X-ray Structure Determination of Human Profilin II: A Comparative ...

Article No. jmbi.1999.3318 available online at http://www.idealibrary.com on J. Mol. Biol. (1999) 294, 1271±1285

X-ray Structure Determination of Human Profilin II:A Comparative Structural Analysis of Human Profilins

Ilana M. Nodelman1, Gregory D. Bowman1, Uno Lindberg2

and Clarence E. Schutt3*

1Department of MolecularBiology, Henry H. HoytLaboratory, PrincetonUniversity, PrincetonNJ 08544, USA2Department of Zoological CellBiology, WGI, ArrheniusLaboratories for NaturalSciences, Stockholm UniversityS-10691, Stockholm, Sweden3Department of ChemistryHenry H. Hoyt LaboratoryPrinceton University PrincetonNJ 08544, USA

E-mail address of the [email protected]

Abbreviations used: Ena, enabledstimulated phosphoprotein; N-WASAldrich syndrome protein; WIP, Wprotein; DMSO, dimethyl sulfoxidephosphatidylinositol 4,5-bisphosphaphosphatidylinositol; PDB, Proteinmean-square; ATCC, American TypNCS, non-crystallographic symmetrglycol; F-actin, ®lamentous actin.

0022-2836/99/501271±15 $30.00/0

Human pro®lins are multifunctional, single-domain proteins whichdirectly link the actin micro®lament system to a variety of signallingpathways via two spatially distinct binding sites. Pro®lin binds to mono-meric actin in a 1:1 complex, catalyzes the exchange of the actin-boundnucleotide and regulates actin ®lament barbed end assembly. Like SH3domains, pro®lin has a surface-exposed aromatic patch which binds toproline-rich peptides. Various multidomain proteins including membersof the Ena/VASP and formin families localize pro®lin:actin complexesthrough pro®lin:poly-L-proline interactions to particular cytoskeletallocations (e.g. focal adhesions, cleavage furrows). Humans express abasic (I) and an acidic (II) isoform of pro®lin which exhibit different af®-nities for peptides and proteins rich in proline residues. Here, we reportthe crystallization and X-ray structure determination of human pro®lin IIto 2.2 AÊ . This structure reveals an aromatic extension of the previouslyde®ned poly-L-proline binding site for pro®lin I. In contrast to serine 29of pro®lin I, tyrosine 29 in pro®lin II is capable of forming an additionalstacking interaction and a hydrogen bond with poly-L-proline which mayaccount for the increased af®nity of the second isoform for proline-richpeptides. Differential isoform speci®city for proline-rich proteins may beattributed to the differences in charged and hydrophobic residues in andproximal to the poly-L-proline binding site. The actin-binding faceremains nearly identical with the exception of ®ve amino acid differences.These observations are important for the understanding of the functionaland structural differences between these two classes of pro®lin isoforms.

# 1999 Academic Press

Keywords: pro®lin; poly-L-proline; isoform; actin; cytoskeleton
*Corresponding author
Introduction

In eukaryotic cells there exists a complex web ofsignal transduction pathways which function toconvey signals from cell surface receptors to theproper intracellular targets. The actin micro®la-ment system is one of the major targets of signal-ling cascades whose activation is essential for

ing author:

; VASP, vasodilator-P, neuronal Wiskott-

ASP-interacting; PIP2,te; PtdIns,

Data Bank; rms, root-e Culture Collection;y; PEG, polyethylene

fundamental cellular processes including motility(Stossel, 1993), endo- and exocytosis (Perrin et al.,1992), cytokinesis (Sanger et al., 1989) and determi-nation of cell shape (Small, 1988). Pro®lin plays acentral role by integrating multiple signalling path-ways and directly affecting actin ®lamentdynamics. Via pro®lin, actin is linked to the phos-phoinositide cycle and to a host of pathways inwhich speci®c proline-rich proteins play a role.

Pro®lin is an essential protein (Verheyen &Cooley, 1994; Witke et al, 1993) which forms a 1:1complex with monomeric actin. Pro®lin was ®rstisolated from spleen and was thought to functionas a sequesterer of monomeric actin (Carlsson et al.,1977); however, subsequent biochemical studieshave shown its role to be more complex. In thepresence of capped F-actin barbed ends, pro®linacts as a sequesterer and causes the depolymeriza-tion of actin ®laments (Pollard & Cooper, 1984;

# 1999 Academic Press

1272 A Comparative Analysis of Human Pro®lins

Pantaloni & Carlier, 1993). In the absence of ®la-ment end cappers, pro®lin complexed to ATP-actinadds to the barbed ends of growing actin ®laments(Pollard & Cooper, 1984; Pring et al., 1992;Pantaloni & Carlier, 1993; Korenbaum et al., 1998).Also, pro®lin catalyzes actin nucleotide exchangein vitro, thereby having the potential to increasethe pool of ATP-actin necessary for barbed endassembly (Mockrin & Korn, 1980; Nishida, 1985;Goldschmidt-Clermont et al., 1991b). In agreementwith its role as a key regulator of actin dynamics,pro®lin localizes to regions in the cell undergoingactive cytoskeletal remodelling in vivo (Buss et al.,1992; Edamatsu et al., 1992; Finkel et al., 1994;Suetsugu et al, 1998; Wills et al, 1999).

Pro®lin binds to proline-rich stretches in a var-iety of proteins including members of the Ena/VASP (Reinhard et al., 1995; Gertler et al., 1996)and formin families (Manseau et al., 1996;Watanabe et al., 1997; Evangelista et al., 1997;Chang et al., 1997), drebrin (Mammato et al., 1998),gephyrin (Mammato et al., 1998), N-WASP(Suetsugu et al., 1998), WIP (Ramesh et al., 1997),dynamin I (Witke et al., 1998), and the p85 subunitof PI3-kinase (Singh et al., 1996). Members of theEna/VASP family localize to focal adhesions andcan interact with ActA on the surface of Listeriamonocytogenes, a bacterial pathogen which utilizesactin to move through the cytoplasm (Chakrabortyet al., 1995; Gertler et al., 1996). Cell polarizationand cytokinesis require formin proteins and viatheir formin homology 1 domains associate withpro®lin (for a review, see Wasserman, 1998). Dis-ruption of the Drosophila formin gene, diaphanous,results in defects in cytokinesis (Castrillon &Wasserman, 1994), and mutations in the humanhomologue of diaphanous, DFNA1, have beenlinked to an autosomal dominant hearing disorder(Lynch et al., 1997). Recruitment of pro®lin:actincomplexes by these proline-rich ligands increasesthe local concentrations of actin necessary for theexecution of various actin-driven processes.

Another way pro®lin links the cytoskeleton tosignal transduction pathways is via a direct inter-action with phosphoinositides. Phosphatidylinositol4,5-bisphosphate (PIP2) causes the dissociation ofpro®lin:actin complexes in vitro which frees actinfor rapid polymerization (Lassing & Lindberg,1985). In addition, the PIP2:pro®lin complex inhibitsthe hydrolysis of PIP2 by phospholipase C-g1 intosecond messengers (Goldschmidt-Clermont et al.,1990) which can be overcome by phosphorylationof the phospholipase (Goldschmidt-Clermont et al.,1991a). After several structural and mutagenicinvestigations (Raghunathan et al., 1992; Haareret al., 1993; Fedorov et al., 1994a; Sohn et al., 1995) itstill remains unclear if pro®lin has a distinct phos-phoinositide binding site(s); yet, pro®lin bindsphosphoinositides with varying af®nities (PtdIns(3,4)P2 > PtdIns(3,4,5)P3 > PtdIns(4,5)P2), suggestingsome speci®city of binding (Lu et al., 1996).

Mammals express two isoforms of pro®lin whichare 62 % identical in amino acid sequence. In 1993,

human pro®lin II was cloned and shown to beexpressed in different tissues than human pro®linI, with pro®lin I apparently the more ubiquitouslyexpressed of the two isoforms (HonoreÂ et al., 1993).The relative af®nities of pro®lin I and II foractin are comparable (Gieselmann et al., 1995;Lambrechts et al., 1995); however, opposing ®nd-ings have been reported for the relative af®nities ofthe pro®lin isoforms for PIP2 (Gieselmann et al.,1995; Lambrechts et al., 1997). In contrast, differ-ences between pro®lin I and II in their af®nitiestowards poly-L-proline rich peptides (Lambrechtset al., 1997; Jonckheere et al., 1999) and proteins(Witke et al., 1998; Suetsugu et al., 1998) have beenclearly established. Studies with continuousstretches of proline as well as VASP-based prolinepeptides have shown pro®lin II to bind moretightly than pro®lin I (Lambrechts et al., 1997;Jonckheere et al., 1999). Nevertheless, these peptidestudies do not re¯ect the af®nities of either isoformfor proline-rich stretches presented in the contextof a full-length protein. Recently, Suetsugu and co-workers showed pro®lin I to exhibit tighter bind-ing to N-WASP compared with pro®lin II(Suetsugu et al., 1998), and studies by Witke andco-workers found that pro®lin I and II associatedwith different functional protein complexes frommouse brain extracts. In particular, pro®lin II wasfound to interact with dynamin I whereas pro®lin Idid not (Witke et al., 1998). These data suggest thatdifferences between the isoforms in and aroundthe poly-L-proline binding site play a critical role inselecting different binding partners, which in turnlink the actin cytoskeleton to distinct functionalpathways.

The structure of pro®lins from human (Metzleret al., 1993; Mahoney et al., 1997; Mahoney et al.,1999), bovine (Schutt et al., 1993; Cedergren-Zeppezauer et al., 1994), Saccharomyces cerevisiae(Eads et al., 1998), Acanthamoeba castellanii(Vinson et al., 1993; Fedorov et al., 1994a), Arabi-dopsis thaliana (Thorn et al., 1997) and birch pol-len (Fedorov et al., 1997) have previously beenreported. Pro®lins have a common fold consist-ing of a central seven-stranded b-sheet ¯ankedby N and C-terminal helices on one side andtwo short helices on the other side. X-ray struc-ture determination of bovine pro®lin I in com-plex with bovine b-actin de®ned the actinbinding site of pro®lin as residues from helix 3,helix 4, and b-strands 4, 5, and 6. Also, crystalstructures of human pro®lin I bound to poly-L-proline peptides (Mahoney et al., 1997; Mahoneyet al., 1999) have con®rmed a previouslydescribed surface patch of aromatic residues(Trp3, Tyr6, Trp31, His133, Tyr139) as the poly-L-proline binding site (BjoÈ rkegren et al., 1993;Schutt et al., 1993; Cedergren-Zeppezauer et al.,1994; Thorn et al., 1997). The uncomplexed struc-tures of bovine pro®lin I and human pro®lin Iare virtually identical with the actin- and poly-L-proline-complexed forms (Cedergren-Zeppezaueret al., 1994; Mahoney et al., 1997). Here, we pre-

A Comparative Analysis of Human Pro®lins 1273

sent the ®rst X-ray crystal structure of thissecond class of mammalian pro®lins, human pro-®lin II. This structure reveals an aromatic exten-sion of the poly-L-proline binding site whichmay explain the increased af®nity of pro®lin IIfor proline-rich peptides. Comparison with pro®-lin I shows the region surrounding the poly-L-proline site to have a distinct electrostatic andhydrophobic character which may explain differ-ential ligand speci®cities.

Results

Structure determination

The asymmetric unit of human pro®lin II con-sists of four molecules related by approximate 222non-crystallographic symmetry (NCS) with thecentral interaction of the tetramer formed by allfour C-terminal helices (Figure 1). Monomersrelated by the NCS 2-fold axis parallel to the crys-tallographic c-axis (A:C and B:D) interact atb-strands 4 and 5, the turn connecting b-strands 4and 5, and the C-terminal helix of each molecule.The A:C and B:D interfaces each bury a total of1171 AÊ 2 of solvent-accessible surface area.A second NCS 2-fold axis (roughly parallel with

the crystallographic a-axis) relates A to B and C toD. The A:B interface includes contacts from the Nand C-terminal helices burying a total of 966 AÊ 2 ofsolvent-accessible surface area. In contrast, a poly-ethylene glycol (PEG) 400 molecule is completelyburied in the C:D interface which restricts directcontacts between chains C and D to include onlythe N-terminal residues. This PEG 400 molecule isstably bound with an average B-factor of 18.2 AÊ 2,and may account for the requirement of PEG 400in the crystallization mixture.

Main-chain Ca atoms of A and B superimposewith an rms deviation of 0.26 AÊ while C and D Ca

atoms superimpose with an rms deviation of0.09 AÊ . Ca superposition of A or B onto C or Dresults in a slightly higher rms deviation of 0.47 AÊ .The ®rst two amino acids are visible only in chainsC and D, and the last two residues are disorderedin all four chains. Also, the electron density is poorfor residues 92-95 of the loop preceding b-strand 6of chain B, whereas it is clearly de®ned for chainsC and D. The presence of a second bound PEG 400molecule and close packing contributes to differ-ences in the local environments of this loop regionin chains C and D. A sulfate molecule from thecrystallization buffer is bound to each molecule in

Figure 1. Backbone worm rep-resentation of the asymmetric unitin human pro®lin II crystals. Thefour chains, A, B, C, and D arecolored pink, brown, gray, andblue, respectively. A PEG 400 mol-ecule (red and gray sticks) is buriedby the C:D interface.

(b)

Figure 2. (a) Sequence alignment of mammalian pro®lins (human pro®lin I (Kwiatkowski & Bruns, 1988); humanpro®lin II (HonoreÂ et al., 1993); bovine pro®lin I (Ampe et al., 1988); bovine pro®lin II (Lambrechts et al., 1995); mousepro®lin I (Widada et al., 1989); mouse pro®lin II (medline accession: AI322548)). Amino acid differences are shaded,and pro®lin I and putative pro®lin II poly-L-proline binding residues are boxed. Sequence alignment was generatedusing AMPS and ALSCRIPT (Barton et al.,1990; Barton et al., 1993). (b) Superposition of human pro®lin I and II. Resi-dues 3 to 136 of 1®k (red) were superimposed onto chain D of pro®lin II (blue). Secondary structural elements arelabelled (a, alpha-helix; b, beta-strand).


the asymmetric unit, coordinated by the side-chains of Arg88, Asn99, His119, the main-chainamide group of Gly120 and the side-chain ofArg74 of a non-crystallographically related mol-ecule. Similarly, two structures of human pro®lin I(PDB accession code: 1®k and 1®l; referenced inMaterials and Methods) have a phosphate and asulfate group positioned in the same location andcoordinated by the same side-chains.

Structural overview of human profilins

Mammals express two isoforms of pro®lin inwhich each isoform class, I or II, is conservedbetween species. Human pro®lin I and II are 62 %identical in primary sequence and share the samefold (Figure 2(a) and (b)). Residues 3 to 136 ofhuman pro®lin I and II superimpose with an aver-age Ca rms deviation of 1.13 AÊ (Ca atoms of chainsA, B, C, and D were each superimposed onto crys-tal structures of human pro®lin I (PDB accessioncode: 1®k; 1®l; 1awi)). Slight deviations in the

main chain are visible around loops preceding b-strands 2 and 6.

Based on sequence alignment, all amino aciddifferences between human pro®lin I and II whichare solvent exposed (>10 AÊ 2 of solvent-accessiblesurface area) are highlighted on a surface render-ing of human pro®lin II in Figure 3. These differ-ences span most of the surface of the protein withthe exception of the majority of the actin and poly-L-proline binding surfaces. A total of ®ve aminoacid differences, S56E, Y59F, V60T, E82D, andL122T (the ®rst and second letters are the aminoacids found at the given positions of human pro®-lin I and II, respectively) are found in the actin-binding face. Residues surrounding the poly-L-pro-line binding site display a wide range of sequencevariation which could result in differential bindingspeci®cities for proline-rich ligands.

The differences in sequence between the humanpro®lin isoforms result in locally unique surfacechemistry and topology without disruption of thepro®lin fold. Presumably, the presence of particu-

Figure 3. (a) and (b) Amino acid residue differences of the human pro®lins. A surface rendering of human pro®linII (chain D) with colored regions highlighting amino acid residues which differ in human pro®lin I (blue, non-conserved amino acid changes; brown, conserved amino acid changes). Sites of the more dramatic differences arelabelled, e.g. S29Y, with the ®rst letter representing the amino acid in human pro®lin I followed by the residuenumber, ending with the amino acid present in human pro®lin II. Human pro®lin II was superimposed with humanpro®lin I and bound proline peptide (PDB accession code: 1awi) to orient the poly-L-proline binding groove. Image(b) is rotated 180 � about the y-axis with respect to (a). Image was generated using the program GRASP (Nichollset al., 1991).


lar intramolecular interactions are selectively main-tained throughout evolution. For example, thesecond isoform of pro®lin has three cysteine resi-dues (Cys12, Cys15, and Cys16) located on a struc-turally conserved loop following the N-terminal

Figure 4. A cysteine-rich loop in human pro®lin II. Twproceeding the N-terminal helix in human pro®lin II comparserved hydrogen-bonding network involving a water molecgen, red; nitrogen, blue; sulfur, green). The Figure was gener

helix (Figure 4). Cys16 is conserved in pro®lin Iand displays the same rotomer conformation as inpro®lin II. The sulfur atom of Cys15 (pro®lin II) ispositioned similarly to the g-oxygen atom of Thr15(pro®lin I), and both form electrostatic interactions

o additional cysteine residues are present in the looped with human pro®lin I. This loop also displays a con-

ule in the human pro®lin isoforms (carbon, yellow; oxy-ated using the program O (Jones et al., 1991).

Figure 5. Stereo representation of structurally conserved interactions at the actin binding interface. Superposition ofhuman pro®lin II (blue; chain D) onto bovine pro®lin I (gray) complexed to b-actin (PDB accession code: 2btf) illus-trates the high degree of structural conservation of residues Phe59, Phe58, Arg55, and Asp75. His173 of b-actin stacksagainst Phe59 of bovine pro®lin I which in turn is stabilized by underlying aromatic and charged residues. TheFigure was generated using the program O (Jones et al., 1991).


with the carboxylate group of Asp13. There is aconserved hydrogen-bonding network in and sur-rounding this loop which includes coordination ofa water molecule by Asp13, the carbonyl oxygenatom of Leu10, and the backbone amides of resi-dues 15 and 16. The side-chain of Asp13 also formsa hydrogen bond with the terminal amino groupof Lys126, found on the second turn of the C-term-inal helix. Additionally, Met11 extends from the®nal turn of helix 1 to pack against the base of thisloop, which positions its d-sulfur atom in proxi-mity to the conserved loop.

Actin binding surface

In the X-ray structure of bovine pro®lin I com-plexed to b-actin, approximately 1050 AÊ 2 of thepro®lin I surface is excluded from solvent. Manypro®lin I residues which either directly interactwith b-actin or act to position side-chains contact-ing actin are conserved in the pro®lin II isoform.Suetsugu et al. (1998) demonstrated dramatic lossof af®nity for actin as a result of pro®lin His119mutation to glutamic acid. In the pro®lin:b-actincomplex, this histidine residue protrudes from thesurface of pro®lin and sits in a pocket largelyformed by Tyr133, Tyr169, Met355, and Phe375 ofactin. Similar to His119, Phe59 contributes to thestabilization of the pro®lin:actin complex. Mutationof Phe59 to alanine results in the disruption of thep-stacking interaction with His173 of actin and a14-fold decrease in af®nity for actin (SchluÈ ter et al.,1998). In contrast to His119, the rotomer confor-mation of Phe59 appears to be stabilized by three

conserved residues in pro®lin (Figure 5). The aro-matic ring of Phe59 participates in a T-shapedstacking arrangement (McGaughey et al., 1998)with Phe58 which in turn participates in anamino/aromatic interaction (Mitchell et al., 1994;Burley & Petsko, 1986) with the d-guanido groupof Arg55. This arginine is partially stabilized by anelectrostatic interaction with the carboxylate oxy-gen atoms of Asp75.

The actin-binding face of bovine pro®lin I orig-inally de®ned by Schutt et al. (1993) was used asthe basis for comparing the putative actin bindingsites of the human pro®lins. Overall, the actin-binding region of human pro®lin I is very similarto that of human pro®lin II with the exception of®ve amino acid differences. As seen in Figure 3(b),two notable differences are hydrophobic residuesVal60 and Leu122 of human pro®lin I which arethreonine in human pro®lin II. In the bovine pro®-lin I:b-actin complex, the closest atom to the side-chain of Met122 of pro®lin is 5.42 AÊ away (Cb ofMet122 to the e-carbon atom of actin Lys373). Theabsence of side-chain contact and indifference tosize at amino acid position 122 suggest that thisresidue contributes little to the stabilization of thepro®lin:actin complex. Val60 of bovine pro®lin Iforms a more involved peripheral interfacial con-tact, packing against Val287 and Arg290 of actin.In human pro®lin II, the threonine residue at pos-ition 60 displays a similar rotomer conformation asthat of bovine pro®lin I Val60. The substitution ofa hydroxyl for a g-methyl group at position 60could allow for an additional hydrogen bond tothe backbone carbonyl group of position 285 or the

Figure 6. Conserved key residues and non-conservedisoform differences highlighted at the pro®lin:actin inter-face. The bovine pro®lin I:b-actin complex (top; 2btf)has been opened and rotated towards the observer toreveal the protein binding surfaces (light blue). Humanpro®lin II (bottom) was superimposed onto bovine pro-®lin I and translated. Residues colored red (Phe59,Arg74, Arg88 and His119) have been shown to contrib-ute signi®cantly to actin binding. The footprints of theseresidues are shown mapped onto b-actin as red trans-parent outlines. The differences between human pro®linI and II at the actin-binding surface are colored darkblue (S56E, V60T, E82D, and L122T; ®rst letter corre-sponds to pro®lin I, the second to pro®lin II). Althoughposition 59 differs between human pro®lin I and II(Y59F) the essential character is conserved.


guanidium group of Arg290 of actin. Additionally,Cedergren-Zeppezauer et al. (1994) noted that thepresence of glutamic acid in position 56 of pro®linII (serine in pro®lin I) could result in the formationof a salt bridge to Lys284 of actin. The two ®naldifferences between human pro®lin I and II areconserved in character. Similar to Glu82 of bovinepro®lin I, the carboxylate group of Asp82 in pro®-lin II would be expected to form a salt bridge withthe amino-terminal group of Lys113 of actin.Amino acid position 59 is phenylalanine in allknown mammalian pro®lin sequences, with theexception of human pro®lin I which encodes atyrosine residue. The additional terminal hydroxylgroup would not be capable of forming anintermolecular hydrogen bond with actin, andthus would not contribute an additional energy ofbinding.

It has been shown in the case of human growthhormone binding to its receptor that residueswhich make signi®cant contributions in bindingenergy, known as ``hot spots'', are surrounded byresidues which contribute little to binding energy

(Clackson & Wells, 1995). Interestingly, all of thedifferences between human pro®lin I and II lie onthe periphery of the actin binding surface(Figure 6). Residues at these positions wouldbe expected to have little contribution to bindingenergy with the exception of the aromatic atposition 59. Mutation of Phe59 to alanine(Kd

mutant � 34 mM, Kdwild-type � 2.3 mM; SchluÈ ter et al.,

1998) corresponds to a binding energy of1.53(�0.28) kcal/mol, or more than 16 % of thetotal binding energy. Additionally, the amino acididentity, dramatic reduction in actin af®nity uponmutagenesis (Sohn et al., 1995; Suetsugu et al.,1998; Korenbaum et al., 1998), and structurallocation in the pro®lin:actin interface of three con-served residues in mammalian pro®lins, Arg74,Arg88 and His119, suggest these residues are alsokey energetic contributors (Figure 6). In contrast,positions 56, 60, 82, and 122 may function in theexclusion of solvent from the key energetic inter-actions (Bogan & Thorn, 1998) due to their locationin the actin-binding face, amino acid identity, andthe comparable af®nities of the human pro®lin iso-forms towards actin.

Poly-L-proline binding site

As determined by site-directed mutagenesis andlater con®rmed by crystallographic studies, resi-dues in pro®lin I which directly interact withstretches of poly-L-proline are Trp3, Tyr6, Trp31,His133, and Tyr139 (BjoÈ rkegren et al., 1993;Mahoney et al., 1997; Mahoney et al., 1999). Super-position of human pro®lin II onto human pro®lin Icomplexed to a pentadeca-prolylpeptide (1cjf)allows for an analysis of the peptide:pro®lin IIinteractions (Figure 7(a)). Comparison of humanpro®lin I poly-L-proline binding residues Trp3,Tyr6, Trp31, and His133 with the analogous pos-itions in human pro®lin II reveals virtually identi-cal rotomer conformations. In human pro®lin II,amino acid positions 133 and 139 are tyrosine andvaline residues, respectively. Analogous to His133of human pro®lin I, Tyr133 of human pro®lin II iscapable of hydrogen bonding to the carbonyl oxy-gen atom of Pro11 and stacking against Pro13 ofthe pentadecapeptide (proline numbering accord-ing to 1cjf complex). Due to the disordering of thelast two amino acids in human pro®lin II, back-bone positioning of Val139 was modelled based onmain-chain positioning of Tyr139 of human pro®-lin I, showing Val139 positioned to make van derWaals contact with Pro10.

Due to the presence of tyrosine at position 29in human pro®lin II, a slight shift in the prolinepentadecamer was introduced in the ®rst sixproline residues to relieve minor clashes. Themost striking feature of the pentadecamer mod-elled onto human pro®lin II is the evident exten-sion of the aromatic region of the poly-L-prolinebinding site in human pro®lin II by Tyr29(Figure 7(a)). This tyrosine residue protrudesfrom the surface of human pro®lin II and

Figure 7. (a) Surface differences of the poly-L-proline binding site between the pro®lin isoforms. Human pro®lin IIwas superimposed onto human pro®lin I complexed to a proline pentadecamer (PDB accession code: 1cjf). Surfaceand bond coloring corresponds to human pro®lin I (brown) and II (blue). Residues comprising the poly-L-prolinebinding site are illustrated as rods and labelled as in Figure 3. The prolylpeptide complexed to human pro®lin I(brown) was modelled (blue) to alleviate minor clashes with the protruding Tyr29 residue of human pro®lin II. Theprotein surfaces within 4 AÊ of the respective prolylpeptide are displayed in transparent (pro®lin I) and mesh (pro®linII) renderings. The Figure was generated using the program GRASP (Nicholls et al., 1991). (b) Architectural differ-ences between the human pro®lin isoforms in or proximal to the poly-L-proline binding site. Stereo ball-and-stickrepresentation of human pro®lin I (gray; PDB accession code: 1awi) superimposed with human pro®lin II (blue; chainD). Residue labels are marked with an asterisk * or parentheses () to indicate pro®lin I or II, respectively. Smallspheres represent hydrogen bonds illustrated for pro®lin II only. The Figure was generated using O (Jones et al.,1991).


accommodates an additional turn of the poly-L-proline helix. Tyr29 has the potential to form astacking interaction with Pro3 and a hydrogenbond with the carbonyl group of Pro4, whereasposition 29 in pro®lin I is a serine residue whichmakes van der Waals contacts to Pro3. Another

unexpected difference occurs at amino acid pos-ition 12. Cys12 of pro®lin II is capable of contri-buting a larger surface area to interact withPro16 than Ala12 of pro®lin I (Figure 7(a)).

Isoform-speci®c interactions contribute to vari-ations in the local architecture of the loop contain-


ing residue 29 (Figure 7(b)). Two charged residuesunique to the pro®lin II isoform, Lys28 and Asp48,participate in an electrostatic interaction in whichthe amino group of Lys28 forms a hydrogen bondwith the carboxylate group of Asp48, a residue onhelix 2. Pro®lin I has proline in amino acid position28 which forms a van der Waals interaction withPro44 of helix 2. A second stabilizing interaction inpro®lin II is formed between the carboxylate groupof Asp26 which participates in an electrostaticinteraction with the guanidinium group of Arg107,a residue found on the b-turn preceding b-strand7. Although the charges at position 26 and 107 areconserved in both human pro®lin isoforms, theydo not form an electrostatic interaction in any ofthe structures of human pro®lin I (1®k, 1®l, 1awiand 1cjf). Instead, the terminal amino group ofLys25 forms a weak salt bridge (4.2 AÊ ) to the car-bonyl oxygen atom of Asp106 (not illustrated inFigure 7(b)). In addition, the positioning of thebackbone in human pro®lin II places the carbonyloxygen atom of Ala27 within hydrogen bondingdistance of the indole nitrogen atom of Trp31. Fur-thermore, the carbonyl oxygen atom of Ala27hydrogen bonds with the backbone amide of Tyr29forming an i to i � 2 hydrogen bond characteristicof g-turns. Residues 27 to 29 of human pro®lin IIform an inverse g-turn with a negative f value forthe (i � 1) position, whereas residues 25-27 ofhuman pro®lin I form a classic g-turn with apositive phi value for the (i � 1) position (Milner-White et al., 1988). The positioning of this loop

Figure 8. Charge and hydrophobic differences betweenbinding site. Electrostatic surface potentials of human pro®lierated using GRASP (Nicholls et al., 1991). The brown patchin human pro®lin I and analogous residues in human pro®Also, the surface area of a ¯anking hydrophobic region diffecolored green and then mapped to the surface). The protcharged residues which differ between the pro®lin isoforms h

region in pro®lin II may be important for presen-tation of Tyr29 in a spatial context competent forbinding to poly-L-proline.

It has been shown that hydrophobicity playsan important role in protein:protein interactions(Janin & Chothia, 1978). A hydrophobic patchon bovine pro®lin I has been previouslydescribed which is separated by a polar strip ofresidues from the poly-L-proline binding site andde®ned by residues: Tyr24, Pro28, Pro44, Val51,Leu78, Phe83, Met104, Ala106, and Tyr128(Cedergren-Zeppezauer et al., 1994). Residuescomprising this hydrophobic patch are largelyconserved between bovine and human pro®lin I,with the exception of positions 104 and 106.This region in human pro®lin II is less con-served and does not form a contiguous hydro-phobic patch. In contrast with the patchdescribed for bovine pro®lin I, human pro®lin IIhas a surface-exposed hydrophobic region whichis contiguous with the poly-L-proline bindingsite. This hydrophobic area, neighboring Tyr29,is made up of side-chains from Pro44, Ile45,Met49, the Cd of Thr43 and the aliphatic portionof Lys28 (Figure 8). The large aliphatic side-chains of Ile45 and Met49 protrude from the sur-face in human pro®lin II in contrast to the smal-ler side-chains of Ala45 and Val49 in humanpro®lin I. These aliphatic regions near the poly-L-proline binding site may contribute to differ-ences in ligand speci®city.

human pro®lin I and II surrounding the poly-L-prolinen I (PDB accession code: 1®k) and II (chain D) were gen-represents side-chains of poly-L-proline binding residueslin II were colored brown and mapped to the surface.

rs between the two human isoforms (only side-chains areeins are displayed in the same orientation. All visibleave been labelled.


Acidic versus basic character of thehuman profilins

Human pro®lin I and II are often noted for theirbasic and acidic isoelectric points, respectively(HonoreÂ et al., 1993). Human pro®lin II has fouracidic residues (Asp48, Glu56, Glu95, Asp138) andtwo basic residues (Lys28 and Lys68) which arenot found in human pro®lin I. Likewise, humanpro®lin I has four charged residues (Lys25, Lys37,Asp106, His133) which are not conserved inhuman pro®lin II. The majority of these non-con-served differences between the two isoforms arelocated in (Asp138-II, His133-I) or proximal to(Lys25-I, Lys28-II, Asp48-II, Asp106-I) the poly-L-proline peptide binding site. The location of thesecharge differences suggests these residues mightcontribute to the differential af®nities displayed bythe pro®lin isoforms for poly-L-proline-rich pro-teins (Figure 8).

The two basic residues found in pro®lin II,but not in pro®lin I, participate in electrostaticinteractions with two of the four acidic residuesunique to pro®lin II. Lys28, on the loop betweenb-strands 6 and 7, forms a hydrogen bond withAsp48 which resides on helix 3 (Figure 7(b)).A second pairwise hydrogen-bonding interactionoccurs between Glu95 of b-strand 6 and Lys68of b-strand 4. The remaining two non-conservedacidic residues in pro®lin II are Glu56 andAsp138. Glu56 is fully exposed and does notparticipate in any intramolecular electrostaticinteractions. The position of Asp138 can only beapproximated in human pro®lin II due to poorelectron density. A molecular surface renderingof human pro®lin II indicates that Asp138 issituated adjacent to an acidic position unique tohuman pro®lin I (Asp106), suggesting that thelocalized acidic character has shifted slightlybetween isoforms. Similarly, each of the basicresidues speci®c to human pro®lin II (Lys28,Lys68) is spatially proximal to the position of abasic residue found only in pro®lin I (Lys25,Lys37).

Discussion

The structure determination of human pro®lin IIhas allowed for a comparative analysis of the twohuman pro®lin isoforms. Amino acid differencesbetween the isoforms are seen throughout the sur-face of the protein with the exception of themajority of the actin and poly-L-proline bindingfaces. These differences contribute to locally uniquechemical environments and topologies, which formthe structural basis for functional distinctionsbetween pro®lin I and II.

Mammalian pro®lin II isoforms display a higheraf®nity for proline-rich peptides than pro®lin I(Lambrechts et al., 1997; Jonckheere et al., 1999).Binding experiments of pro®lin I and II to a VASP-based peptide containing the (GP5)3 repeat demon-strated that bovine pro®lin II bound the VASP

peptide with a dissociation constant of 0.5mMwhereas pro®lin I bound with a Kd value of150 mM (Jonckheere et al., 1999). Elution of pro®linII from poly-L-proline Sepharose requires higherDMSO (this study; see Materials and Methods)or urea (Lambrechts et al., 1995) concentrationsthan pro®lin I, also indicating that pro®lin II has atighter association with proline-rich stretches. Thehigher af®nity of pro®lin II for proline-rich pep-tides may be attributed to the aromatic extensionof the poly-L-proline binding site by tyrosine 29.The presence of tyrosine at this position, in lieu ofserine as found in pro®lin I, may further stabilize abound proline stretch through hydrogen bondingand accommodating an additional turn of thepoly-L-proline type II helix via stacking and vander Waals interactions.

Other pro®lin surface features proximal to the``core'' poly-L-proline binding site need to beconsidered when the proline stretch is presentedin the context of a folded domain. Suetsugu andco-workers recently demonstrated pro®lin I bindsone order of magnitude (Kd � 60 nM) moretightly to N-WASP over pro®lin II (Kd � 400 nM;Suetsugu et al., 1998). Notably, the proline-richregion of N-WASP harbors several of the (GP5)repeats albeit not consecutively as in VASP. Thediscrepancy between the tighter binding of pro®-lin II to (GP5)3 and the higher af®nity of pro®linI to full-length N-WASP strongly suggests thatregions ¯anking the poly-L-proline binding siteare important for these differences in speci®city.As was shown in the case of the SH3 domains,the unusually tight binding of HIV-1 nef proteinto the SH3 domain of hck is due to a regionoutside the aromatic poly-L-proline binding site.A region on the hck SH3 domain known as theRT loop contains isoleucine in position 96 whichis critical for tight binding to HIV-1 nef protein(Lee et al., 1995). A crystal structure complex ofthe conserved core of HIV-1 nef protein boundto a high af®nity mutant of fyn SH3 domain(Arg96! Ile) shows the critical region of bind-ing to be located outside of the poly-L-prolinebinding site and increases the solvent-excludedsurface area from 780 AÊ 2 (poly-L-proline peptidebinding region only) to 1200 AÊ 2 (Lee et al., 1996;Lim, 1996). Comparative structural analysis ofthe human pro®lin isoforms shows that six outof ten charged differences are in or proximal tothe poly-L-proline binding face. Although theprecise elements of structural speci®city cannotbe addressed without mutagenesis and/or struc-tural studies of the particular pro®lin:ligandcomplex, the differences bordering the poly-L-proline binding site may play a critical role inrecognition of the region surrounding the pro-line-rich target.

It has been shown that a fraction of residues in aprotein:protein interface contribute the majority ofthe binding energy (Cunningham & Wells, 1993;Clackson & Wells, 1995; Bogan & Thorn, 1998).These key residues, referred to as hot spots, are


often located near the center of the interface andare most often tryptophan, arginine, or tyrosineresidues. In contrast, residues which lie on the per-iphery of the interface typically make insigni®cantcontributions to binding energy, and are thoughtto function primarily in the exclusion of solvent(for a review, see Bogan & Thorn, 1998). Withrespect to the pro®lin:actin interaction, both pro®-lin isoforms show comparable af®nity to actinwhich is consistent with the maintenance of keyactin binding residues and the location and natureof the amino acid differences between isoforms(Figure 6). Although systematic alanine-scanningof the actin binding surface of pro®lin along withthe respective dissociation constants has not beenreported, mutagenesis studies of Phe59 (SchluÈ teret al., 1998), Arg74 (Korenbaum et al., 1998), Arg88(Sohn et al., 1995) and His119 (Suetsugu et al.,1998) show dramatic effects on actin af®nity, andsuggest that these residues may be hot spots. Theperipheral differences between the human pro®linisoforms (positions 56, 60, 82, and 122) may resultin kinetic differences in the association rates of pro-®lin I and II with actin, as was seen in the case ofhuman growth hormone and its receptor that per-ipheral electrostatic residues function to increasethe rate of association (Cunningham & Wells,1993).

The binding of PIP2 to either of the mamma-lian pro®lin isoforms disrupts the pro®lin:actincomplex, however there is disagreement as tothe relative af®nities of each isoform for poly-phosphoinositides (Gieselmann et al., 1995;Lambrechts et al., 1997). It has been suggestedthat pro®lin is capable of associating with sev-eral PIP2 molecules via pro®lin's positive electro-static surface potential (Goldschmidt-Clermontet al., 1990; Fedorov et al., 1994a). There are tencharged differences between human pro®lin Iand II and structural comparison shows thatmany of these differences are spatially close tothe position of the charged residue in the otherisoform (Asp138-II and Asp106-I; Lys28-II andLys25-I; Lys68-II and Lys37-I) such that on aglobal scale the human pro®lin isoforms appearto share the same electrostatic patterns. Similarto Acanthamoeba pro®lin I and II (Fedorov et al.,1994a), the electrostatic surface of each of thetwo human isoforms presents two large basicpatches that are separated by a common borderof acidic residues (data not shown). Mutagenesisof single amino acids has been carried out tolocate residues critical for PIP2 interaction(Haarer et al., 1993; Sohn et al., 1995), yet noneabolish binding. Mutation of Arg88 to leucine inhuman pro®lin I results in a threefold decreasein PIP2 binding (Sohn et al., 1995). Interestingly,in three of the human pro®lin crystal structures(1®k, 1®l, and here), a negatively charged phos-phate or sulfate anion is bound to a basic pocketconsisting of Arg88, Asn99, His119 and themainchain amide of residue 120. Notably, 1®kwas solved in low salt, 30 % PEG 8000 and

50 mM potassium phosphate (Fedorov et al.,1994b), which would correspond to a 1:100 ratioof pro®lin I to phosphate. The general propen-sity of pro®lin to accommodate a negativelycharged group in this pocket lends support tothe idea that these residues participate in pho-phoinositide binding.

Overall, pro®lin isoforms from many differentspecies share the same global biochemical charac-teristics even with a considerable degree ofsequence variation. The two human isoforms havea higher level of sequence similarity, and yet differ-ences on the atomic level re¯ect the distinct signal-ling and biochemical pathways speci®c to thetissues in which each isoform is expressed. The®nding that pro®lin I and pro®lin II associate withdifferent functional complexes from brain lysatesindicates that these isoforms have distinct roles intissues where they are coexpressed (Witke et al.,1998). Sequence variation between pro®lin I and IIalso suggests the ability to bind to proline-richligands is differentially regulated by phosphoryl-ation. Pro®lin I is phosphorylated on Tyr139 in anEGF-stimulated assay which leads to the inabilityto bind to poly-L-proline (BjoÈ rkegren-SjoÈgren et al.,1997); whereas pro®lin II encodes a valine orphenylalanine residue in position 139. An atomicresolution structure of pro®lin in complex with afull-length proline-rich protein will be necessary toelucidate the structural determinants not evident inthe structural studies of pro®lin:peptide complexes.The comparative analysis presented here will aidcharacterization of the pro®lin:ligand interfacewith respect to isoform speci®city, and may pro-vide insight into the basis for two distinct pro®linisoforms.

Materials and Methods

Expression and purification of human profilin II

The cDNA for human pro®lin II was obtained fromATCC #84635, subcloned into a pET/T7 plasmid (Nova-gen) and transformed into Escherichia coli strainBL21(DE3). Low protein expression due to two unpre-ferred arginine codons was boosted using an ArgU plas-mid (courtesy of Professor J. Walker), resulting inapproximately 20 mg of protein from a ten liter fermen-tor growth. Human pro®lin II was puri®ed according toLindberg et al. (1988) with modi®cations. Brie¯y, thefreeze-thaw technique (Johnson & Hecht, 1994) was usedto extract human pro®lin II without cell lysis into thepoly-L-proline Sepharose loading buffer A (10 mM Tris-HCl (pH 8.0), 100 mM KCl, 100 mM glycine). The puri®-cation was performed in batch and human pro®lin IIwas eluted from the poly-L-proline Sepharose in severalwashes of buffer A with 50 % DMSO in contrast to 30 %for the pro®lin I isoforms. Protein was dialyzed into15 mM Tris (pH 7.5), 50 mM KCl under nitrogen andconcentrated by ultra®ltration to 30 mg/ml. Human pro-®lin II was passed over an S-100 column (Pharmacia),desalted using a PD-10 column (Pharmacia) into 15 mMTris-HCl (pH 7.5), 10 mM DTT and concentrated byultra®ltration (Amicon/centricon) to 18 mg/ml. Proteinpurity was monitored by silver and Coomassie stain as

Table 1. Data collection and statistics

A. Data setSpace group P21

Cell dimensions (AÊ ; deg.) a � 71.43b � 44.80c � 89.60b � 98.59

Molecules per asymmetric unit 4Solvent content (%) 56Resolution (AÊ ) 50-2.2No. of reflectionsObserved 430,124Unique 28,050hI/s(I)i 10.3Completeness (%)

Overall 96.9Highest shell 83.5

Rmergea (%) 8.9

B. Refinement statisticsResolution (AÊ ) 20-2.2Rcryst

b (%) 21.51Rfree

c (%) 26.78No. of water molecules 398Average B-factor (AÊ 2)

All atoms 23.00Overall (protein) 17.57Main-chain 17.40Side-chain 17.78Water molecules 27.82

rms deviations from idealityBonds (AÊ ) 0.007Angles (deg.) 1.34

a Rmerge � �jI ÿ hIij/�I , where hIi is the average of sym-metry-equivalent re¯ections and the summation extends overall observations for all unique re¯ections.

b Rcryst � �jjFoj ÿ jFcjj/�jFoj, where Fo and Fc are theobserved and calculated amplitudes.

c Rfree set contains 5.3 % of total re¯ections.


well as electrospray mass spectroscopy which showed amolecular mass corresponding to human pro®lin II lack-ing an amino-terminal methionine residue. This recombi-nantly expressed pro®lin lacked the N-terminalacetylation commonly observed on pro®lins puri®edfrom mammalian tissue. Protein concentration wasmeasured using an extinction coef®cient of 1.4 at280 nm.

Crystallization and data collection

Human pro®lin II crystals grew in hanging drops con-sisting of 2 ml of protein solution mixed 1:1 with andequilibrated against a well solution of 1.35 M MgSO4,1.5 % (v/v) PEG 400, 100 mM Hepes (pH 7.3) at 4 �C.Crystals were bathed in increasing concentrations of eth-ylene glycol plus mother liquor and ¯ash frozen at a®nal concentration of 12 % (v/v) ethylene glycol. Datawere collected at 100 K at Brookhaven National Labora-tories on beamline X9B. Human pro®lin II crystallized inthe space group P21 with four molecules in the asym-metric unit. Data reduction and scaling were performedusing DENZO/HKL suite (Otwinowski & Minor, 1997;see Table 1).

Structure determination

Bovine pro®lin I (PDB accession code 1pne) wasemployed as a molecular replacement search model,with all non-conserved residues modi®ed to alanine.Molecular replacement solutions for all four molecules inthe asymmetric unit were determined using AMoRe(Navaza, 1994) as part of the CCP4 suite of programs(Collaborative Computational Project, 1994). The ASU isa dimer of dimers with roughly 222 NCS symmetry.Cycles of building and re®nement were performed usingO (Jones et al., 1991) and XPLOR with bulk solvent cor-rection (BruÈ nger, 1993). During the ®rst cycles of re®ne-ment NCS restraints grouped all four molecules. In laterstages NCS restraints grouped A to B and C to D, and inthe ®nal rounds of re®nement all NCS restraints werelifted. Solvent molecules were added to stereochemicallysensible positions and a sulfate group was placed inidentical positions for each of the four chains. Also, twoPEG 400 molecules of varying length were modelled asdescribed in the text.

Analysis

Structures of mammalian pro®lin I used in structuralcomparisons with human pro®lin II were: PDB accessioncode 1®k (human pro®lin I crystallized in low salt;Fedorov, A.A., Pollard, T.D., Almo, S.C.), 1®l (humanpro®lin I crystallized in high salt; Fedorov, A.A., Pollard,T.D., Almo, S.C.), 1cjf (Mahoney et al., 1999), 1awi(Mahoney et al., 1997), 1pne (Cedergren-Zeppezauer et al.,1994), and 2btf (Schutt et al., 1993). All solvent-accessiblesurface area calculations were performed using GRASPwith a probe radius of 1.4 AÊ (Nicholls et al., 1991). Pro-tein secondary structure was analyzed using PROMOTIF(Hutchinson & Thornton, 1996) and protein superposi-tions were performed using O (Jones et al., 1991).

Coordinates

The coordinates and experimental amplitudes havebeen deposited in the Protein Data Bank (PDB accessioncode 1D1J).

Acknowledgments

We are indebted to Kevin Parris for his crystallo-graphic advice and support. We thank Robert Sweet fororganizing DataCol99, the workshop in which this data-set was collected, and Zbigniew Dauter for assistance onbeamline X9B. We are grateful to Elspeth Garman fortechnical advice and to Herwig SchuÈ ler for critical com-ments on the manuscript. We thank Professor J. Walkerfor the use of the ArgU plasmid for protein expression.I.M.N. was supported by an NIH pre-doctoral traininggrant in molecular biophysics (GM08309). G.D.B. andC.E.S. were supported by grants from the National Insti-tutes of Health (GM44038) and U.L. from the SwedishCancer Foundation and the Swedish Natural ScienceResearch Council.

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Edited by R. Huber

(Received 27 July 1999; received in revised form 13 October 1999; accepted 13 October 1999)

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