COMMUNICATION

The Structure of an FF Domain from HumanHYPA/FBP11

Mark Allen, Assaf Friedler, Oliver Schon and Mark Bycroft*

MRC Centre for ProteinEngineering, Hills RoadCambridge CB2 2QH, UK

The FF domain is a 60 amino acid residue phosphopeptide-bindingmodule found in a variety of eukaryotic proteins including the transcrip-tion elongation factor CA150, the splicing factor Prp40 and p190RHOGAP.We have determined the structure of an FF domain from HYPA/FBP11.The domain is composed of three a helices arranged in an orthogonalbundle with a 310 helix in the loop between the second and third a helices.The structure differs from those of other phosphopeptide-bindingdomains and represents a novel phosphopeptide-binding fold.

q 2002 Elsevier Science Ltd. All rights reserved

Keywords: NMR structure; transcription; phosphopeptide recognition;RNA polymerase II carboxyl-terminal domain*Corresponding author

Phosphorylation is used as a control signal inmany important biological processes. The activityof eukayrotic RNA polymerase II (RNAP II), forexample, is regulated by phosphorylation of thecarboxyl-terminal domain (CTD) of its largestsubunit.1 Unphosphorylated CTD is required fortranscription initiation. Once transcription hascommenced, a variety of kinases phosphorylatethe CTD to produce an elongation-competentenzyme. The CTD consists of 17–52 tandem hep-tad repeats with the consensus sequence YSPTSPS.Phosphorylation occurs predominantly at serine 2and serine 5. The phosphorylated repeats recruit avariety of proteins that regulate mRNA synthesisand processing. The yeast protein Prp40 and thehuman protein CA150 have both been shown tobind to phosphorylated CTD.2,3 These proteinsbind also to the splicing factor BBP/SF1, andprovide a link between RNA processing and tran-scription. Prp40 and CA150 both contain a seriesof WW domains followed by an array of FFdomains. In addition to CA150, the human genomecodes for two more WW and FF domain-containingproteins; HYPA/FBP11 and HYPC. HYPA/FBP11,HYPC and CA150 have all been shown to interactwith huntingtin, the protein encoded by thegene mutated in Huntington’s disease,4,5 and ithas been proposed that the disruption of these

interactions may contribute to the pathology ofthe disease.6

The interaction between CA150 and phosphoryl-ated RNAP II CTD is mediated by the FF domainsof this protein.7 As well as being components ofthe CA150/Prp40 family of proteins, FF domainsare found singly or in multiple copies in a numberof other proteins.8 In order to try and learn moreabout this module, we have determined the struc-ture of an FF domain from human HYPA/FBP11.

Structure description

The structure of a 71 amino acid residue frag-ment of human HYPA/FBP11 encompassing thefirst FF domain was determined using standardNMR methods (Figure 1, Table 1). The sequencesof the first FF domain of human and mouseHYPA/FBP11 are identical. Residues are numberedaccording to the mouse sequence, as this proteinhas been characterised in more detail. The FFdomain structure is composed of three a helicesarranged in an orthogonal bundle with a 310 helixin the loop between the second and third a helices(Figure 1(a)). The N and C termini of the domainare at opposite ends of the structure, a featurefound often in modules that form tandem arrays.A few FF domain sequences have insertions anddeletions compared to the sequence of the HYPA/FBP11 domain. These are restricted to the loopbetween helices 1 and 2 and the loop between the310 helix and the final a helix (Figure 2(b)). Thetwo highly conserved phenylalanine residues,from which the domain takes its name, are in the

0022-2836/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved

E-mail address of the corresponding author:mb10031@cus.cam.ac.uk

Abbreviations used: RNAP II, RNA polymerase II;CTD, carboxyl-terminal domain.

doi:10.1016/S0022-2836(02)00968-3 available online at http://www.idealibrary.com onBw

J. Mol. Biol. (2002) 323, 411–416

middle of the first and third a helices, and formpart of the hydrophobic core of the protein (Figure2(a)). The first of these residues is part of a con-served FXXLL sequence. It has been noted thatthis is a potential nuclear receptor (NR) box8 asequence motif present in a number of transcrip-tion co-activators that is involved in ligand-depen-dent binding to nuclear receptors.9 In the FFdomain structure, the two leucine residues of thismotif are at the C terminus of the first a helix andform part of the hydrophobic core (Figure 2(a)). Itis unlikely that the structure could rearrange suchthat these residues became available for an inter-action with a ligand and the sequence similarity isprobably not functionally significant. The onlyother residue that is conserved in more than 80%of FF domains is a tryptophan residue correspond-ing to Trp411 in this structure. This residue is at thestart of the second a helix and packs into thehydrophobic core of the domain (Figure 2(a)). Anumber of positions are occupied by hydrophobicor aromatic amino acid residues in the majority ofFF domains. Most of these are in the a helices andare involved in helix/helix packing. The excep-tions are Tyr424 and Leu427, which are in the 310

helix and anchor this element of secondary struc-ture into the main hydrophobic core. The strongpreference for aromatic or hydrophobic aminoacid residues at these positions suggests that the310 helix is a feature of most FF domains. Residueson the surface of the domain are less well con-served. The most conserved solvent-exposedresidues are Asp421 and Arg423. They are con-served in approximately 50% of FF domainsequences and, where present, the residue between

them is usually proline. These residues are situatedin the turn between the second a helix and the 310

helix. Analysis of the ensemble of structuresrevealed the potential formation of a salt-bridgebetween these residues and it appears that theymay be conserved for stability reasons rather thanfor function. Residues 393 and 433 are conservedas lysine or arginine in over 50% of FF domains.They are situated at the start of a-helix 1 and inthe middle of the final a helix (Figure 3(b)). Theyare close together in space and could be function-ally important.

Structural similarities

The structures of several other phosphoserine/threonine domains have been determined.10 TheFF domain structure differs significantly fromthese and represents a novel phosphopeptide-binding fold. When the FF domain structure wascompared to other known protein structures usingthe program DALI,11 the highest score (Z ¼ 5) wasto the C-terminal region (residues 304–368) of thea isoform of human protein serine/threoninephosphatase 2C (Figure 1(c)).12 The region of simi-larity is found only in a sub-family of mammalianphosphatases and forms a distinct unit that foldsindependently of the catalytic domain. Compari-son of protein structures can potentially revealdistant evolutionary relationships. For small, all-helical modules such as the FF domain, however,structural similarities may be only the result of theconstraints imposed by the rules governing thepacking of helices and must be viewed with

Table 1. Summary of conformational constraints and statistics for the accepted 25 structures of human FF domain

A. Structural constraintsIntra-residue 603Sequential 361Medium-range (2 # li 2 jl # 4) 353Long-range (li 2 jl . 4) 440Dihedral angle constraints 20TALOS constraints 112Distance constraints for 36 H bonds 72Total 1961

B. Statistics for accepted and minimized average structureStatistics parameter (^SD) Accepted structures Minimized average structure

Rms deviation for distance constraints (A) 0.0113 ^ 0.0009 0.0092Rms deviation for dihedral constraints (deg.) 0.628 ^ 0.040 0.76

Mean X-PLOR energy term (kcal mol21 ^ SD)E (overall) 137.97 ^ 5.26 219.08E (van der Waals) 11.67 ^ 1.88 23.76E (distance constraints) 3.18 ^ 0.42 15.39E (dihedral and TALOS constraints) 16.34 ^ 0.60 4.56

Rms deviations from the ideal geometry (^SD)Bond lengths (A) 0.0018 ^ 0.0001 0.0027Bond angles (deg.) 0.543 ^ 0.006 0.657Improper angles (deg.) 0.424 ^ 0.007 0.539

Average atomic rms deviation (A) from the average structure (^SD)Residues 145–203 (N, Ca, C atoms) 0.271 ^ 0.078 0.200Residues 145–203 (all heavy-atoms) 0.757 ^ 0.065 0.404

412 Structure of an FF Domain

caution. The fact that the region of the phosphatasesimilar to the FF domain forms a discrete domainand is found in a protein of related function is sug-gestive of a potential homology. The role of thephosphatase C-terminal domain is unknown, how-ever, and it is not clear if the structural similarity tothe FF domain reflects an evolutionary or func-tional relationship.

Phosphopeptide binding

The FF domains of CA150 have been shown tobind to phosphorylated RNAPII CTD.7 To deter-mine if the HYPA/FBP11 FF domain could interactwith the RNAP II CTD, we used isothermaltitration calorimetry to study the binding of thedomain to a 16 residue peptide corresponding totwo CTD repeats phosphorylated on serine 2 andserine 5 of the consensus sequence. An interactionbetween the protein and the peptide was detectedwith a binding constant of approximately 50 mM.No binding could be detected with a non-phosphoylated peptide. We next tried to localisethe binding site by monitoring the changes inchemical shifts produced in the 2D 1H–15N-hetero-nuclear single quantum coherence (HSQC) spectraof the domain when an excess of the peptide wasadded. The biggest changes in chemical shift werefor residues at the N terminus of the first a helixand the N terminus of the third a helix (Figure

3(a)). The changes in chemical shift form an arc onthe protein surface centred on a shallow cleftbetween the N termini of a helices 1 and 4. Thisregion of the protein contains a cluster of positivelycharged amino acid residues (Figure 3(b)) includ-ing the two conserved lysine residues and isprobably the peptide-binding site. The CTD repeatcontains two SPXX motifs, SPTS and SPSY, whichhave the potential to form b turns and the patternof chemical shifts changes seems consistent withthe peptide binding in a turn-like conformation.Unfortunately, transfer nuclear Overhauser effect(NOE) experiments could not be used to provideinformation about the bound state conformation,due to the low molecular mass of the FF domain–peptide complex.

Conclusions

The CTD of RNAP II plays a central role incoordinating transcription and RNA processing.13

The FF domain is one of several protein modulesthat have been found to bind to phosphorylatedRNAP II CTD. The best-characterised interactionis that between the WW domain of Pin1 andRNAP II CTD.14 Pin1 binds to a CTD-derivedphoshopeptide about an order of magnitudetighter than the HYPA FF domain. Pin1 has only asingle CTD-binding site, whilst HYPA/FBP11 andrelated proteins have a series of FF domains that

Figure 1. (a) An overlay of thebackbone atoms of the 25 lowest-energy NMR structures. The codingsequence for residues 357–425 ofSPTREMBL O75400 correspondingto the FF domain was amplified byPCR from IMAGE cDNA clone731611 (obtained from the MRCHGMP Resource Centre) by stan-dard methods and cloned into apRSET-derived pHisGro vector.This was used to over-express a

soluble histidine-tagged GroEL apical domain/FF domain fusion protein in Escherichia coli. The fusion protein waspurified under native conditions using NTA agarose (Qiagen). After cleavage with thrombin, the FF domain was puri-fied using ion-exchange chromatography and gel-filtration. The samples for NMR spectroscopy typically contained2.5 mM human FF domain in 90% H2O/10% 2H2O containing 50 mM KCl, 50 mM potassium phosphate (pH 6.0) at298 K. The NMR spectra were assigned using standard NMR methods.18,19 The assignments have been deposited inthe BioMagResBank under accession numbers PDB 1H40 BMRDB 5537. A set of distance constraints were derivedfrom a series of NOESY spectra recorded in H2O and 2H2O with mixing times of 150 ms. The NOESY spectrum wasintegrated according to the cross-peak strengths and calibrated by comparison with NOE connectivities obtained forstandard inter-residue distances within an a helix. After calibration, the NOE constraints were classified into thefollowing categories: strong, medium, weak and very weak, corresponding to inter-proton distance constraints of1.8–2.8 A, 1.8–3.5 A, 1.8–4.75 A, and 2.5–6.0 A, respectively. Hydrogen bond constraints were included for a numberof backbone NH groups whose signals were observed in a 2D 1H–15N-HSQC recorded in 99.996% 2H2O at 298 K (pH5.0). For hydrogen bond partners, two distance constraints were used where the distance (D)H–O(A) corresponded to1.5–2.5 A and (D)N–O(A) to 2.5–3.5 A. Torsional angle constraints were obtained from an analysis of C0, N, Ca Ha andCb chemical shifts using the program TALOS.20 The three-dimensional structure of the FF domain was calculatedusing a dynamic simulated annealing protocol based upon the work of Nilges et al.21 in the program XPLOR (Brunger,A. T. (1992). X-PLOR Version 3.1: a system for cystallography and NMR, Yale University, New Haven, CT). The coordi-nates have been deposited in the protein structure database, entry. (b) A ribbon representation of the lowest-energystructure prepared using the program MOLSCRIPT.22 (c) A ribbon representation of the C-terminal region of humanphosphatase 2C alpha prepared using the program MOLSCRIPT.22 Note there is a break in the electron density in theloop between the first and second helices.

Structure of an FF Domain 413

could potentially bind to several CTD repeats. Theincreased avidity produced by the multiple bind-ing sites would lead to a tighter interaction and itis possible that HYPA/FBP11-like proteins bindpreferentially to hyper-phosphorylated CTD.

Are FF domains specialised phosphopeptide-binding modules that just recognise phosphoryl-ated RNAPII CTD or are they capable of bindingto other phoshopeptide motifs? FF domain-containing proteins fall into three categories.Proteins containing WW and FF domains havebeen found in all eukaryotic genomes sequencedso far. The members of this family that have beencharacterised to date have all been found to havea role in RNA metabolism. The FF domains ofthree of these proteins CA150, PRP40 and HYPA/FBP11 have been shown to bind phosphorylated

RNAPII CTD, suggesting that the FF domains ofthe WW/FF protein family all recognise thismotif. FF domains are present in a number ofhypothetical proteins identified by various genomesequencing projects for which no biochemical dataare available. Interestingly, one of these, theDrosophila protein CG1078, contains, in additionto a single FF domain, a region similar in sequenceto FIP1, a component of yeast pre-mRNA poly-adenylation factor.15 RNAP II CTD has beenshown to be required for pre-mRNApolyadenylation,16 suggesting that the FF domainof CG1078 could be involved in RNAP II CTDbinding. The mammalian p190RhoGAP family ofproteins contain FF domains. These proteinsmodulate the activity of the small GTPase Rho,and have been implicated in the regulation of

Figure 2. (a) Ribbon representation of the FF domain with the side-chains of the structural ensemble for the con-served residues in the hydrophobic core of the domain. (b) Structure-based sequence alignment of selected FFdomains. Proteins included are the WW/FF domain-containing proteins that have been shown to bind to RNAP IICTD via their FF domains (mouse HYPA/FBP11(Q9R1C7), Saccharomyces cerevisiae PRP40 (PR40_YEAST), humanCA150 (O14776)), human RHOGAP 190-A (Q9NRY4) and human RHOGAP 190-B (Q13017). Residues that areconserved in 50% of FF domains are indicated.

414 Structure of an FF Domain

actin rearrangement in response to a variety ofextracellular signals.17 The FF domains of theseproteins are more divergent in sequence and con-tain distinct patterns of conserved residues (Figure2(b)). The second of the conserved lysine/arginineresidues is generally not present, and many havea histidine residue at end of the last helix.p190RhoGAP proteins have not been reported tobind to the CTD of RNAP II. They are, however,known to be involved in signal transduction path-ways that are controlled by phosphorylation, andit is likely that FF domains of these proteins bindto a different phosphopeptide motif.

We clearly have a lot more to learn about the FFdomain. The structure described here will providea structural basis for experiments aimed at obtain-ing a more detailed understanding of the functionsof this module and proteins that contain it.

Acknowledgements

O.S. is supported by a fellowship from theFriedrich-Ebert-Foundation (GER). A.F. is sup-ported by a long-term fellowship, no. LT00056/2000-M, from the Human Frontier Science ProgramOrganisation.

References

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Figure 3. (a) The molecular surface of the FF domain in the same orientation as in Figure 1(a) and (b) calculatedusing the program GRASP23 with residues whose backbone amide proton or nitrogen chemical shifts alter upon thebinding of the peptide SYpSPTpSPSYpSPTpSPSY are coloured in purple. The residues indicated are those whose back-bone amide 1H shift changes by more than ^0.05 ppm or whose backbone amide 15N chemical shift changes by morethan ^0.20 ppm when a threefold excess of the CTD peptide was added to a 0.5 mM solution of the domain in25 mM Mes (pH 6.5). The phosphopeptide was synthesized using a pioneer peptide synthesizer (Perseptive biosys-tems). Standard Fmoc chemistry was used, with double coupling of every amino acid. Phosphoserine residues wereincorporated using the protected derivative Fmoc-Ser (PO(Obzl)OH)-OH (NOVAbiochem). The peptide was cleavedfrom the resin using trifluoroacetic acid (TFA)/water/triisopropylsilane (95:2.5:2.5, by vol.) for four hours at room tem-perature. Purification of the peptide was performed on a Waters 600 HPLC using a preparative C8 column (Vydac).The peptide was characterized by analytical HPLC and matrix-assisted laser desorption/ionisation time-of-flight(MALDI-TOF) mass spectrometry. The dissociation constant for the peptide FF domain interaction was determinedusing an OMEGA isothermal titration calorimeter (Microcal, Northampton, USA). Peptide was titrated into a solutionof the FF domain in 25 mM Mes (pH 6.5) at 298 K. (b) The electrostatic potential at the molecular surface of the FFdomain. Potentials less than 10 kT and greater than 10 kT are indicated. The two conserved lysine residues are labelled.

Structure of an FF Domain 415

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Edited by M. F. Summers

(Received 29 April 2002; received in revised form 4 September 2002; accepted 4 September 2002)

416 Structure of an FF Domain