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Solution structure of the orphan PABC domain from

Saccharomyces cerevisiae poly(A) binding protein

Guennadi Kozlov1, Nadeem Siddiqui1, Stephane Coillet-Matillon1, Jean-François Trempe1, Irena

Ekiel2,3, Tara Sprules1, Kalle Gehring1,4

1Departments of Biochemistry and 4Chemistry, McGill University, 3655 Promenade Sir William

Osler, Montreal, QC H3G 1Y6 Canada

2Biotechnology Research Institute, National Research Council of Canada, 6100 Royalmount

Ave., Montreal, QC H4P 2R2 Canada and 3Department of Chemistry and Biochemistry,

Concordia University, Montreal, QC Canada

Running title: PABC domain from yeast

Address correspondence to: Kalle Gehring, Dept. of Biochemistry, McGill University, 3655

Promenade Sir William Osler, Montreal, QC H3G 1Y6, Canada. Fax (514) 398-7384. E-mail:

kalle@bri.nrc.ca

Copyright 2002 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on April 8, 2002 as Manuscript M201230200 by guest on February 15, 2018

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Summary

We have determined the solution structure of the PABC domain from Saccharomyces cerevisiae

Pab1p and mapped its peptide binding site. PABC domains are peptide binding domains found in

poly(A) binding proteins (PABP) and a subset of HECT-family E3 ubiquitin ligases (also known

as hyperplastic disks proteins, HYD). In mammals, the PABC domain of PABP functions to

recruit several different translation factors to the mRNA poly(A) tail. PABC domains are highly

conserved with high specificity for peptide sequences of roughly 12 residues with conserved

alanine, phenylalanine and proline residues at positions 7, 10 and 12. Compared to human PABP,

the yeast PABC domain is missing the first alpha helix, contains two extra amino acids between

helices 2 and 3, and has a strongly bent C-terminal helix. These give rise to unique peptide

binding specificity wherein yeast PABC binds peptides from Paip2 and RF3 but not Paip1.

Mapping of the peptide binding site reveals that the bend in the C-terminal helix disrupts binding

interactions with the N-terminus of peptide ligands and leads to greatly reduced binding affinity

for the peptides tested. No high affinity or natural binding partners from Saccharomyces

cerevisiae could be identified by sequence analysis of known PABC ligands. Comparison of the

three known PABC structures shows that the features responsible for peptide binding are highly

conserved and responsible for the distinct but overlapping binding specificities.

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Introduction

The yeast poly(A) binding protein (Pab1p or yPABP) is an essential protein that functions

as a scaffold to organize the mRNA ribonucleic acid protein (RNP) complex around the mRNA

poly(A) tail. Pab1p contains 570 amino acids arranged as four N-terminal RNA-recognition

motifs (RRM) and a C-terminal PABC domain of ~70 amino acids. The two parts are separated

by a largely unstructured region of ~100 amino acids. The N-terminal RRMs bind the mRNA

poly(A) tail and interact with the eIF4F complex at the mRNA 5' cap. This Pab1p-eIF4F

interaction is important for the circularization of the mRNA in actively translating complexes (1).

At the C-terminus, the PABC domain acts as a peptide/protein binding domain, recruiting various

translation or mRNA processing factors to the mRNA RNP complex. In yeast, Pab1 is an

essential gene whose deletion leads to inhibition of translation initiation, poly(A) shortening and

delay in the onset of mRNA decay (2-4), but those effects can be suppressed by mutations that

alter the 60S subunit of the ribosome, as well as those that inhibit mRNA decay (2,5,6).

In metazoans, several protein binding partners of PABC have been identified. These

include the PABP-interacting proteins Paip1 and Paip2, hnRNPE (or αCP1 and 2) as well as

eRF3/GSPT (7-9). A number of potential interacting agents have also been identified in plants

and yeast: Pab1p-binding protein (Pbp1p), eIF4B, Rna15p and a viral RNA-dependent RNA

polymerase (4, 10, 11). We recently showed that a large number of potential binding partners can

be identified by sequence analysis based on the presence of a consensus PABC recognition site

(12). Finally, it is notable that in addition to poly(A) binding proteins, PABC domains also occur

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in a subclass of ubiquitin E3 protein ligases that contain a HECT (homologous to E6-AP C-

terminus) domain. The function of the PABC domain in these ubiquitin ligases is unknown.

The structures of PABC domains from human PABP (hPABP) and HYD (a human

ubiquitin ligase) have recently been determined by NMR spectroscopy and X-ray crystallography

(12, 13). The two structures are largely similar and consist of 75 or 60 amino acid residues

arranged as bundle of five or four alpha helices. Sequence conservation is highest in helices 2, 3

and 5 which correspond to the peptide-binding site determined by NMR spectroscopy (12).

Here, we report the structure of the PABC domain from the yeast poly(A) binding protein,

Pab1p. The yeast sequence shows 40% and 57% identity with the domains from hPABP and

HYD (themselves 52% identical). Together the three proteins span much of the sequence

variation in PABC domains. The yeast structure shows several distinct features which result in

unique specificity and affinity of peptide binding.

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Experimental Procedures

Sequence comparison of PABC domains

Forty sequences of PABC domains were obtained from a Ψ-BLAST search (14) of the

NCBI non-redundant database with the yPABC sequence (gi417441, residues 490-563) as query.

These unique sequences were analyzed by ClustalW to generate an alignment and Neighbor

Joining (NJ) tree (15). To simplify comparisons between different PABC domains, we have

adopted a numbering scheme which is anchored on the KITGMLLE motif common to all PABC

domains. The PABC domain was defined to begin 34 residues prior to this motif (36 residues in

the case of yPABC). The two additional residues in the loop between alpha helices 2 and 3 that

are unique to the Saccharomyces and Caenorhabditis proteins were numbered 30a and 30b.

yPABC expression and purification

The C-terminal domain of Pab1p, residues 491 to 577, was amplified by PCR using

genomic DNA from S. cerevisiae (gift from Malcolm Whiteway) with primers PAByC-F 5'-

C C C A C A A G G T G G A T C C C C A A G A A A T G C - 3 ' a n d P A B y C - R 5 ' -

GTGATTACATGAATTCTTAAGCTTGCTCAG-3'. The PCR product was cloned into the

BamHI and EcoRI restriction sites of the vector pGEX-6P-1 (Amersham Pharmacia Biotech).

pPYC was transformed into E. coli expression host BL21 Gold (DE3) (Stratagene) and grown at

37°C in Luria Broth or M9 media supplemented with 100µg/ml Ampicillin. Expression of the

GST-yPABC fusion protein was induced at 30° C by 1 mM isopropylthio-β-galactoside (IPTG)

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for 3 hours and purified by affinity chromatography using a Glutathione sepharose 4B column

(Amersham Pharmacia Biotech). The N-terminal GST-tag was cleaved from yPABC by

treatment for 20 hours at 4° C with PreScission Protease (Amersham Pharmacia Biotech) on the

column at 2.5 units per mg fusion protein to yield a 92 residue protein fragment, consisting of the

87 C-terminal residues of Pab1p plus a five-residue (Gly-Pro-Leu-Gly-Ser) N-terminal extension.

Glutathione sepharose was used to remove the PreScission Protease. The sequence composition

of purified yPABC was confirmed by mass spectrometry. For NMR analysis, the protein was

exchanged into NMR buffer (50 mM K·HPO4 , 100 mM NaCl, 1 mM NaN3 , pH 6.3). The final

yield of purified yPABC domain was 6 mg per liter of culture (M9) media including 6 g of

Na2HPO4, 3 g of KH2PO4, 0.5 g of NaCl, 0.5 g of 15N-NH4Cl (Isotec, Inc) and 2 g of 13C6-

glucose (Cambridge Isotope Laboratory).

Peptide preparation and purification

Unlabeled peptides were synthesized by Fmoc solid-phase peptide synthesis and purified

by reverse phase chromatography on a Vydac C18 column (Hesperia, CA). The composition and

purity of the peptides was verified by ion-spray quadrupole mass spectroscopy.

The C-terminal domain of human Paip2, residues 106-127, was amplified by PCR using a

plasmid template (gift of Nahum Sonenberg) with primers P2C-F 5'-

T C T T C T C T G G A A G G A T C C G T G G T C A A G A G C - 3 ' a n d P 2 C - R 5 ' -

CAGATGCACGACGAATTCTCAAATATTTCC-3'. The PCR product was cloned into the

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BamHI and EcoRI restriction sites of the vector pGEX-6P-1 (Amersham Pharmacia Biotech) to

make plasmid pP2C. An 15N labeled peptide of this construct was made by expressing pP2C in E.

coli expression host BL21 Gold (DE3) grown in M9 media containing 15NH4Cl and purifying the

fusion protein as described previously for yPABC. Digestion with PreScission Protease, using the

same conditions as described above, yielded a 27 residue peptide consisting of the 22 C-terminal

residues of human Paip2 plus a 5 residue (Gly-Pro-Leu-Gly-Ser) N-terminal extension. The

peptide was desalted using C18 reverse phase chromatography and then lyophilized. The

composition and purity of the peptides was verified by ion-spray quadrupole mass spectroscopy.

Peptide titrations were carried by adding either labeled or unlabeled Paip2 peptide into

unlabeled or labeled yPABC respectively. Titrations were monitored by 15N-1H heteronuclear

single quantum correlation (HSQC) spectra of the labeled species (either peptide or protein) and

were brought to a final protein concentration of 1 mM.

NMR spectroscopy

NMR resonance assignments of yPABC were determined using standard triple-resonance

techniques on a 13C,15N-labeled sample (16) on a Bruker DRX500 NMR spectrometer. All NMR

experiments were recorded at 303K. Main-chain Cα, N, HN and side-chain Cβ resonances were

assigned using HNCACB and CBCA(CO)NH experiments (17, 18). Hα resonance assignments

and 3JHN-Hα coupling constants were obtained from an HNHA experiment (19). 15N-1H dipolar

couplings were measured with an IPAP-HSQC experiment on an isotropic sample (without

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phage) and on a sample containing 18 mg/ml Pf1 phage (20, 21). Other backbone and side-chain

signal assignments were obtained from three-dimensional heteronuclear NOESY and TOCSY

experiments at 500 and 750 MHz and homonuclear two-dimensional NOESY experiment at 500

MHz. NOESY constraints for the structure determination were obtained from 15N-edited NOESY

and 13C-edited NOESY 3D experiments and 2D homonuclear NOESY. The 15N-NOESY

spectrum was recorded on a Varian Unity Plus 750 MHz spectrometer at the Pacific Northwest

National Laboratory. Assignments of amide resonances in the yPABP-Paip2 complex were based

on an 15N-1H edited NOESY spectrum obtained at 500 MHz. The 15N-1H heteronuclear NOEs

were measured at 500 MHz on an 15N-labeled Paip2 (106-127) complexed with unlabeled

yPABC (22). NMR spectra were processed with GIFA (23) and XWINNMR software version 2.5

(Bruker Biospin) and analyzed with XEASY (24).

Structure calculations

For the structure determination, a set of 971 NOEs were collected from homonuclear and

15N-edited NOESY spectra of Pab1p (491-577) acquired at 500 and 750 MHz, respectively.

Following determination of the protein fold using manual NOE assignments (25), automated

NOE assignments were made using ARIA (26) and the structure refined using standard protocols

in CNS v.0.9 (27). PROCHECK-NMR was used to check protein stereochemical geometry and

generate the Ramachandran plot of Fig. 2 (28). The coordinates have been deposited in the RCSB

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under PDB accession code 1IFW and the NMR assignments under BMRB accession number

5053.

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Results

The C-terminal fragment, yPABC, of Pab1p (residues 491-577; Fig. 1) from

Saccharomyces cerevisiae was prepared as an isotopically labeled, recombinant protein fragment

for structural studies by NMR spectroscopy at 500 and 750 MHz. The yPABC domain gave

excellent quality spectra and a large number of structural constraints were determined (Table 1).

The secondary structure and NOEs were very similar to human PABP with the notable absence ot

the first alpha helix (Fig. 2a). In addition to NOE and dihedral angle constraints, a small set of 48

residual dipolar couplings (RDC) were measured on a sample of 15N-labeled yPABC in Pf1

phage (Fig. 2b). These RDCs dramatically improved the precision of the structures, particularly

in the region of helix 4. The backbone RMSD in the absence of RDCs was almost twice (0.61 Å)

the final value for the 30 accepted structures (0.34 Å) calculated with RDCs. Inclusion of the

RDCs also improved the Ramachandran plot statistics (Fig. 2).

The folded domain of yPABC includes approximately 65 residues (502 to 567 of Pab1p)

as a bundle of four helices (Fig. 3). The overall fold is similar to the recently determined PABC

structures from human PABP (12) and HYD (13). The four helices (numbered 2 to 5) form a

compact structure with a well-packed hydrophobic core consisting of residues L17, L21, V25,

A32, A33, I36, I40, L43, V48, F49, L51, L52, F58, Y62, A65, A68, and Y69. yPABC contains a

unique two amino acid insertion which is accommodated in the loop between helices 2 and 3

(Fig. 1). The most unusual feature of yPABC is the strong bend in the last alpha helix. This helix

shows a roughly 50° bend centered around Tyr62 and terminates antiparallel with helix 3. Helix 5

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contributes three aromatic residues (F58, Y62, Y69) to the hydrophobic core. The bend can be

detected in the RDC data; parallel RDC experiments on hPABP confirm that this is a unique

feature of the yeast domain (G.K., unpublished results). Surprisingly, all the φ/ψ angles in helix 5

fall in the most favored region of the Ramachandran plot for alpha helices (Figure 2d).

As is the case for the HYD PABC domain, the yPABC domain is missing the first helix.

Instead the fourth helix in yPABC is raised and replaces a number of the contacts between helix 1

and 2 in hPABP (Fig. 3d-f). The first N-terminal helix of PABC from hPABP is itself dispensable

and a shortened fragment (PABP residues 554-636) gave a 15N-1H correlation spectrum similar to

that of the full length domain (data not shown). Overall, the PABC domain from yeast PABP

appears to be more closely related to the PABC domain from human HYD than human PABP.

This is a consequence of the greater sequence relatedness of yPABC and HYD but is more

clearly evident in the three dimensional structures. A pairwise overlay of the most conserved Cα

residues in the three proteins gives over twice the RMSD for hPABP compared to HYD (Fig 3c).

Peptide binding studies were used to determine the specificity and position of the peptide-

binding site on yPABC. The initial choice of peptides was based on the consensus binding

sequence determined for the human PABC (12). The list of peptides studied is shown in Table 2.

Four peptides were found to bind to yPABC. The 22 residue C-terminal peptide from Paip2

demonstrated one of the highest affinities. Chemical shift mapping was used to identify yPABC

residues that participate in peptide binding (Fig. 4). Residues with the largest chemical shift

changes (((∆1H shift)2 + (∆15N shift x 0.2)2 )1/2) on binding of Paip2 were K35 (0.6 ppm), Y22

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(0.47), E19 (0.45), M39 (0.39), Q20 (0.39), and I40 (0.37). Only small chemical shift changes

were observed in helix 5 (Fig. 3) in contrast to previous results with hPABP (12). Instead almost

all the chemical shift changes occur in helices 2 and 3 around the hydrophobic binding pocket

which is the presumed binding pocket for F118 of Paip2. Additional changes occur at the N-

terminus of helix 2 and likely reflect interactions with the C-terminal portion of the peptide (see

below).

Yeast does not have a protein homologous to Paip2. Using the previously published

consensus for human PABC (Table 3), we searched the Saccharomyces cerevisiae genome for

related sequences which might bind to yPABC. Among proteins known to interact with Pab1p,

we identified residues 234-250 from Pan1p (29, 30), two regions from Pbp1p (residues 308-327

and 376-399) (11) and a peptide derived from the N-terminus of RF3 (residues 106-122) (Table

2). None of these peptides bound to yPABC as determined by the absence of chemical shift

changes in 15N-1H correlation spectra. Similar negative results were obtained for a peptide

derived from human Paip1. This was unexpected since Paip1 has been shown to bind to HYD

(13). Of nine peptides tested, the four that bound were the Paip2 peptide, a peptide from the N-

terminus of human RF3, a peptide similar to Pichia pinus RF3, and a peptide from the

Drosophila shuttle craft protein (Table 2).

From titration experiments, we measured the dissociation constant of the Paip2

peptide/yPABC complex to be approximately 1 mM. Based on the similar amounts of line

broadening in spectra with other peptides, we estimate that the affinities of all the ligands tested

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are in the millimolar range. This is three to four orders of magnitude higher than measured for

Paip2/hPABP (7) and likely reflects the fact that the peptides do not interact with the C-terminal

helix of yPABC.

We also monitored complex formation from the peptide side by cloning and expressing an

15N-labeled fragment of Paip2 (residues 106-127). The 15N-1H correlation spectrum of the

unbound peptide showed the small dispersion of signals characteristic of an unfolded peptide

(Figure 4c). Addition of unlabeled yPABC to the 15N-labeled peptide caused changes in roughly

half of the signals. The complex was in intermediate exchange so many of the NMR peaks

broadened or disappeared upon the addition of yPABC.

We identified peptide residues involved in PABC binding by an 15N-1H heteronuclear

NOE (hNOE) experiment (Figure 4d). The hNOE is a measure of the reorientation rate of the

amide nitrogen-hydrogen internuclear vectors and thus of the mobility of the peptide residues. At

500 MHz, the hNOE varies between -3.6 and 0.82 for mobile and immobile residues (22). For

Paip2 residues L103 to L111 and K123 to I127, the hNOE was negative, which indicates a lack

of binding. Residues N112 to V122 gave small or zero hNOEs and identified these residues as

binding to yPABC. The absence of small hNOEs is a reflection of the weak binding of Paip2 to

yPABC. For several residues in the middle of the binding region, no hNOE signal could be

detected. This was a consequence of exchange broadening and constitutes independent evidence

for PABC binding by these residues. Residue F118 of Paip2 showed the largest hNOE which is

consistent with its key role in complex formation. .

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Previous studies identified a 12-residue consensus PABC-site between Ser109 and Pro120

of Paip2 (7). Our hNOE results suggest that this motif is shifted toward the C-terminus and

includes Gly121 and Val122. This agrees with on-going studies with PABC from human PABP

which suggest that Paip2 binds hPABP as a series of beta-turns (G.K., unpublished results). For

yPABC, small negative hNOE values were observed for residues S109 through L111. These

negative hNOEs likely reflect the differences in the structure and position of the last alpha helix

in yPABC and hPABP. Residues S109 through L111 do bind hPABP but via helix 5 (G.K.,

unpublished). These results suggest that the major specificity differences between PABC

domains in human and yeast PABP occur in the N-terminal residues of the peptide ligands due to

the altered structure of the terminal helices.

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Discussion

PABCs are highly conserved eukaryotic protein domains of 64-72 amino acids in length

and sequence identities of 40% in interspecies comparisons. Three subfamilies can be

distinguished in a phylogenetic tree of PABC sequences (Fig. 1b). The first group encompasses

PABCs of animal origin, with overall pairwise identities of 80% or more (62% for C. elegans).

Tissue-specific (testis) or inducible (activated platelets or T cells) isoforms have been described

in humans (31-33) and these sequences show a slightly lower level of identity when compared to

other forms. This is likely the result of subcellular or tissue specialization, with conserved but

distinct specificity among the different PABC domains within one organism.

A second branch groups a family of more divergent PABC domains of vegetal origin, with

pairwise sequence identities of about 70%. PABC from the parasite Trypanozoma brucei is

branched with its homologues in plants which is supported by recent work that has hinted at a

weak phylogenetic link between the euglenozoan lineage (to which trypanozomids belong) and

plants (34). The third, most divergent group contains the PABC domains of the ubiquitin ligases

of the hyperplastic discs (HYD) family, as well as Saccharomyces cerevisiae and

Schizosaccharomyes pombe PABPs. The relationship between HYD and PABP proteins has not

yet been established but it hints at a role for ubiquitination in the regulation of protein synthesis.

Structurally, the absence of the first alpha helix in yPABC and HYD seems to be a feature of the

third group of PABC domains. Secondary structure predictions using the Multivariate Linear

Regression Combination (MLRC) software (35), as well as sequence conservation, indicate the

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likely presence of helix 1 in the wheat PABC domain and most other plant PABCs, including

trypanosomids, while the fungal PABC from Aspergillus nidulans was predicted to harbor only

four helices.

Limited data is available on the binding specificity of PABC domains. PABC from hPABP

is known to bind a number of different proteins and peptides which were used to derive a

consensus binding sequence or PABC-site (12). The PABC domain of HYD has been shown to

bind Paip1 (13). Here, we show that yPABC binds peptides from Paip2 and human RF3 but not

Paip1. The failure of yPABC to bind Paip1 is surprising given the close similarity of Paip1 and

Paip2 around the critical Phe-X-Pro sequence at the C-terminus of the consensus motif (Table 3).

The phenyalanine is highly conserved and essential for binding (G.K., unpublished data). The

weak affinities of the tested peptides suggest that a novel specificity exists for yPABC.

Among the proteins/peptides which bind to yPABC (Table 2), only RF3 occurs in yeast. In

Saccharomyces cerevisiae, RF3 was first identified as the stop codon suppressor mutations

Sup35 and Sup2 (36, 37). More recently, this protein has received considerable attention as it

mediates non-Mendelian inheritance through a prion-like mechanism. The yeast [PSI+] prion

phenotype results from self-propagating aggregation of RF3 through its N-terminal domain (38).

This behavior is thought to be related to the large number of glutamine residues at the N-

terminus.

Comparison of RF3 sequences from 11 different yeast species allowed us to identify

potential PABC-binding sequences in all but 4 species: Saccharomyces cerevisiae,

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Zygosaccharomyces rouxii, Saccharomycodes ludwigii, and Kluyveromyces lactis (Table 3). As

is the case for human RF3, several of the RF3 proteins contain two or three potential PABC-

binding sites. Yarrowia lipolytica RF3 contains three overlapping, putative PABC-sites. It is

unknown if all these sites are functional or if cooperativity exists between them. The absence of

an evident PABC-site in Saccharomyces cerevisiae RF3 suggests that this interaction may be

absent in baker's yeast and related strains. The four species missing PABC-sites are most closely

related to each other based on phylogenetic grouping of yeast using 23S RNA sequences which

suggests that the site was lost relatively recently (38, 39).

Mangus, Amrani and Jacobson used the C-terminal portion of Pab1p as bait in a two-hybrid

screen for interacting yeast proteins (11). Surprisingly, none of the proteins identified contain a

consensus PABC-site. Instead, mutagenesis studies indicate that the region preceding yPABC

(Pab1p residues 406-494) is required for the binding of Pbp1p (11). This preceding region is not

a structured part of the C-terminal domain of yPABP (12, 13). Although Pbp1p does not bind

PABC, it is related to ataxin-2, the human protein responsible for type 2 spinocerebellar ataxia

(SCA2), which does contain a PABC-site (11, 12). Perhaps co-incidentally, the origin of SCA2 is

a polyglutamine expansion in ataxin-2 which leads to protein aggregation as for RF3.

In conclusion, the structure of the PABC domain from the yeast poly(A) binding protein

shows similarities to previous structures but contains a very different C-terminal helix (Fig. 3).

This gives rise to a distinct binding specificity for yPABC particularly toward the N-terminus of

the bound peptides (Fig. 4). A hydrophobic pocket between helices 3 and 5, which is unique to

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yPABC, could bind aromatic residues that occur in the N-terminal end of PABC-sites in fungal

RF3 proteins (Table 3) but no PABC-site was detected in RF3 from Saccharomyces cerevisiae.

Future work will be directed towards understanding the function of yPABC in Saccharomyces

cerevisiae and in the identification of physiological binding partners.

Acknowledgements

We thank Malcolm Whiteway for the gift of S. cerevisiae genomic DNA, Nahum

Sonenberg for the Paip2 plasmid, and David Mangus for helpful discussions. We acknowledge

the Pacific Northwest National Laboratory for access to the Environmental Molecular Sciences

Laboratory High Field Magnetic Resonance Facility. This study was supported by the Canadian

Institutes of Health Research grant 14219 to K.G. NRC publication no. 00000.

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References

1. Wells, S. E., Hillner, P. E., Vale, R. D., and Sachs, A. B. (1998) Mol. Cell 2, 135-1402. Sachs, A. B., and Davis, R. W. (1989) Cell 58, 857-8673. Sachs, A. (1990) Curr. Opin. Cell. Biol. 2, 1092-10984. Amrani, N., Minet, M., Le Gouar, M., Lacroute, F., and Wyers, F. (1997) Mol. Cell. Biol.

17, 3694-37015. Hatfield, L., Beelman, C. A., Stevens, A., and Parker, R. (1996) Mol. Cell. Biol. 16, 5830-5838

6. Wyers, F., Minet, M., Dufour, M. E., Vo, L. T., and Lacroute, F. (2000) Mol. Cell. Biol.

20, 3538-35497. Khaleghpour, K., Kahvejian, A., De Crescenzo, G., Roy, G., Svitkin, Y. V., Imataka, H.,O'Connor-McCourt, M., and Sonenberg, N. (2001) Mol. Cell. Biol. 21, 5200-52138. Hoshino, S., Hosoda, N., Araki, Y., Kobayashi, T., Uchida, N., Funakoshi, Y., andKatada, T. (1999) Biochemistry (Mosc) 64, 1367-13729. Craig, A. W., Haghighat, A., Yu, A. T., and Sonenberg, N. (1998) Nature 392, 520-52310. Le, H., Tanguay, R. L., Balasta, M. L., Wei, C. C., Browning, K. S., Metz, A. M., Goss,D. J., and Gallie, D. R. (1997) J. Biol. Chem. 272, 16247-1625511. Mangus, D. A., Amrani, N., and Jacobson, A. (1998) Mol. Cell. Biol. 18, 7383-739612. Kozlov, G., Trempe, J. F., Khaleghpour, K., Kahvejian, A., Ekiel, I., and Gehring, K.

(2001) Proc. Natl. Acad. Sci. U S A 98, 4409-441313. Deo, R. C., Sonenberg, N., and Burley, S. K. (2001) Proc. Natl. Acad. Sci. U S A 98,4414-441914. Altschul, S. F., Madden, T. L., Schäffer, A. A., Zhang, J., Zhang, Z., Miller, W., andLipman, D. J. (1997) Nucleic Acids Res. 25, 3389-340215. Thompson, J. D., Higgins, D. G., and Gibson, T. J. (1994) Nucleic Acids Res. 22, 4673-468016. Bax, A., and Grzesiek, S. (1993) Acc. Chem. Res. 26, 131-13817. Grzesiek, S., Dobeli, H., Gentz, R., Garotta, G., Labhardt, A. M., and Bax, A. (1992)Biochemistry 31, 8180-819018. Constantine, K. L., Goldfarb, V., Wittekind, M., Friedrichs, M. S., Anthony, J., Ng, S. C.,

and Mueller, L. (1993) J. Biomol. NMR 3, 41-5419. Kuboniwa, H., Grzesiek, S., Delaglio, F., and Bax, A. (1994) J. Biomol. NMR 4, 871-87820. Ottiger, M., Delaglio, F., and Bax, A. (1998) J. Magn. Reson. 131, 373-37821. Hansen, M. R., Mueller, L., and Pardi, A. (1998) Nat. Struct. Biol. 5, 1065-107422. Peng, J. W., and Wagner, G. (1994) Methods Enzymol. 239, 563-59623. Malliavin, T., Pons, J.-L., and Delsuc, M.-A. (1996) J. Biomol. NMR 8, 445-452

by guest on February 15, 2018http://w

ww

.jbc.org/D

ownloaded from

20

24. Bartels, C., Xia, T.-H., Billeter, M., Guntert, P., and Wüthrich, K. (1995) J. Biomol. NMR

5, 1-1025. Wüthrich, K. (1986) NMR of Proteins and Nucleic Acids, Wiley & Sons, New York26. Nilges, M., Macias, M. J., O'Donoghue, S. I., and Oschkinat, H. (1997) J. Mol. Biol. 269,408-42227. Brünger, A. T., Adams, P. D., Clore, G. M., Gros, P., Grosse-Kuntsleve, R. W., Jiang, J.-S., Kuszewski, J., Nilges, M., Pannu, N. S., and Read, R. J. (1998) Acta Crystallogr. D54, 905-92128. Laskowski, R. A., Rullmannn, J. A., MacArthur, M. W., Kaptein, R., and Thornton, J. M.(1996) J. Biomol. NMR 8, 477-48629. Boeck, R., Tarun, S., Jr., Rieger, M., Deardorff, J. A., Muller-Auer, S., and Sachs, A. B.

(1996) J. Biol. Chem. 271, 432-43830. Sachs, A. B., and Deardorff, J. A. (1992) Cell 70, 961-97331. Feral, C., Guellaen, G., and Pawlak, A. (2001) Nucleic Acids Res. 29, 1872-188332. Houng, A. K., Maggini, L., Clement, C. Y., and Reed, G. L. (1997) Eur. J. Biochem. 243,209-21833. Yang, H., Duckett, C. S., and Lindsten, T. (1995) Mol. Cell. Biol. 15, 6770-677634. Baldauf, S. L., Roger, A. J., Wenk-Siefert, I., and Doolittle, W. F. (2000) Science 290,972-97735. Guermeur, Y., Geourjon, C., Gallinari, P., and Deleage, G. (1999) Bioinformatics 15, 413-42136. Stansfield, I., Jones, K. M., Kushnirov, V. V., Dagkesamanskaya, A. R., Poznyakovski,

A. I., Paushkin, S. V., Nierras, C. R., Cox, B. S., Ter-Avanesyan, M. D., and Tuite, M. F. (1995)EMBO J. 14, 4365-437337. Zhouravleva, G., Frolova, L., Le Goff, X., Le Guellec, R., Inge-Vechtomov, S., Kisselev,L., and Philippe, M. (1995) EMBO J. 14, 4065-407238. Santoso, A., Chien, P., Osherovich, L. Z., and Weissman, J. S. (2000) Cell 100, 277-28839. Kushnirov, V. V., Ter-Avanesyan, M. D., Didichenko, S. A., Smirnov, V. N., Chernoff,Y. O., Derkach, I. L., Novikova, O. N., Inge-Vechtomov, S. G., Neistat, M. A., and Tolstorukov,II. (1990) Yeast 6, 461-47240. Page, R. D. M. (1996) Computer Applications in the Biosciences 12, 357-35841. Cornilescu, G., Marquardt, J. L., Ottiger, M., and Bax, A. (1998) J. Am. Chem. Soc. 120,6836-6837

42. Clore, M. G., and Garrett, D. S. (1999) J. Am. Chem. Soc. 121, 9008-9012

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Figure legends

Figure 1. Sequence conservation in PABC domains from poly(A) binding proteins (PABP) and

hyperplastic disks proteins (HYD). (a) PABC sequences from structures of human poly(A)

binding protein (hPABP) (12), human hyperplastic disks protein (hHYD) (13), and

Saccharomyces cerevisiae Pab1p (yPABP). Residue numbers in the intact protein are given along

with the proposed numbering scheme for residues within PABC. The domain from yeast contains

a two-residue insertion after residue 30. Above the sequence alignment, the positions of the four

alpha helices in yPABP are shown. hPABP contains an additional first helix (light gray) which is

absent in hHYD and yPABP. (b) Unrooted phylogenetic tree of 40 PABC domains showing the

grouping of plant, vertebrate and HYD sequences. Sequences labeled HYD are PABC domains

from HECT E3 ubiquitin ligases (hyperplastic disks proteins), all other sequences are poly(A)

binding proteins. The three proteins in panel (a) are underlined. Figure generated with ClustalW

(15) and TreeViewPPC (40).

Figure 2. Structure determination of the yeast PABC domain. (a) Histogram of long-range (open

bars), medium-range (light gray), sequential (dark gray) and intraresidue NOEs (filled bars). Only

residues comprising the PABC domain as defined in Fig. 1a are shown. Yeast specific residues

30a and 30b are indicated by a gray bar. (b) Correlation of experimental and back-calculated

values for 48 1H-15N residual dipolar couplings (RDCs). (c) Plot of root-mean-squared deviation

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of Cα atoms in the 30 final structures calculated without (gray) and with (black) dipolar

couplings. Inclusion of the RDCs doubles the precision of the final ensemble. Alpha helices in

yeast PABP are shown as black rectangles; the missing N-terminal helix found only in human

PABC is in gray. (d) Ramachandran plot of the five lowest energy structures calculated with

RDCs.

Figure 3. Structure of PABC domains. (a) Stereoview of thirty yPABC structures showing

residues in the aromatic-rich hydrophobic core (light blue) and peptide binding site (green balls).

(b) Ensemble turned 90° to show the binding site in helices 2 and 3 (numbered as for hPABP). (c)

Enlargement of the peptide binding site showing conserved residues in yPABP (white), HYD

(green) and hPABP (blue). In spite of two extra amino acids in yPABC (between helix 2 and 3)

the positions of residues involved in peptide binding are highly conserved. Pairwise comparison

of these same residues shows that yPABP is more like HYD than hPABP. (d-f) Comparison of

the helix arrangement of PABC domains from hPABP (12), hHYD (13), and yPABP. For hPABP

and yPABC, the peptide-binding sites identified by chemical shift mapping with a Paip2 peptide

are shown (gray surfaces). Peptide binding to yeast PABP likely occurs through the same

mechanism as identified for human PABP (12). The conserved peptide phenyalanine residue

(Table 3) inserts into a hydrophobic pocket between helices 2 and 3 and stacks with PABC

residue F/Y22. In yeast, the strong bend in the C-terminal (red) helix gives rise to an additional

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hydrophobic pocket (adjacent to residue F72) which may give rise to different specificity for

peptide N-terminal sequences (Table 3).

Figure 4. Mapping the PABC - ligand interaction. (a) 15N,1H-HSQC of 15N-labeled yPABC in the

absence (left panel) and presence (right panel) of a 22 residue Paip2-derived peptide (see Table

2). (b) Plot of chemical shift changes in yPABC as a function of residue number. Residue

numbering is as in Fig. 2 but includes the entire protein fragment studied by NMR. Residues Y22

and K35 in helices 2 and 3 showed the largest chemical shift changes. Helix 5 showed only minor

changes. (c) 15N,1H-HSQC of an 15N-labeled 27 residue Paip2-derived peptide in the absence (left

panel) and presence (right panel) of unlabeled yPABC. (d) 15N,1H-heteronuclear NOEs of Paip2

peptide when bound to yPABC. The weak or absent signals between residues 9 and 23 of the

peptide indicate that these residues interact with yPABC.

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Table 1. Structural statistics

Mean energies (std. dev.) for 30 structures (kcal/mol)Lennard-Jones VdW energy -442.1 ±15.2

Pairwise rmsd analysis for residues 11-74 of PABC (Å):backbone pairwise rmsd:

average=0.34 (0.07), min= 0.22, max= 0.50non-hydrogen (heavy) atom pairwise rmsd:

average=0.95 (0.10), min= 0.81, max= 1.21

Total number of constraints 1127intraresidue (n=0) NOE's 394sequential (n=1) range NOE's 245medium (n=2,3,4) range NOE's 181long (n>4) range NOE's 151dihedral angles constraints 68hydrogen bonds 40NH residual dipolar couplings 48

Ramachandran plot for 30 structures:residues in most favored regions 84.0 %residues in additional allowed regions 16.0 %

Deviation from idealized covalent geometrybonds 0.0019 ± 0.0001angles 0.353 ± 0.007impropers 0.258 ± 0.009

Deviation from experimental NMR restraints

distances (Å) 0.011 ± 0.001dihedral angle (º) 0.39 ± 0.05

Dipolar couplingsa

rmsd (Hz) 2.36 ± 0.10Qf (%) 13.1 ± 0.5

a references (41,42)

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Table 2. Peptides used in yPABC binding assays.

NO. PEPTIDE ORIGIN SEQUENCE BINDING1

P1913 Human RF3 AFSRQLNVNAKPFVPNVHAAEFVPSFLR +P1895 Human Paip2 VVKSNLNPNAKEFVPGVKYGNI +P1934 Shuttle craft protein SKLQASAPEFVPNFAKL +P1954 Pichia pinus RF3-related SYIPNTAKAFVPSAQPY +P1914 Human Paip1 VLMSKLSVNAPEFYPSGYSSSY -P1986 S.cerevisiae RF3 NNLQGYQAGFQPQSQGM -P1933 S.cerevisiae Pan1p EPLKPTATGFVNSFANN -P1955 S.cerevisiae Pbp1p SNSKPNSNKGNRYVPPTLRQ -P1956 S.cerevisiae Pbp1p SLSSKEAQIEELKKFSEKFKVPYD -

1based on amide chemical shift changes in NMR titrations.

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Table 3. Potential PABC-sites in yeast RF3 sequences.

Consensus alignment1 S-LN-NA-EF-P

Candida albicans KQQQQQQQQQQQQQNYYNPNAAQSFVPQGGYQQFQ-----Candida maltosa QSKQPQQQQPQQQQPYFNPNQAQAFVPTGGYQQFQPQQQQDebaryomyces hansenii QQQSYQQYQQQPQQNNFNANSAPTFTPSGQQGGY------Yarrowia lipolytica GQGYGQYQAPQQFVPGQSFVPGQSFVPGQSFAP-------Yarrowia lipolytica QGQGQQGQGYGQYQAPQQFVPGQSFVPGQSFVPGQSFAP-Yarrowia lipolytica -----GQGQGQQGQGYGQYQAPQQFVPGQSFVPGQSFVPGPichia pastoris KNEQRFNPNSASSFQPSFNPQAQNFVPGQYQESQSYQNY-Pichia pinus --INLNAPAYDPAVQSYIPNTAQAFVPSAQPYIPGQQEQQSchizosaccharomyces pombe SMSAKAPTFTPKAAPFIPSFQRPGFVPVNNIAGGYPYA--Schizosaccharomyces pombe -------KLSMSAKAPTFTPKAAPFIPSFQRPGFVPVNN-

1from (12).

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hPABP 544-LTASMLASAPPQEQKQMLGERLFPLIQAMHP--TLAGKITGMLLEhHYD 2379-EGNPSDDPEPLPAHRQALGERLYPRVQAMQP--AFASKITGMLLEyPABP 491-RNANDNNQFYQQKQRQALGEQLYKKVSAKTSNEEAAGKITGMILD 0 . 1 . 2 . 3-- . 4Conserved la p Q+Q LGE L*P V p-- A KITGMLLE

hPABP IDNSELLHMLESPESLRSKVDEAVAVLQAHQAK-619hHYD LSPAQLLLLLASEDSLRARVDEAMELIIAHGRE-2454yPABP LPPQEVFPLLESDELFEQHYKEASAAYESFKKE-568 . 5 . 6 . 7 .Conserved e L L S E L+ +V EA VL

1 2 3

54

a

Schizosaccharomyces

Yeast PABP

Arabidopsis "PABP-like"

Leshmania

Drosophila HYDHuman HYD

Trypanosoma

Fern

Arabidopsis

Tobacco

Wheat

Carrot

Arabidopsis

Arabidopsis

Aspergillus FabMChloroplast

Caenorhabditis DrosophilaVertebrate C1 isoforms

Vertebrate

Human PABP

0.1

(Saccharomyces)

Rat HYD

b

Kozlov et al, Figure 1

pg 27

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0 10 20 30 40 50 60 700

0.5

1

1.5

2

2.5

3

RM

SD

(Å

)

542 3

Residue in PABC

1c

0

10

20

30

40

50

60

70

Num

ber

of N

OE

s

0 10 20 30 40 50 60 70Residue in PABC

a b

Calculated RDCs

Mea

sure

d R

DC

s

-40

-30

-20

-10

0

10

20

30

40

-40 -30 -20 -10 0 10 20 30 40

R =0.98slope=0.98

2

-180 -90 0 90 180Phi (degrees)

-180

-90

0

90

180

Psi

(de

gree

s)

d

Kozlov et al, Figure 2

pg 28

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K35A33

F/Y22

T37

F72

a

e

b

d

c

f

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0 10 20 30 40 50 60 70 80

Che

mic

al s

hift

chan

ge

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7 542 31

Residue in PABC

b

8.0 6.07.09.0 8.0 6.07.09.0

110

120

130

a

1 1H (ppm)H (ppm)

15N

(ppm

)

Het

eron

ucle

ar N

OE

d GPLGSVVKSNLNPNAKEFVPGVKYGNI

-1.2

-1

-0.8

-0.6

-0.4

-0.2

0

0.2c G21

G4 G25

S5S9

N26V22K16N12

N10

L3

E17L11

V6Y24

A15I27

V19V7

K23K8

8.0 6.07.09.0 8.0 6.07.09.0

110

120

130

1H (ppm)

G4G25

N14S5

S9

N26

E17

N10

L3

K8K23

V7

V19

A15

V22

L11

F18

N6N12

Y24

1H (ppm)

I27

15N

(ppm

)

F18

N14

Kozlov et al, Figure 4

pg 30

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Irena Ekiel, Tara Sprules and Kalle GehringGuennadi Kozlov, Nadeem Siddiqui, Stephane Coillet-Matillon, Jean-François Trempe,

poly(A) binding proteinSolution structure of the orphan PABC domain from Saccharomyces cerevisiae

published online April 8, 2002J. Biol. Chem. 

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