?, respectively. However, the dimeric interfaces or ?ASA
in B-FABP and cTHAP4 are 470 and 1310 A˚2, respectively,
corresponding to about 7 and 15% of the surface area of each
monomer. Interestingly, cTHAP is found as a dimer both in
the crystal and in the solution state (Bianchetti et al., 2011),
while B-FABP is only found as a dimer in the crystal
(Balendiran et al., 2000); this can be understood from the
?ASA values of the two dimers. The ?ASA of 1340 A˚2for
YLR301w is about the same as that of cTHAP. However, since
this value includes the interaction provided by the residues
from the linker loop (460 A˚2), it was of interest to determine
whether YLR301w is a dimer in solution. In order to check
this, we carried out DLS and gel-filtration analysis. As shown
in Fig. 6(d), both DLS measurements (data not shown) and
analytical gel-filtration chromatography suggested amolecular
weight of 56–60 kDa, suggesting that YLR301w exists as a
dimer in solution. Therefore, the dimeric YLR301w observed
in the crystal structure may be biologically relevant and
required for proper functioning of the YLR301w protein.
3.6. Interaction with Sec72
Sec72 has been reported to contribute to the selective
recognition of signal peptides by the secretory polypeptide
translocation complex (Fang & Green, 1994; Rapoport, 2007).
Recently reported cryo-electron microscopic analysis of the
Sec62–Sec63 complex showed that Sec72 and Sec71 form a
stable complex with Sec63, although the exact mechanism
requires further characterization (Harada et al., 2011). Yeast
two-hybrid library screening using Sec72 as bait previously
gave YLR301w as an interaction partner (Willer et al., 2003).
To confirm this, we analyzed the interaction between the two
by measuring the amount of YLR301w bound to immobilized
Sec72 and measuring the binding kinetics of GST-YLR301w to
His-Sec72 protein. Application of GST-YLR301w to surface-
immobilized His-Sec72 yielded a saturated binding curve, as
shown in Fig. 7. The dissociation constant (Kd) between the
two proteins is estimated to be 6.2 mM. It is worth mentioning
that YLR301w only appears to be stable, and perhaps func-
tional, in its full form, since all attempts to obtain soluble
forms of the individual domains separately failed (data not
shown). Nonetheless, the biological significance of the inter-
action between YLR301w and Sec72 requires further inves-
tigation. Interestingly, recent investigation of yeast protein
Acta Cryst. (2012). D68, 531–540Kim et al.
Interaction between GST-YLR301w and His-Sec72. The amount of GST-
YLR301w bound to a His-Sec72-coated plate was measured using a
modified ELISA method as described in x2.
kinases using protein microarrays revealed that YLR301w Download full-text
was one of the interacting proteins for Hrr25 along with Crz1,
Pcl10 and Tmt1; based on this, YLR301w was proposed to be
named Hri1 (Hrr25-interacting protein 1). Hrr25 is a member
of the casein kinases that is required for normal cellular
growth, nuclear segregation, DNA repair and meiosis (Fasolo
et al., 2011).
The crystal structure of S. cerevisiae YLR301w has been
determined. The structure of YLR301w shows a unique
?-structure composed of a ten-stranded ?-barrel with two
short ?-helices at the N-terminus connected by a 23-residue
linker to a seven-stranded ?-sandwich with two short helices at
the C-terminus. The N-terminal barrel with two PEG mole-
cules bound has hydrophobic residues lining the channel and
highly conserved residues leading to the channel. It appears
that YLR301w represents a new class of proteins that have yet
to be functionally characterized.
We thank the beamline staff for assistance in data collec-
tion. This work was supported financially by the Functional
Proteomics Center, the 21C Frontier Research and Develop-
ment Program and the Global Research Laboratory Program
of the Korea Ministry of Science and Technology and an
institutional grant from Korea Institute of Science and Tech-
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