Molecular Cell, Vol. 7, 855–865, April, 2001, Copyright 2001 by Cell Press

Crystal Structure of a Pumilio Homology Domain

protein acts with DmPUM to produce a complementaryXiaoqiang Wang,* Phillip D. Zamore,†gradient of Hunchback protein, most concentrated atand Traci M. Tanaka Hall*‡

the anterior pole of the fly embryo where it acts to pro-*Laboratory of Structural Biologymote development of head and thoracic structuresNational Institute of Environmental Health Sciences(Schroder et al., 1988; Tautz, 1988; Irish et al., 1989;National Institutes of HealthStruhl, 1989; Tautz and Pfeifle, 1989; Hulskamp et al.,Research Triangle Park, North Carolina 277091990, 1994). Translational repression of hbmat requires a†Department of Biochemistry and Molecularthird protein, Brain Tumor (BRAT), which forms a quater-Pharmacologynary complex with NOS, PUM, and NRE-containing RNAUniversity of Massachusetts Medical School(Sonoda and Wharton, 2001). BRAT is a member of theWorcester, Massachusetts 01655conserved NHL family of proteins, which includes theC. elegans developmental regulator, LIN-41.

In worms, the Puf proteins FBF-1 and FBF-2 mediateSummarythe sperm/oocyte switch in hermaphrodites by bindingthe 39 UTR of the fem-3 mRNA and repressing its expres-Puf proteins regulate translation and mRNA stabilitysion (Zhang et al., 1997). Like DmPUM, FBF interactsby binding sequences in their target RNAs through thewith a NOS protein, NOS-3 (Kraemer et al., 1999). Xeno-Pumilio homology domain (PUM-HD), which is charac-pus PUM also interacts with a NOS protein, Xcat-2 (Na-terized by eight tandem copies of a 36 amino acidkahata et al., 2001). In both worms and frogs, Puf pro-motif, the PUM repeat. We have solved the structureteins interact with the cytoplasmic polyadenylationof the PUM-HD from human Pumilio1 at 1.9 A resolu-element binding protein, CPEB, suggesting that Puf fam-tion. The structure reveals that the eight PUM repeatsily members may have distinct functions dependingcorrespond to eight copies of a single, repeated struc-upon their protein binding partners (Luitjens et al., 2000;tural motif. The PUM repeats pack together to form aNakahata et al., 2001). In Dictyostelium, the Puf protein,right-handed superhelix that approximates a half dough-PufA, represses expression of protein kinase A mRNA,nut. The distribution of side chains on the inner andand appears to be a key developmental regulator in thatouter faces of this half doughnut suggests that theorganism (Souza et al., 1999). In yeast, the Puf3p proteininner face of the PUM-HD binds RNA while the outeris a transcript-specific regulator of mRNA degradationface interacts with proteins such as Nanos, Brain Tu-and binds the 39 UTR of the COX17 mRNA in vitro (Olivasmor, and cytoplasmic polyadenylation element bind-and Parker, 2000). Similarly, the yeast Puf protein MPT5ping protein.binds the 39 UTR of HO mRNA and regulates HO expres-sion posttranscriptionally (Tadauchi et al., 2001). BothIntroductionDmPUM and Puf3p promote deadenylation of their tar-get mRNAs (Wreden et al., 1997; Olivas and Parker,Members of the Puf family of RNA binding proteins regu-2000), and it is possible that all Puf proteins share alate translation and mRNA stability in a wide variety ofcommon mechanism of action, although in yeast, theeukaryotes including flies, worms, slime mold, andoutcome of deadenylation is mRNA turnover (Olivas andyeast. The first Puf protein identified, Drosophila mela-Parker, 2000), whereas in flies, deadenylation appearsnogaster Pumilio (DmPUM), acts maternally to repressto correlate with translational repression (Wreden et al.,translation of maternal hunchback (hbmat) mRNA in the1997).

posterior half of the Drosophila embryo, thereby permit-Puf family members are readily identified by the pres-

ting abdominal development (Lehmann and Nusslein-ence of eight tandem copies of an imperfectly repeated,

Volhard, 1987; Barker et al., 1992; Macdonald, 1992). In 36 amino acid sequence motif, the PUM repeat (Barkeraddition to its role in abdominal development, DmPUM et al., 1992; Macdonald, 1992; Zamore et al., 1997; Zhangfunctions in the development of germline stem cells (Lin et al., 1997), and typically contain evolutionarily con-and Spradling, 1997; Forbes and Lehmann, 1998; As- served sequences that are amino- and carboxy-terminalaoka-Taguchi et al., 1999). DmPUM binds hbmat at two to the repeated region of the protein (Zamore et al.,sequence motifs, called Nanos Response Elements 1997; Zhang et al., 1997). The eight repeats, together(NREs), in the hbmat 39 untranslated region (UTR; Murata with the terminal conserved sequences, form a se-and Wharton, 1995). DmPUM acts in conjunction with quence-specific RNA binding domain, the Pumilio ho-the CCHC zinc finger protein, Nanos (NOS; Curtis et mology domain (PUM-HD; Zamore et al., 1997) or Pufal., 1997), which is translated in a posterior-to-anterior domain (Zhang et al., 1997). To date, as many as 59 Pufgradient from a pool of posterior-localized nanos mRNA proteins can be identified by BLAST searching Gen-deposited in the egg during oogenesis (Wang and Leh- Bank. Six Puf proteins have been directly shown to bindmann, 1991; Gavis and Lehmann, 1992). NOS binds di- specific RNA sequences: DmPUM (Murata and Wharton,rectly to DmPUM in the presence of NRE-containing 1995; Zamore et al., 1997), human Pumilio1, which isRNA (Sonoda and Wharton, 1999). The gradient of NOS 80% identical to the fly protein within the PUM-HD

(HsPUM1; Zamore et al., 1997), C. elegans FBF (Zhanget al., 1997), the yeast proteins Puf3p (Olivas and Parker,‡ To whom correspondence should be addressed (e-mail: hall4@

niehs.nih.gov). 2000) and MPT5p (Tadauchi et al., 2001), and Xenopus

Molecular Cell856

PUM (Nakahata et al., 2001). Furthermore, the isolated of approximately 85 A, and a thickness of approximately30 A. The structure reveals the three-dimensional basisDrosophila PUM-HD (DmPUM-HD) retains some func-for the repeated primary structure motifs that character-tion in translational repression in vivo (Wharton et al.,ize Puf proteins: each PUM repeat corresponds to a1998). The function and biological target of HsPUM1compact structural unit comprising three helical seg-remain unknown.ments. The protein contains eight repeats, each withThe DmPUM-HD has been studied in the most detail.three helices (repeats 1–8) and two imperfect repeatsDmPUM-HD is a monomer in solution, and a single(repeats 19 and 89), one at each terminus, which approxi-DmPUM-HD monomer binds to each NRE with subnano-mate the shape of repeats 1–8 but only have one or twomolar affinity (Zamore et al., 1999). Based on estimateshelices. The imperfect repeats include the Csp1 andof the intraembryonic concentration of DmPUM, it isCsp2 motifs of FBF-1 and FBF-2 (Zhang et al., 1997).likely that in vivo, most NREs are occupied by DmPUMBoth of these regions in FBF are required for RNA bind-monomers (Zamore et al., 1999). The DmPUM-HD recog-ing; the Csp2 motif is also required for binding NOS.nizes sequences in both the 59 and 39 halves of each

The eight central repeats are very similar structurally,NRE (Zamore et al., 1997; Sonoda and Wharton, 1999).and the structures of repeats 2–8 can be superimposedIn particular, UGU triplets in both the 59 and 39 half siteson that of repeat 1 with an average root mean squareof the NRE are critical for binding (Zamore et al., 1997).(rms) deviation of 1.0 A for 36 Ca positions (Figure 1B).The RNA sequences bound by fly, worm, and yeastIn each repeat, the three helices (a1, a2, and a3) formPUM-HD proteins contain a common core motif, UUGU,roughly a triangle. The a2 helices on the concave facealthough sequences outside the core element can playand a3 helices on the convex face are each three toa role in RNA recognition (Zhang et al., 1997; Zamorefour turns in length, whereas the a1 helices are shorter,et al., 1999; Tadauchi et al., 2001). The effects of muta-approximately two turns long, and run along the side oftions in the RNA sequence have been extensively stud-the protein, following the curve of the doughnut. Adja-ied both in vitro and in vivo, but little is known aboutcent repeats in the molecule are related by an averagewhat elements of PUM-HD proteins are important forrotation of 208 (range, 148–238) about an axis parallel totheir exquisite binding specificity.the helical axis of the a2 helix. The relationship betweenHere, we present the structure of the PUM-HD fromrepeats 5 and 6 differs from that of the others, and hasHsPUM1, the HsPUM-HD. Our structure reveals that thea much smaller rotation angle of 148 and a 148 angleeight PUM repeats correspond to eight copies of a singlebetween a2rep5 and a2rep6. In contrast, the other adjacentrepeated structural motif. These eight copies, along witha2 helices are roughly parallel (average angle of 0.88).conserved amino- and carboxy-terminal sequences,

The repeats pack together to form a right-handedpack together to form a right-handed superhelix thatsuperhelix that creates a continuous hydrophobic coreapproximates a half doughnut. The distribution of sidethroughout the domain (Figure 2A). A network of hydro-chains on the inner and outer faces of this half doughnutgen bonds links the repeats. The structure predicts hy-suggest that the inner face binds RNA while the outerdrogen bonding between the side chains of residues atface interacts with proteins such as NOS, BRAT, andposition 33 (Figure 2A, green side chains) and the mainCPEB.chain amide nitrogens in the last position of the previousrepeat and at position 2 within the same repeat for re-Results and Discussionpeats 1, 3, 5, and 7 (see Figure 2B for the numbering ofresidue positions). For repeat 8, the side chain of posi-Structure Determinationtion 33 makes a hydrogen bond with the main chain

We expressed a 349-amino acid fragment of thecarbonyl oxygen of position 35 of the previous repeat.

HsPUM1 protein (Gly-828 to Gly-1176) correspondingFor repeat 6, hydrogen bonds to water molecules in-

to the HsPUM-HD domain. This fragment, which retains terposing between the side chain of the Glu residue atthe ability to bind to hb NRE–containing RNA (data not position 33 of repeat 6 and the main chain carbonylshown), produced single crystals that diffract to greater oxygens of positions 35 and 36 of repeat 5 link thethan 1.9 A Bragg spacings. The structure of the protein repeats. These water molecules are accommodated be-was determined by multiple isomorphous replacement cause of the larger angle between the a2 helices ofwith anomalous scattering using two mercury deriva- repeats 5 and 6. Hydrogen bonds between the sidetives (Table 1). A model comprising amino acid residues chains of residues at position 26 and side chains ofGly-828 to Ala-1149 was built and refined to a crystallo- residues at position 28 in the previous repeat link repeatsgraphic R value of 19.78% and a free R value of 24.22% 1–2, 3–4, 4–5, and 6–7; repeat 6 is linked to repeat 5with data extending to 1.9 A Bragg spacings. Weak instead by a hydrogen bond between the side chain atdensity for an additional 13 residues (Thr-1150 to Ala- position 26 and the side chain at position 35 in repeat1162) was observed, and a model including this region 5 (Figure 2A, blue side chains).was built and refined to a crystallographic R value of Basic residues at position 17 and acidic residues at19.55% and a free R value of 24.16% with data extending position 20 in each repeat form an alternating patternto 1.9 A Bragg spacings. of positively and negatively charged surface potential

that extends the length of the molecule on the concaveOverall Structure surface of the molecule (Figure 2A, yellow side chains).The HsPUM-HD protein comprises 26 a helices and two For repeat 7, the residue at position 20 is a Thr, and ashort 310 helices (Figure 1A). The protein has a curved Glu at position 16 replaces the position 17 basic residue.shape overall, reminiscent of a half doughnut, with an It has been previously observed that the seventh repeats

of PUM-HDs are more similar to each other than toinner diameter of approximately 40 A, an outer diameter

Structure of the Human Pumilio RNA Binding Domain857

Table 1. Summary of Data Collection, Phasing, and Refinement Statistics

Data statistics

Data set Native Thimerosal1 MeHgCl2

Space group R32 R32 R32Unit cell a 5 142.88 A a 5 908 a 5 142.96 A a 5 143.22 A

b 5 142.88 A b 5 908 b 5 142.96 A b 5 143.22 Ac 5 111.31 A g 5 1208 c 5 111.25 A c 5 111.19 A

Resolution (A) 1.9 2.3 2.3Number of reflections3 34328 (3405) 19512 (1957) 17558 (1839)Completeness (%)3 99.7 (99.2) 99.8 (100) 89.6 (93.6)Rsym (%)3,4 6.3 (32.6) 4.2 (12.3) 6.0 (22.6)I/sI3 21.2 (3.2) 36.8 (11.4) 24.9 (5.9)

Phasing statistics

Number of sites 2 6Rcullis acentric/centric 0.79/0.73 0.67/0.68(Rcullis anomalous) (0.90) (0.81)Phasing power 1.24/0.83 1.87/1.24acentric/centricFigure of merit5 0.5981 (0.4878)

Refinement statistics6

Model 1 Model 2(G828–A1162) (G828–A1149)

R factor (%) 19.55 19.78Rfree (%) 24.16 24.22Number of protein atoms 2712 2606Number of solvent molecules 392 392Number of ligand atoms (bME) 4 4Rmsd from ideal values

Bond length (A) 0.009 0.009Bond angle (8) 1.29 1.29

Average B factor, all atoms (A2) 38.20 (93.1 for T1150–A1162) 36.50

1 Thimerosal, mercury-[(o-carboxyphenyl)thio]ethyl sodium salt, C9H9HgO2SNa, FW 5 404.82 Methylmercury (II) chloride, CH3HgCl, FW 5 251.13 Numbers in parentheses are for the highest resolution shell (1.97–1.90 A for native and 2.38–2.30 A for derivatives).4 Rsym 5 ShSi|Ii(h) 2 ,I(h).|/ShSiIi(h)5 Number in parentheses is for the highest resolution shell (2.62–2.30 A).6 All data with I . 0 were used in the refinement. A subset of the data (6%) was excluded from the refinement and used to calculate the freeR value (Rfree). R factor 5 S||Fo| 2 |Fc||/S|Fo|

the other repeats within the same protein (Zhang et al., Fasman, 1977), presumably because it prevents a clashwith the carbonyl oxygen of the previous amino acid1997). Many of these basic/acidic pairs are spaced 4–5

A apart, longer than would be expected for true salt (Richardson, 1981).bridges, but the acid–base interactions may nonethelesscontribute to the stability or function of the protein. Similarity to Other Structures

A search for structurally homologous proteins using theThe most highly conserved sequences among theeight repeats of the HsPUM-HD are contained in the program DALI (Holm and Sander, 1993) revealed that the

topology of the PUM repeats is similar to the topology ofa2 helices facing the concave surface of the molecule(Figure 2B) and include residues that are part of the Armadillo (ARM) repeats that are observed in structures

of the multifunctional protein b-catenin and the nuclearhydrophobic core or residues that we propose to playa role in RNA binding (see Surfaces for RNA and Protein transport factor karyopherin a (also known as importin

a; Huber et al., 1997; Conti et al., 1998; Figure 3A). ARMInteractions, below). Other conserved residues appearto be important structurally. The residue at position 8 repeats also comprise three a helices, but the ARM

repeats pack together differently from PUM repeats andin each repeat is either an Asp, Gln, or His, and the sidechains of these residues form hydrogen bonds with the consequently produce a protein with a roughly cylindri-

cal topology. When the a2 and a3 helices of the PUMmain chain amide nitrogens of residues at positions 10and 11, capping the N-terminal ends of the a2 helices repeats are aligned with the H3 and H1 helices of an

ARM repeat, residues in the two aligned helices occupy(Aurora and Rose, 1998). The Gly or Ala residues thatoccur at position 11 in each repeat occupy a space in analogous positions in their hydrophobic cores (Figure

3B). Furthermore, the a2 helix of the PUM repeat isthe hydrophobic core near residues at position 5 thatwould not be expected to accommodate a larger resi- structurally equivalent to the H3 helix of the ARM repeat.

In the ARM repeat–containing proteins b-catenin anddue. Gly residues are found at position 36 in repeats 2,3, 4, and 6. In the structure, these glycines occupy the karyopherin a, the H3 helices line a groove that extends

the length of the molecule, formed by the superhelicali 1 2 position of a type II b turn. Gly is the most favoredamino acid for this position in such a turn (Chou and twist of the repeats. This groove is analogous to the

Molecular Cell858

Azotobacter vinelandii (Peters et al., 1996), show pairsof helices that correspond to the a2 and a3 helices ofthe PUM repeats according to analysis with DALI. Theoverall packing of repeats in the LRV protein mostclosely resembles that observed in the HsPUM-HD do-main. The LRV repeats contain alternating a helices thatline the concave surface of the molecule and 310 helicesthat form the convex surface. The overall structure ofthe a helices of the first 8 of the 9 repeats in the LRVprotein can be aligned with the a2 helices of repeats1–8 of the HsPUM-HD structure with an rms deviationof 2.3 A for 103 Ca positions (Figure 3C). The 310 helicesof the LRV protein are shorter than the a3 a helices of theHsPUM-HD structure and do not align well. A structure-based alignment of the amino acid sequences of the a2helices from the PUM repeats with the a helices of theLRV repeats shows that the pattern of hydrophobic coreresidues is comparable (Figure 3B), except that the hy-drophobic core residues at position 15 of the PUM re-peats are replaced by Arg at the corresponding positionof the LRV repeats. These Arg residues are involved ina ladder of hydrogen bond and salt bridge interactionsthat extends throughout the LRV protein. Although theLRV protein is thought to be expressed in A. vinelandiiunder nitrogen-fixing conditions (Jacobson et al., 1989),little is known about the function of the LRV protein, sowe cannot determine whether its structural relationshipto the PUM-HD reflects a common function.

Surfaces for RNA and Protein InteractionsThe amino acid side chains presented on the surface ofthe HsPUM-HD suggest that the inner, concave surfaceinteracts with RNA while the outer, convex surface inter-acts with the protein partners needed for Puf protein–mediated regulation of mRNA stability and translation.Strikingly, the distribution of charge on the surface ofFigure 1. Structure of a Human Pumilio Homology Domainthe PUM-HD structure is asymmetric. The concave sur-(A) Ribbon diagram of the HsPUM-HD. Residues Gly-828 to Ala-face of the molecule is highly basic (Figure 4A), while1162 are shown. Residues Lys-1150 to Ala-1162 are shown withthe convex surface of the molecule is acidic (Figure 4B).dashed lines and are lighter in color. Repeats are colored alternately

blue and yellow. The N and C termini are indicated. The loop con- Basic residues at positions 13, 17, and 21 of each PUMtaining Gly-1107 to His-1109 is colored red. repeat all lie on the concave surface of the molecule,(B) Structural alignment of the PUM-HD repeats. Repeats 2–8 were producing a positively charged surface that we envisionaligned with respect to repeat 1. Repeats 1–8 are colored sequen-

to bind RNA. The 22 basic residues on this surface mighttially: 1, blue; 2, green; 3, yellow; 4, red; 5, magenta; 6, cyan; 7,make multiple contacts with phosphates in the RNAorange; 8, purple. The N and C termini are indicated. This figurebinding site. Furthermore, this surface contains 16 ex-was prepared with the program MOLSCRIPT (Kraulis, 1991).posed polar and 18 hydrophobic residues (Figure 4C).Polar residues on the surface at positions 9 and 16 ofeach PUM repeat may interact with specific bases, andinner, concave surface of the HsPUM-HD structure. In

karyopherin a, the groove binds nuclear localization solvent-exposed hydrophobic residues at positions 10,13, and 21 of each repeat may stack against the bases insignals on karyopherin a cargo (Conti et al., 1998). In

b-catenin, the groove is positively charged and has been the RNA, providing opportunities for sequence-specificRNA recognition (Figures 2B and 4C).proposed to interact with acidic binding partners: cad-

herin cell adhesion molecules, the tumor suppressor Three DmPUM mutants do not appear to alter RNAbinding: the point mutant, PUM680, in which Gly-1067 isgene product, Adenomatous Polyposis Coli (APC), and

Tcf family transcription factors (Huber et al., 1997). A replaced by Asp (numbering as in Figure 2B for thecorresponding positions in HsPUM1; Wharton et al.,recent crystal structure of a complex of b-catenin with

the b-catenin binding domain of a Tcf protein, together 1998) and two insertional mutants, PUMSphI and PUMMluI

(Sonoda and Wharton, 1999). In a fourth point mutant,with mutagenesis data, confirm this hypothesis (Grahamet al., 2000). Other repeated motifs, such as the HEAT PUM21, Arg-1066 is changed to a His (numbering as in

Figure 2B for the corresponding positions in HsPUM1; D.repeats of the PR65/A subunit of protein phosphatase 2A(Groves et al., 1999), the repeated unit of the a subunit of Chagnovich and R. Lehmann, personal communication).

Each of these mutations are located in helix a3 on thefarnesyl transferase (Park et al., 1997), and the repeatedunit of the leucine-rich repeat variant (LRV) protein of convex surface of the domain (Figures 2B and 4C), oppo-

Structure of the Human Pumilio RNA Binding Domain859

Figure 2. Roles of Conserved Residues inPUM Repeats

(A) Stereo diagram of a Ca trace of the struc-ture of the HsPUM-HD showing the hydrogenbond network that links the repeats and thealternating pattern of acidic and basic resi-dues. Every tenth Ca atom is indicated with ablack sphere. Every fiftieth residue is labeled.Repeats are rendered alternately with thickand thin lines. Side chains of residues in-volved in hydrogen bonding with residues atposition 33 are colored green. Side chains ofresidues involved in hydrogen bonding withresidues at position 26 are colored blue. Sidechains of alternating acidic and basic resi-dues are colored yellow. Hydrogen bondsand salt bridges shorter than 3.2 A are indi-cated with red dashed lines. Black dashedlines indicate distances greater than 3.2 A.The N and C termini are indicated.(B) Sequence alignment of the PUM repeats.The amino acid sequences of repeats arealigned based on the structural alignment inFigure 1B. Secondary structural elements areindicated and colored as in Figure 1A. a heli-ces are shown in solid color and 310 helicesare shown in lighter color. Residues that wesuggest may bind RNA are highlighted withcolor. Basic residues (Arg, His, and Lys) areblue, acidic residues (Asp and Glu) are red,residues that may form stacking interactionsare orange, and polar residues are yellow.Residues at conserved hydrophobic core po-sitions are shown in gray. Green boxes indi-cate positions of mutations in DrosophilaPUM protein (open triangle, pumSphI insertion;open circle, pum21; filled triangle, pum680; filledcircle, pumMluI insertion; open square,pumC1365R; filled square, pumT1366D). The loca-tion of the pumMluI insertion (Gly-1107 to His-1109) is colored red. Numbers in parenthesesindicate the number of the first amino acidresidue in each line. Numbers above repeat1 indicate the amino acid positions within re-peats 1–8. (A) was prepared with the programMOLSCRIPT (Kraulis, 1991). (B) was preparedwith ALSCRIPT (Barton, 1994).

site the surface we propose to bind RNA. The concave of hbmat translation (Irish et al., 1989; Lehmann and Nuss-lein-Volhard, 1991; Wang and Lehmann, 1991). Sonodasurface is extensive (greater than 2000 A2), and could

bind at least 17 nucleotides if the RNA were in a fully and Wharton have identified a three–amino acid inser-tion in HsPUM1 that blocks its interaction with NOSextended conformation. If the NRE adopts a more struc-

tured conformation when bound to PUM, the concave (Sonoda and Wharton, 1999). Deletion of these threeamino acids, Gly-1107, Pro-1108, and His-1109, restoressurface of the HsPUM-HD might accommodate the en-

tire 25 nucleotides that have been proposed to form a PUM–NOS interactions and restores PUM function inDrosophila–human PUM chimeras in flies (Sonoda andminimal high-affinity binding site (Wharton et al., 1998).

The NRE comprises two conserved sequence motifs, a Wharton, 1999). The Gly–Pro–His triplet forms a loop onthe convex surface of the HsPUM-HD (Figure 4C). Thisfive-nucleotide A box and a six-nucleotide B box, which

are separated by six nucleotides. Such an extensive loop links repeats 7 and 8. Furthermore, the Csp2 motif,which follows repeat 8, is required for the interaction ofbinding surface is consistent with previous observations

that the DmPUM-HD recognizes nucleotides in both FBF-1 with NANOS-3 (Kraemer et al., 1999). The convexsurface is acidic, suggesting that it might interact withboxes (Zamore et al., 1997; Wharton et al., 1998), al-

though only B box sequences are important for HsPUM- the relatively basic NOS protein (calculated pI 9.6; thepI for residues 208–401, the subdomain of NOS that isHD binding to the NRE (Zamore et al., 1997).

With the RNA occupying most if not all of the concave required for binding PUM, is 10.4). Both the worm proteinFBF (Luitjens et al., 2000) and the Xenopus PUM proteinsurface, the convex face of the protein might interact

with other proteins required for PUM function, such as (Nakahata et al., 2001) interact with homologs of thecytoplasmic polyadenylation element binding proteinNOS and BRAT. NOS protein binds PUM (Sonoda and

Wharton, 1999), and is required in vivo for the repression (CPEB), another relatively basic protein (calculated pI

Molecular Cell860

Figure 3. Structural Similarity of the HsPUM-HD to Armadillo Repeats and Leucine-RichRepeat Variant Protein

(A) Stereo diagram of a structural alignmentof a PUM repeat with an ARM repeat. Repeat1 (His-852 to Gln-887) from the HsPUM-HDstructure (yellow) is shown in superpositionwith H2 and H3 of repeat 2 and H1 of repeat3 (Val-131 to Asp-173) from the yeast kary-opherin a structure (blue, PDB ID 1bk5). Heli-ces are labeled, and N and C termini are indi-cated.(B) Structure-based alignment of the aminoacid sequences of HsPUM-HD, yeast kary-opherin a repeat 2 H2 and H3, and LRV pro-tein B helices. Conserved hydrophobic corepositions for each protein that lie in commonstructural positions are highlighted with gray.Numbers in parentheses indicate the numberof the first amino acid residue in each line.(C) Stereo diagram of a structural alignmentof the HsPUM-HD structure with the structureof the LRV protein. The HsPUM-HD structure(yellow) is shown in superposition with the A.vinelandii LRV protein (green, PDB ID 1lrv).The N and C termini are indicated. (A andC) were prepared with MOLSCRIPT (Kraulis,1991). (B) was prepared with ALSCRIPT (Bar-ton, 1994).

9.1). It has not yet been shown whether Puf proteins can domain (Sonoda and Wharton, 2001). Mutant DmPUM-HD proteins bearing the PUM680 mutation (Gly-1330 tointeract simultaneously with NOS and CPEB, or whether

these interactions are mutually exclusive. Asp), a Cys-1365 to Arg mutation, or a Thr-1366 to Aspmutation, all fail to repress translation of hbmat in vivoIn addition to NOS, BRAT is required for DmPUM func-

tion and forms a quaternary complex with NOS, and fail to recruit BRAT to the quaternary complex inyeast (the numbering corresponds to positions inDmPUM, and NRE-containing RNA (Sonoda and Whar-

ton, 2001). Binding of BRAT is mediated by its NHL DmPUM, Figure 2B; Barker et al., 1992; Wharton et al.,

Structure of the Human Pumilio RNA Binding Domain861

Figure 4. Possible PUM-HD RNA and Protein Interaction Surfaces

(A) Electrostatic surface representation of the HsPUM-HD. The molecule is rotated approximately 908 about the vertical axis with respect toFigure 1A and shows the concave surface proposed to bind RNA. Blue represents regions of positive potential and red represents regionsof negative potential at the 10 kT/e level.(B) Electrostatic surface representation as in (A), but rotated 1808 about the vertical axis relative to (A) to show the convex surface proposedto interact with NOS, BRAT, or CPEB.(C) Stereo ribbon diagram of the HsPUM-HD showing the side chains that lie on the proposed RNA binding and protein binding surfaces.Side chains on the concave surface are colored as in Figure 2B. Acidic side chains (Asp and Glu) in a1 and a3 are colored red. Side chainscorresponding to the location of mutations in DmPUM are colored green. The protein is oriented as in Figure 1A.(D) Molecular surface representation as in (B), but rotated 608 about the horizontal axis with respect to (B) to show the proposed NOS- andBRAT-interacting surfaces. Indicated on the HsPUM-HD are the positions of mutations in DmPUM that disrupt interaction with BRAT (pink),the loop in HsPUM that blocks its interaction with NOS in flies and that corresponds to the MluI insertion in DmPUM (green), and sites ofmutations that disrupt neither DmPUM function nor BRAT binding (black). Mutation positions are numbered according to the sequence ofDmPUM, not HsPUM. The position of the DmPUM680 mutation corresponds to HsPUM Gly-1067, DmPUM Cys-1365 to HsPUM Cys-1102, andDmPUM Thr-1366 to HsPUM Thr-1103. Likewise, DmPUM Gly-1186 is HsPUM Gly-923, Gly-1222 is Gly-959, Gly-1258 is Gly-995, and Lys-1331 corresponds to Asn-1068. (A, B, and D) were prepared with the program GRASP (Nicholls et al., 1991). (C) was prepared with MOLSCRIPT(Kraulis, 1991).

1998; Sonoda and Wharton, 2001). Notably, the PUM680 1103, define a surface on the PUM-HD, which we pro-pose to interact with the NHL domain of BRAT andmutation does not prevent recruitment of NOS (Sonoda

and Wharton, 2001). The wild-type positions of these potentially other NHL-containing proteins (pink in Figure4D). This surface is adjacent to a loop containing themutations in HsPUM1, Gly-1067, Cys-1102, and Thr-

Molecular Cell862

Figure 5. Conservation of Residues in PUM Repeats of Puf Family Members

(A) Conservation of amino acid residues in helix a2. For each position in helix a2, the residue or residue type (1, basic; 2, acidic; φ, hydrophobic;GA, Gly or Ala; ST, Ser or Thr) that occurs most frequently in the PUM repeats aligned in the Pfam database is listed with the percentage ofsequences in which it occurs. Residues or residue types occurring in 80%–100% of the sequences are colored red; 50%–79%, orange; and30%–49%, yellow, as indicated on the color bar. Positions 14, 15, 18, and 19 are buried hydrophobic core residues and are colored gray.(B) Ribbon diagram of repeats 1–8 (His-852 to Pro-1146) of HsPUM-HD with residues in helix a2 colored as in (A). Residues that show significantvariability in positions 9, 20, and 21 are indicated. (B) was prepared with MOLSCRIPT (Kraulis, 1991).

three amino acid residues in the HsPUM-HD that block of the PUM-HD domain likely performs the same func-tion, which we envision to be binding RNA.its interaction with NOS in flies (green in Figure 4D;

Sonoda and Wharton, 1999). It is tantalizing to speculate The acidic character of the convex surface is likewiseconserved. 56% of repeats in the Pfam alignment havethat NOS binds the PUM-HD at or near this loop in a

manner that permits it to interact with BRAT bound to two or more acidic residues in the sequences that corre-spond to a3, the helix of the PUM repeat that lies on thethe PUM-HD. The positions of four additional mutations

that do not disrupt PUM function in vivo or PUM–BRAT convex surface. Therefore, the function of the convexsurface of PUM-HD domains may be shared amonginteractions in yeast are indicated in black in Figure 4D

(Sonoda and Wharton, 2001). These residues lie outside many Puf proteins. Alternating basic and acidic residueson the concave surface at positions 17 and 20 are alsothe BRAT interaction surface defined by Gly-1067, Cys-

1102, and Thr-1103. well conserved. Arg, His, or Lys is present at position17 in 69% of the Pfam sequences, and an Asp or Gluoccupies position 20 in all but repeat 7 in 70% of theFunctional Implications for Other Puf Family Members

We analyzed the PUM repeats of the 38 Puf family pro- Pfam sequences. At position 16 in repeat 7, which lacksan acidic residue at position 20, the acidic residue Glutein sequences listed in version 6.0 of the Pfam data-

base, a collection of protein families and domains (Bate- is present in 91% of the repeat 7 sequences among the38 Puf proteins listed in Pfam (Figure 5A). Thus, theman et al., 1999), by placing a total of 255 PUM-HD

repeats into categories corresponding to repeats 1–8. function of these residues seems likely to be conservedamong PUM-HD domains. One notable exception is theThe resulting alignment reveals that the basic nature of

the concave surface observed in the HsPUM-HD struc- FBF protein, which seems to lack alternating acidic andbasic residues, suggesting that they are not essentialture is conserved among most Puf proteins. The basic

residues Arg, His, or Lys usually occupy position 13 in for the overall structure of the domain and may reflecta functional difference between FBF and other Puf pro-repeats 1, 3, 4, and 5, position 17 in all but repeat 1,

and position 21 in repeat 5 (more than 50% of the time teins.Zhang et al. noted patterns of amino acid residuefor each position; Figure 5A). More than 50% of the time,

the polar residues Asn, Gln, Ser, or Thr occupy position changes at positions 5 and 6 of their core consensussequence (Zhang et al., 1997). These positions corre-9 in repeats 1, 4, 6, and 8 and position 16 in all but

repeat 7, and hydrophobic residues (Ile, Leu, Met, Phe, spond to positions 12 and 13 in our alignment. Basedon analysis of the Pfam alignment, the pattern of N, C,Trp, Tyr, and Val) at position 10 in repeats 2, 3, 5, 6, 7,

and 8, position 13 in repeats 2, 6, and 8, and position N, C, N, S, N at position 12 of repeats 2 through 8appears to be conserved among most PUM-HD domains21 in repeats 2 and 8 (Figure 5A). All of these positions

lie on the concave face of the PUM-HD (Figure 5B). and may be expanded to include a Ser for the first repeatto give, successively, S, N, C, N, C, N, S, N at positionThus, in the majority of Puf proteins, the concave surface

Structure of the Human Pumilio RNA Binding Domain863

by sonication in a dry ice/ethanol bath. The fusion protein was12 of the eight repeats (Figure 5A). In the PUM-HD struc-purified from the soluble fraction by incubation with chitin agaroseture, the side chains of these residues appear to beresin (New England Biolabs) for 40 min at 48C. The resin was washedimportant structurally, forming side chain to main chainextensively with sonication buffer, then with sonication buffer con-

carbonyl oxygen contacts in repeats 2, 4, 6, 7, and 8, taining 500 mM NaCl, and finally with sonication buffer containingor appear to position the side chain at position 16 in 150 mM NaCl. The resin was then transferred to a 50-ml conical

tube and equilibrated with 50 mM dithiothreitol to initiate cleavagerepeats 1, 2, 4, and 6, where it may interact with RNA.by the intein domain. The air above the slurry of beads was purgedTypically, Y, R, H, R, Y, x, Y occupy position 13 inwith argon, the tube sealed, and the resin incubated for 3–4 daysrepeats 2 through 8 (Zhang et al., 1997). Our analysis ofat room temperature. The beads were next packed into a 10-mlthe Puf protein sequences listed in the Pfam databasedisposable column and the cleaved HsPUM-HD protein was col-

shows that a Lys or Arg occupies position 13 in all lected. The beads were washed 2–3 times with a one-fourth columnsequences of repeat 1 (FBF has a Gln at position 13 of volume of sonication buffer with 150 mM NaCl to collect additional

cleaved protein. The eluted HsPUM-HD protein was concentratedrepeat 1, but its first repeat is not in the Pfam database).to z1 mg/ml and chromatographed on a Superdex 200 gel filtrationAn Asn is found at position 13 of repeat 7 for 59% ofcolumn (Amersham Pharmacia Biotech) equilibrated with 10 mMthe Pfam sequences, and repeat 2 may also contain PheTris-HCl (pH 7.4), 150 mM NaCl, and 5 mM b-mercaptoethanol.(24% of the Pfam sequences) in addition to Y (62% ofMonomeric protein was pooled and concentrated to z5.5 mg/ml.

the Pfam sequences). Thus, the consensus for position13 of each of the eight repeats is, successively, R/K,

Crystallization and Data CollectionY/F, R, H, R, Y, N, Y (Figure 5A). As noted above, de-Crystals of the HsPUM-HD were grown from sitting drops by thepending on the repeat, position 13 is either a basic,method of vapor diffusion. One ml of a 5.5 mg/ml solution of protein

hydrophobic, or polar residue that could interact with was mixed with 2 ml of reservoir solution (12% [w/v] PEG 6000, 100RNA. Since this pattern is so well conserved, these resi- mM Li2SO4, and 100 mM sodium citrate [pH 5.6]) and equilibrated

over the reservoir solution at 48C. Crystals typically grew over 2–5dues may interact with the backbone (basic residues)days with the dimensions 0.2 mm 3 0.2 mm 3 0.2 mm.or form sequence-independent stacking interactions

Prior to data collection, the crystals were transferred to a solution(hydrophobic residues) and may not contribute to theof 18% (w/v) PEG 6000, 100 mM Li2SO4, 100 mM sodium citrate (pHRNA binding specificity of individual Puf proteins. Alter-5.6), 150 mM NaCl, and 15% (v/v) ethylene glycol and flash cooled

natively, their high degree of conservation may reflect to 21808C. Data from a crystal of the HsPUM-HD were measureda common core sequence that is present in the RNA to 1.9 A Bragg spacings using a conventional x-ray source (Rigaku

RU-H3R) and an R axis IV detector. To obtain heavy atom deriva-sequences of all Puf protein binding sites, as has beentives, the crystals were soaked in mother liquor containing 1 mMsuggested previously (Zamore et al., 1997).thimerosal or 2% of a saturated solution of methylmercury chlorideThe amino acid positions that appear most likely toand flash cooled for data collection. Data from crystals soaked inbe involved in sequence-specific RNA binding lie at po-thimerosal or methylmercury chloride were measured to 2.3 A Bragg

sition 9 in repeats 2, 3, 5, 6, and 8, position 20 in repeat spacings.7, and position 21 in all eight repeats. At each of thesepositions, the side chains are located on the concave

Model Building and Refinementsurface of the PUM-HD (Figure 5B), and there is greaterThe asymmetric unit of the crystal contains one molecule with a

variability among polar, charged, and hydrophobic side solvent content of z54% (Matthews, 1968). The structure was deter-chains than at other positions in the Pfam alignments. mined using multiple isomorphous replacement with anomalous

scattering (MIRAS). Heavy atom sites were determined with theFor example, at position 21, hydrophobic groups areprograms CNS (version 1.0; Brunger et al., 1998), RSPS (Bailey,present in 34% of the sequences, but Arg/Lys, Ser/1994), and FFT (Bailey, 1994). Heavy atom parameters were refinedThr, and His are present in 13%, 17%, and 18% of theand the phases were calculated with MLPHARE (Bailey, 1994). Den-sequences, respectively, and at position 20 in repeat 7,sity modification was carried out to improve the electron density

Ser/Thr, Lys/Arg, Leu/Met, and Glu are present in 31%, with RESOLVE (Terwilliger, 1999). Model building was carried out22%, 19%, and 16% of the sequences, respectively. with the program O (Jones et al., 1991). The resulting atomic model

was refined iteratively against data to 1.9 A Bragg spacings usingDetermining the molecular basis by which the PUM-the program CNS (Brunger et al., 1998). B factors were refinedHD achieves its exquisite RNA binding specificity andindividually. The best model that could be fit for residues Thr-1150by which it recruits NOS, BRAT, CPEB, and perhapsto Ala-1162 was included for one model (Lys-1158 is modeled asother proteins to the RNA remain major challenges forAla), but the electron density in this region is weak and many other

the future. The three-dimensional structure of the PUM- models are consistent with the data. Water molecules were addedHD presented here should aid in achieving these goals. conservatively. A bulk solvent correction was applied. The program

PROCHECK (Laskowski et al., 1993) was used to check the models.All backbone φ-c torsion angles are within allowed regions of theExperimental ProceduresRamachandran plot and 93.5% of the residues were in the energeti-cally most-favored regions.Protein Expression and Purification

A cDNA encoding the PUM-HD from Homo sapiens Pumilio1 protein(HsPUM-HD; Gly-828 to Gly-1176) was amplified by the polymerase Acknowledgmentschain reaction (PCR) from a pGEX-2T plasmid containing a repairedBamHI to DarI fragment from human cDNA KIAA0099 (Zamore et We are grateful to J. Krahn for computer support and advice onal., 1997), and cloned into the pTYB3 expression plasmid (New crystallography; G. Mueller for advice on calculating relative rotationEngland Biolabs) using NcoI and SapI restriction sites. Codons for angles; K. Knight, R. Lehmann, L. Pedersen, and K. Weeks for criticalArg-829 and Arg-831 were changed from AGG to CGC during PCR comments on the manuscript; and R. Lehmann, D. Chagnovich, andto enhance the expression of the protein in E. coli. M. Yamashita for sharing data prior to publication. P. D. Z. is a Pew

The protein, in which the HsPUM-HD was fused to an intein and Scholar in the Biomedical Sciences.a chitin binding domain, was expressed in E. coli strain BL21(DE3).Bacterial pellets were resuspended in sonication buffer (20 mMsodium phosphate [pH 8.0], 1 M NaCl, and 0.1 mM PMSF) and lysed Received March 2, 2001; revised March 30, 2001.

Molecular Cell864

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Protein Data Bank ID Codes

The coordinates and structure factors for the two structures havebeen deposited with the ID codes 1ib2 (Gly-828 to Ala-1149) and1ib3 (Gly-828 to Ala-1162).