DOI: 10.1002/cbic.200900616

Structural Diversity of PDZ–Lipid InteractionsRodrigo Gallardo,[a, b] Ylva Ivarsson,[a] Joost Schymkowitz,[b] Fr�d�ric Rousseau,[b] andPascale Zimmermann*[a]

Dedicated to the Seefeld meeting community and Mario Gimona.

456 � 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ChemBioChem 2010, 11, 456 – 467

Introduction

PDZ domains are globular domains that have a typical foldcomprising six b strands (bA/bF) and two a helices (aA andaB). PDZ domains were first discovered in the postsynapticdensity protein PSD-95 as three repeats of approximately 90amino acids containing a conserved Gly-Leu-Gly-Phe motif. Thename is an acronym derived from the first three proteinsfound to contain such domains, namely PSD-95, Drosophiladiscs large tumor suppressor, and zonula occludens-1. Thestructural elements form a b sandwich capped by the a helices(Figure 1 A). These domains are found in single or multiplecopies in a wide variety of intracellular proteins. Proteins con-

taining PDZ domains generally act as scaffolds that control theassembly and the subcellular location of signaling complexes.[1]

PDZ domains are over-and-above recognized as protein-in-teracting modules. The PDZ–peptide interactions have beenunder intense studies the last decade, and general features ofthese interactions were reviewed recently.[2] Canonical PDZ–peptide interactions are achieved by b addition of short pep-tides motifs of cognate proteins. These peptide motifs, calledPDZ-binding motifs (PDZBM), usually correspond to the last 4

to 6 residues of membrane receptors, but PDZ domains canalso interact by b addition with internal peptide stretches.[3]

Some deviations of this binding mechanism have been report-ed in which the bound peptide only interacts through its lasttwo residues, and no extension of the b sheet is observed. Thisdeviation is referred to as noncanonical peptide binding andhas been reported for few proteins, such as the PDZ1 domainof syntenin-1 and the PDZ domain of tamalin.[4, 5] The PDZ in-teractions are highly variable and versatile; one PDZ domaincan bind different PDZBM, and one PDZBM can be recognizedby different PDZ domains. The average dissociation constant(KD) for the canonical PDZ–protein interaction is in the low-mi-cromolar range, which is appropriate for systems under tight

[a] R. Gallardo, Dr. Y. Ivarsson, Prof. P. ZimmermannDepartment of Human Genetics, K.U.LeuvenHerestraat, 49 Box 602, 3000 Leuven (Belgium)Fax: (+ 32) 16-347 166E-mail : pascale.zimmermann@med.kuleuven.be

[b] R. Gallardo, Dr. J. Schymkowitz, Dr. F. RousseauVIB Switch LaboratoryDepartment of Molecular and Cellular InteractionsVrije Universiteit Brussel, Pleinlaan 2Building E, 1050 Brussel (Belgium)

PDZ domains are globular protein modules that are over-and-above appreciated for their interaction with short peptidemotifs found in the cytosolic tail of membrane receptors, chan-nels, and adhesion molecules. These domains predominate inscaffold molecules that control the assembly and the locationof large signaling complexes. Studies have now emergedshowing that PDZ domains can also interact with membranelipids, and in particular with phosphoinositides. Phosphoinosi-tides control various aspects of cell signaling, vesicular traffick-ing, and cytoskeleton remodeling. When investigated, lipid

binding appears to be extremely relevant for PDZ protein func-tionality. Studies point to more than one mechanism for PDZdomains to associate with lipids. Few studies have been fo-cused on the structural basis of PDZ–phosphoinositide inter-actions, and the biological consequences of such interactions.Using the current knowledge on syntenin-1, syntenin-2, PTP-Bas, PAR-3 and PICK1, we recapitulate our understanding ofthe structural and biochemical aspects of PDZ–lipid interac-tions and the consequences for peptide interactions.

Figure 1. Peptide binding and putative PtdIns-4,5-P2 binding sites partially overlap in the PDZ1 and the PDZ2 domain of syntenin-1 (PDB ID: 1N99). A) Tubediagram of the PDZ1 domain of syntenin-1 highlighting the peptide binding groove (dark gray). The residues Lys119, Ser171, Asp172, and Lys173, correspond-ing to the quadruple mutation abolishing PtdIns-4,5-P2 binding, are shown as sticks. B) Accessible surface representation of the PDZ2 domain of syntenin-1highlighting the peptide binding groove (dark gray) and the putative PtdIns-4,5-P2 binding site (black) as determined by mutational analysis performed onthe PDZ1 domain (in panel A). C) Accessible surface area representation of the PDZ2 domain of syntenin-1 highlighting the peptide binding groove (darkgray) and the putative PtdIns-4,5-P2 binding site (black) as concluded from the tamalin-phosphate bound model.

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regulation that must quickly respond to changes in their envi-ronmental conditions. Yet, KD values ranging from nanomolarto micromolar have been observed.

Many PDZ proteins present more than one PDZ domain. Insome cases, PDZ domains are connected through a short pep-tide linker to form tightly packed PDZ tandems. In certaincases, such tandems should be considered as single functionalunits.[6] For example, the GRIP1 PDZ4–5 tandem binds the pep-tide partner with a stoichiometry of one peptide-binding part-ner per tandem PDZ4–5. This surprising finding was explainedby the GRIP1 PDZ4 peptide-binding pocket being deformedand nonfunctional, and its function was found to be to providecrucial structural support for the adjacent PDZ5 domain, which

is completely unfolded in isolation.[7] In other cases, the func-tions of PDZ tandems have been shown to be intricately regu-lated by autoinhibition.[6, 8, 9] An interesting case is the PDZtandem of X11 in which the first PDZ domain is autoinhibitedby binding to the C terminus of the second PDZ domain.[10]

However, upon a Tyr-to-Glu mutation of the �1 position of theC-terminal tail, the tail shifts its preferred binding site to thesecond PDZ domain, thus suggesting that phosphorylation ofthe C-terminal tail might regulate the binding target of theX11 PDZ12 module.

PDZ interactions are regulated by several ways, as expectedfrom their critical functions in assembling protein complexes.One of the best-documented ways of regulation is phosphory-lation of residues (serine, threonine, or tyrosine) within thePDZBM. In some cases, phosphorylation abolishes PDZ–pep-tide interactions, as in the case of the Kir2.3 channel with thePDZ domains of PSD-95.[11] In other cases, phosphorylationincreases the affinity, as observed for the C terminus of MRP2with three different PDZ proteins (PDZK1, IKEPP, and EBP50).[12]

PDZ interactions can also be regulated by changes in pH orionic strength. Such environmental variations can directlyaffect the KD of PDZ–PDZBM interactions and might abrogatethe formation of PDZ–protein complexes. For instance, theaffinity of the PDZ3 domain of PSD-95 for the peptide KQTSV

Rodrigo Gallardo is an engineer in mo-

lecular biotechnology from the Univer-

sity of Chile, Santriago, Chile. In 2006

he joined the Zimmermann group for

predoctortal studies on the structural

biology of PDZ–lipid interactions. In

2008 he moved to the group of Fr�d-

�ric Rousseau and Joost Schymkowitz

to do a PhD on protein misfolding and

aggregation kinetics.

Ylva Ivarsson was trained (Master of

Science) at the University of Uppsala,

Sweden, where she obtained her PhD

in biochemistry with Bengt Mannervik

in 2006. She moved to Italy as a post-

doctoral fellow, studying folding and

peptide binding of PDZ domains with

Maurizio Brunori in the Department of

Biochemical Sciences at the Sapienza

University of Rome. In 2009 she

moved to Belgium for a second post-

doctoral term and is now working with

Pascale Zimmermann on the integration of lipid and peptide bind-

ing by PDZ domains.

Joost Schymkowitz obtained his PhD

in 2001 from the University of Cam-

bridge where he worked on mecha-

nisms of protein folding in the labora-

tory of Sir Alan Fersht. He then moved

to the European Molecular Biology

Laboratory in Heidelberg where he did

postdoctoral work in the laboratory of

Luis Serrano. There he contributed to

the development of several structural

modeling tools. He is now a group

leader at the Flanders Institute for Bio-

technology at the Free University of Brussels where he conducts

research on mechanisms of protein misfolding and aggregation.

Fr�d�ric Rousseau also obtained his

PhD in 2001 from the University of

Cambridge where he worked on

mechanisms of protein folding in the

laboratory of Sir Alan Fersht. He then

moved to the European Molecular

Biology Laboratory in Heidelberg

where he carried out postdoctoral

work in the laboratory of Luis Serrano.

There he contributed to the develop-

ment of several structural modeling

tools. He is now a group leader at the

Flanders Institute for Biotechnology at the Free University of Brus-

sels where he conducts research on mechanisms of protein mis-

folding and aggregation.

Pascale Zimmermann obtained her

PhD at the Christian de Duve Institute,

UCL, Brussels, Belgium in 1995 for her

work on the role of chromatin struc-

ture in the control of gene expression

with Guy Rousseau. She carried out

postdoctoral studies with Guido David

on proteoglycans (K.U.Leuven, Leuven,

Belgium), with Jo�l Vandekerckhove

on the cytoskeleton (RUG, Gent, Bel-

gium), and with Vic Small on live-cell

imaging at the Austrian Academy of

Science, Salzburg, Austria. She is currently a research professor at

the K.U.Leuven Belgium, and investigates how PDZ scaffolds and

signaling lipids control cell fate.

458 www.chembiochem.org � 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ChemBioChem 2010, 11, 456 – 467

P. Zimmermann et al.

decreases when the acidity of the medium drops belowpH 7.0. Furthermore, depending on the relative abundance ofthe protein partners, a rise in chloride concentration from 30to 150 mm might compete out a large fraction of cognate pro-teins bound in cells.[13]

More recently, it has become evident that at least some PDZdomains also interact with membrane and even nuclear lipids,and in particular with signaling lipids, namely phosphoinosi-tides (PIPs).[14–18] Phosphatidylinositol is, together with phos-phatidylcholine, phosphatidylethanolamine, phosphatidylser-ine, and phosphatidic acid, one of the main phospholipids inmammalian cell membranes. A small proportion of the phos-phatidylinositol is phosphorylated at one, two or three hydrox-yl groups of the inositol headgroup (the 3-, 4-, and/or 5-posi-tion) generating a total set of seven PIPs that can either serveas precursors for second messengers, or exert a function asmembrane-bound signaling molecules regulating the localiza-tion and function of protein-signaling complexes.[19] Althoughthey constitute a small fraction of the cellular PIPs, they controla whole range of cellular processes like the activity of ionchannels and transporters, vesicular trafficking, membrane andactin dynamics, cell metabolism, growth and differentiation,immune response, chromatin remodeling, and RNA metabo-lism.[20] Moreover, PIPs are key factors in cell polarization.[21]

The level of PIPs can be tightly regulated by the many PI kinas-es and phosphatases that are distributed in the different intra-cellular compartments. For example, whereas PtdIns-4,5-P2 andPtdIns-3-P are constitutively present on the plasma membraneand endosomes, respectively, PtdIns-3,4,5-P3 is transiently in-duced at the plasma membrane in response to external stimuli.Protein domains capable of interacting with both lipids andpeptide motifs, or proteins containing distinct domains inter-acting with lipids and peptides, might serve as hubs for inte-gration of lipid and peptide signaling. Members of severalmodular protein domain families, such as PH, FYVE, PX, ENTH,CALM, PTB, and FERM domains are well-known PIP interac-tors.[22] Even though their structure and PIP-binding sites arediverse, they exploit a common set of mechanisms to interactwith cell membranes, such as electrostatic recruitment, mem-brane penetration, and PIP binding.[23] Many of these domainshave low affinity for PIPs, and are therefore not able on theirown to recruit their target proteins to proper membrane locali-zation. To achieve these goals, they often act together withother protein domains to build complexes with higher avidityfor membranes. Such complexcan be exclusively built by onekind of domain that is repeatedin the same polypeptide chainor that oligomerizes, as in thecase of the PH domains of dyna-min,[24] or by different types ofdomains in the same polypep-tide chain or protein complex, asin the case of sortin nexin pro-teins that contains a PX and aBAR domain.[25] By extension,PIPs might thus play a pivotal

role in controlling the localization of PDZ complexes as well as,for example, in sorting and assembly.

Diverse techniques have been used to study PDZ–PIP inter-actions, most of which have been validated in the study ofother PIP-interacting domains, and the advantages and disad-vantages of the different techniques have been reviewed else-where.[26, 27] In general, care should be taken not to over-inter-pret electrostatic interactions as specific PIP binding. In thefield of PDZ–PIP interactions, sedimentation assays with lipo-somes prepared from total bovine brain lipid extracts and size-exclusion chromatography of PDZ domains binding to micellesof pure lipids have been used for screening purposes.[15, 16, 18, 28]

However, the poor characterization of constituents of thebovine brain lipid extracts and the extremely high concentra-tion of PIPs in the micelles make these techniques suitableonly for initial screening. The PIP-binding selectivity has beenaddressed by the use of so-called PIP strips in which purelipids are spotted on hydrophobic membranes. Furthermore,analytical techniques such as surface plasmon resonance (SPR)have been used to quantitatively compare the affinity of inter-action with the different PIPs. The use of composition-definedliposomes, in which different PIPs can be incorporated in anenvironment that mimics the cytosolic leaflet of cellular mem-branes, appears to be particularly interesting.[14, 18]

An overview of the PDZ–PIPs interactions characterized sofar and of the techniques used is given in Table 1. In thisreview we recapitulate these studies, highlight the commonand divergent features of the lipid/PIP-binding sites in the dif-ferent PDZ domains, and summarize our current understand-ing of lipid/PIPs regulation of PDZ–peptide interactions.

The Syntenins

Syntenin-1–PtdIns-4,5-P2 interaction

Syntenin-1 contains a PDZ tandem connected by a short fouramino acid linker, a 110 amino acid N-terminal domain with noobvious structural motif, and a short 24 amino acid C-terminaldomain. This protein was first identified as an intracellular part-ner for the syndecans.[29] Syndecans constitute a family of hep-aran sulfate proteoglycans that are implicated in cell adhesionand growth factor signaling. Syntenin-1 was later shown tobind a plethora of target proteins, which suggests that thisscaffolding protein might be involved in a variety of cellular

Table 1. Different biochemical approaches used for the characterization of PDZ–PIP interactions.

PDZ Biochemical assay Mutationanalysis

PIPstrips

Liposomesedimentation

SPR NMR[a]

syn1 1 + 2 size-exclusion chromatography + + + +

syn2 1 + 2 + + +

PTP-Bas 2b size-exclusion chromatography + +

PAR-32 membrane insertion + + + +

PICK1 membrane protection + +

[a] Most of the NMR spectroscopy studies have been carried out in phosphate buffer. Phosphate ions in thebuffer system might introduce noise in the determination of the binding site, as the PDZ–PIP interaction islikely to be mediated by the recognition by basic positively charged amino acids of the phosphates groups ofthe PIPs.

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PDZ–Lipid Interactions

processes.[30] The structural basis of peptide binding has beenestablished in several biochemical and/or X-ray crystallographystudies,[4, 29, 31, 32] demonstrating that the two PDZ domains ofsyntenin-1 have distinct peptide-binding specificities.

The interaction between PIPs and PDZ domains was first dis-covered in studies on the biology of syntenin-1.[17, 18] It wasshown in cell experiments that syntenin-1 concentrates at theplasma membrane. As for many other PDZ domain proteins, itwas assumed that this localization only relies on the interac-tion with cognate membrane receptors. However, studies re-vealed that over-expressed syntenin-1 strongly colocalizes withpools of PtdIns-4,5-P2. Moreover, this localization is lost uponPtdIns-4,5-P2 breakdown, which is induced by ionomycin/calci-um; this indicates that membrane localization of over-ex-pressed syntenin-1 is PtdIns-4,5-P2 dependent. The structuralbasis of syntenin-1–PtdIns-4,5-P2 binding is far from completelyunderstood (see below). Several cellular, biochemical, and mu-tational studies have indicated that both PDZ domains cooper-ate for PtdIns-4,5-P2 binding and membrane localization. ThePDZ1 and PDZ2 domain of syntenin-1 appear to have respec-tively medium and low affinity for PtdIns-4,5-P2 in isolation.The PDZ tandem corresponds to a high-affinity module that isable to target the protein to the plasma membrane. This addsPDZ domains to the list of domains combining low-affinity PIP-binding sites for efficient targeting.[23, 33] The syntenin-1–PtdIns-4,5-P2 interaction has also been proven to be essential for thesorting of the syndecans to endosomes, recycling to theplasma membrane, and for cell spreading.[34] This indicates thatPIP binding constitutes a mechanism for sorting PDZ proteinsand PDZ–protein complexes to different subcellular domains.

Syntenin-2–PtdIns-4,5-P2 interaction

Syntenin-2 is the closest homologue of syntenin-1. The overallsequence identity is 58 %, but increases up to 69 % identity(87 % similarity) if considering only the tandem of PDZ do-mains. The cognate peptide partners of syntenin-1 do not in-teract with syntenin-2, and so far only one protein, the tetra-spanin L6 antigen, has been proposed to interact with synte-nin-2.[35] Besides these differences in protein binding, syntenin-2 shares the ability to interact with PIPs.

The interaction of syntenin-2 with PIPs was characterized byMortier and co-workers by surface plasmon resonance (SPR)experiments with reconstituted liposomes.[14] The strongest in-teractions were observed with PtdIns-4,5-P2 and PtdIns-3,4,5-P3,for which the KD values were in the low-micromolar range. Insyntenin-2 both PDZ domains have similar affinity for PtdIns-4,5-P2 in isolation. In cell localization assays the tandem of PDZdomain, fused to eGFP, was capable of targeting the protein-to-membrane and nuclear PtdIns-4,5-P2 pools, but the isolatedsyntenin-2 PDZ domains displayed diffuse localization. Thus,syntenin-2 combines two domains of moderate affinity to gen-erate a supramodule with high affinity for PtdIns-4,5-P2. Therelevance of the syntenin-2–PtdIns-4,5-P2 interaction was fur-ther tested by an in vivo loss-of-function experiment in cul-tured cells. In such experiments syntenin-2 depletion disrupts

nuclear speckles PtdIns-4,5-P2 distribution. It also reduces cellviability and the rate of cell division.

Syntenin-1–PtdIns-4,5-P2 versus syntenin-1–peptideinteraction

The effect of PtdIns-4,5-P2 on the syntenin-1 PDZ–peptide in-teraction has been tested by several biochemical approaches.By mutational analysis it was determined that the peptide andPtdIns-4,5-P2-binding sites partially overlap. By using pull-downassays with C-terminal peptides containing the PDZBM fromcognate receptors, it was demonstrated that PtdIns-4,5-P2, butnot phosphatidylinositol, can compete with the interaction be-tween the PDZ tandem and the C-terminal peptides;[18] thiscasts doubt on the idea that both peptide and PtdIns-4,5-P2

binding can occur simultaneously. However, in retrospect, theexperimental setup might not have been optimal. In the assay,the cognate peptides were closely linked to Sepharose beads,whereas PtdIns-4,5-P2 was presented on liposomes or micelles.It is difficult to envision how a PDZ domain, in this experimen-tal set-up, could simultaneously interact with the two bindingtargets, and there was hence a bias in the experimental set-upfor a competitive outcome. What really the situation is at thecellular membrane where both ligands are presented in thesame phase remains to be investigated.

Structural biology of syntenins–PtdIns-4,5-P2 interaction

A quadruple mutant of syntenin-1 known to have impairedpeptide-binding abilities was shown to also display a loss ofPtdIns-4,5-P2 binding by both in vivo localization studies andin vitro SPR binding studies.[18] The positions probed by muta-genesis correspond to the peptide-binding loop (Lys119Ala)and the N-terminal region of aB helix (Ser171-Asp172-Lys173to His-Glu-Gln) in the PDZ1 domain (Figure 1 A). Structurallyequivalent quadruple mutants in the second PDZ domain ofsyntenin-1 also affect PIP binding, but to a lesser extent, anddo not abolish the plasma membrane recruitment of thetandem in localization assays. Yet, the regions probed by muta-genesis are located at distinct ends of the peptide-bindinggrooves (Figure 1 B) of PDZ1, and do not cluster to form theexpected basic polar pocket as observed in other PIP-bindingdomains.[22] It is thus unlikely that all four residues directly con-tribute to the PIP binding, but rather likely that one of the twodistinct regions corresponds to the binding site of PtdIns-4,5-P2. Similarly, Meerschaert and co-workers[36] studied a triplemutant of syntenin-1, Lys124Ala-Arg128Ala-Lys130Ala, in whichall mutations are located in the first PDZ domain. This mutantdisplayed reduced PIP binding in PIP strip assays and a loss ofmembrane localization. Yet the same remarks as for the above-described mutations can be formulated. All three positions aremost likely not involved directly in the PtdIns-4,5-P2 binding.

A similar mutational strategy was used by Mortier and co-workers to characterize the interaction of syntenin-2 withPtdIns-4,5-P2. The same regions, the peptide-binding loop andthe N terminus of aB helix, were studied by introducing themutations Lys113Ala and Lys167Ala. A structurally equivalent

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P. Zimmermann et al.

double mutant was constructed in the PDZ2 domain (muta-tions Lys197Ala and Lys244Ala). In both cases the mutatedPDZ domains in isolation lost their binding to composite lipo-somes containing PtdIns-4,5-P2.[14] Once again mapping theprobed positions on the surface of a structural model of thePDZ domains of syntenin-2 revealed that the residues are lo-cated at the most distinct regions of the peptide-bindinggrooves and do not cluster to form a polar PIP-binding site. Asin the syntenin-1 case, it may be speculated that one of thetwo probed regions is responsible for PtdIns-4,5-P2 binding,but further structural biology and biochemical studies are nec-essary to clarify this point.

A computational docking study suggested a structuralmodel of the syntenin-1–PtdIns-4,5-P2 interaction based on acrystal structure of the PDZ domain of the nonhomologous ratprotein tamalin, or GRP1-associated scaffolding protein. Thetamalin PDZ, fused with a C-terminal peptide ligand, was crys-tallized in a phosphate buffer, and two phosphate ions werefound in the structure. The distance between the phosphateions and their position in the N-terminal region of the aB helixwere claimed to reflect the positions of the phosphate groupsof a hypothetically bound PtdIns-4,5-P2.[37] Specifically, thephosphate ions directly interact with the residues Arg166,His167, and Arg168. The peptide was found to be bound inboth canonical and noncanonical binding modes, but thephosphate ions were found only in the subunits binding thepeptide in a noncanonical mode; this suggests that in thiscase, phosphate ion binding is not compatible with canonicalpeptide binding. A docking model of the syntenin-1–PtdIns-4,5-P2 interactions was constructed for the second PDZ (Fig-ure 1 C). In the model, the residue Ser252 of syntenin-1 (at theposition of Arg168 of tamalin) forms H-bonds with the 4’ and5’ phosphates groups of PtdIns-4,5-P2. The Lys250 of syntenin-1 (corresponding to Arg166 of tamalin) forms an H bond withthe 4’ phosphate group. Both of these residues are close tothe N-terminal domain of the aB helix. However, the crucial in-teractions between His175 of tamalin and the phosphate ionsare not provided by the structurally equivalent Asp251 of syn-tenin-1. An additional interaction between the Lys214 at theend of bB strand and the 3’ OH group of the inositol ringmight be established according to the docking model. Themodel thus suggests that the binding site for the phosphory-lated inositol headgroup is close to the exit of the peptide-binding groove. In the model, the diacylglycerol moiety ofPtdIns-4,5-P2 was assumed to be bound and was manuallyplaced on the partially hydrophobic peptide-binding groove ofsyntenin-1. According to this model, the aliphatic chains of thelipid would cover most of the peptide-binding groove (Fig-ure 1 C); this implies that PtdIns-4,5-P2 binding is not compati-ble with any mode of peptide binding, even though the dock-ing was originally based on a complex of the PDZ domain oftamalin in which phosphate ion binding and noncanonicalpeptide binding occurred simultaneously.

In conclusion, our understanding of syntenin–PtdIns-4,5-P2

interaction and its influence on peptide binding is still amatter of debate. Complementary experiments, in particularcrystallographic data, should provide better insight.

The Protein Tyrosine Phosphatase-Basophil

PIP interaction

Protein tyrosine phosphatase-basophil (PTP-Bas) is a large non-receptor-type phosphatase that modulates diverse cellularfunctions in a context-dependent manner. The PDZ2 domainof PTP-BL, the mouse homologue of PTP-Bas, was identified asa weak PtdIns-4,5-P2 interactor in gel filtration experimentswith PtdIns-4,5-P2 micelles.[18] One year later the interactionbetween the PDZ2 domain of PTP-Bas, which closely resemblesPTP-BL (more than 95 % identity), and PIPs was characterizedby NMR spectroscopic analysis.[18, 28] This domain has two splic-ing variants, PDZ2a and PDZ2b, which differ by the insertion offive amino acids between the end of bB and the bB-bC loop inPDZ2b. Whereas the PDZ2a variant is able to interact with C-terminal cognate peptides, PtdIns-4,5-P2 and PtdIns-3,4,5-P3,the spliced version interacts with PIPs but seems to lose theability to interact with cognate proteins. The GST-PDZ2a fromPTP-Bas has a micellar KD of about 55 mm for PtdIns-4,5-P2. Thesplicing variant PDZ2b has a higher affinity than the PDZ2avariant, with a KD of 21 mm. This affinity is comparable to thatobtained for the GST-syntenin-1 PDZ1 in the same assay. Theinteraction of PDZ2b with a soluble form of PtdIns-3,4,5-P3

yielded a KD of about 200 mm as calculated from NMR spectro-scopic titration assays. The KD obtained by using the solublevariant of PtdIns-4,5-P2 was of the same magnitude. The signifi-cant differences between the KD obtained by gel filtrationassays and NMR spectroscopy assays might come from the dif-ferent buffer system used (Tris-HCl in the gel filtration assaysand 50 mm phosphate buffer in the NMR spectroscopy assays),but might also be explained by additional interactions estab-lished between the micelles and the PDZ domain, which arenot established when soluble PIPs are dispersed in the solutionof the NMR spectroscopic analysis.

PTP-Bas–PtdIns-3,4,5-P3 versus PTP-Bas–peptide interaction

The insertion of five amino acids in the bB strand of the splic-ing variant PTP-Bas PDZ2b has no impact over the overall foldof the domain, but introduces two structural modifications: itshortens the bB strand and it reorients both a helices with re-spect to the protein core (Figure 2 A). Shortening the bB strandaffects the peptide binding by narrowing the peptide-bindinggroove. In contrast to peptide binding, the modifications posi-tively affect PtdIns-4,5-P2 and PtdIns-3,4,5-P3 binding.[28] Nocompetition experiments were conducted for peptide bindingof PTP-Bas PDZ2 in presence of PtdIns-4,5-P2 or PtdIns-3,4,5-P3,leaving the relation between peptide and lipid binding in thePTP-Bas PDZ2 an open question.

Structural biology of PTP-Bas–PtdIns-3,4,5-P3 interaction

The binding site for PIPs in the PDZ2b domain of PTP-Bas wasdetermined by NMR spectroscopy by measuring the changesof the chemical shifts in the 15N HSQC spectra upon titrationwith a soluble, C8 fatty acid version of PtdIns-4,5-P2 and PtdIns-

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PDZ–Lipid Interactions

3,4,5-P3. Chemical shift changes of diverse intensity were ob-served over a broad collection of residues, which might reflectthe low affinity of the interaction between soluble isolatedPIPs and PDZ2 of PTP-Bas. Titration with both PtdIns-3,4,5-P3

and PtdIns-4,5-P2 produced chemical shift changes that spreadover the bB strand, the bD-bE loop and both ends of the aBhelix (Figure 2 B). The authors interpreted the data to meanthat these residues (Ile24, Val26, Val70, Asn71, Gly77, Ala78,Glu85, Arg88, Gly91, and Gln92 of the PDZ2b) form a pocketthat corresponds to the binding site for PIPs. However, it is dif-ficult to realize how these residues cluster together when map-ping their distribution on the surface of PTP-Bas PDZ2b (Fig-ure 2 C). One explanation for the broad distribution of chemicalshift changes is that some of them correspond to residues thatdo form a binding site for the phosphorylated PtdIns head-group (perhaps Val26, Glu76, Gly77, Ala78, and Glu85), whereasothers correspond to a rearrangement induced by the bindingof the phospholipid in residues further away from the peptide-binding groove (Val70, Arg71, Gly91, and Gln92). Indeed, aninduced-fit mechanism has been described for PDZ–peptidebinding affecting residues far away from the peptide bindinggroove.[38–40]

From the study by Kachel and co-workers one might con-clude that there are no conserved residues between the synte-nin PIP-binding sites and the PTP-Bas PIP-binding site. Yet, thesurface area of the PDZ domains implicated in the binding isroughly the same: the N and C-terminal regions of the aB helixand bB strand, which are structural elements of the peptide-binding groove. This evidence again links both peptide andPIP-binding events.

The PAR-3 Protein

PIP interaction

PAR-3 is a scaffold protein of about 150 kDa consisting of anN-terminal oligomerization domain and three PDZ domainsknown to be responsible for multiple protein–protein interac-tions. PAR-3 forms a complex, with PAR-6 and aPKC, which dis-tributes asymmetrically in the cell and plays a crucial role incell polarization and asymmetric cell division. How the asym-metrical distribution of the PAR-3–PAR-6–aPKC complex is ach-ieved is not fully understood, but PtdIns-4,5-P2 and/or PtdIns-3,4,5-P3 might be essential players.[41–43] The hypothesis is fur-

Figure 2. Peptide binding and putative PtdIns-4,5-P2 binding sites in the PDZ2b domain of PTP-Bas. A) Tube diagram of the PDZ2b (PDB ID: 1Q7X) domain ofPTB-Bas highlighting the changes in the peptide-binding groove (dark gray) introduced by the insertion of the five amino acids (black) in the splicing variant,compared to the PDZ2a variant (darker gray; PDB ID: 3PDZ). B) Tube diagram of the PDZ2b domain showing the peptide binding groove (dark gray) and theresidues that display strong chemical shift changes upon titration with PtdIns-3,4,5-P3 (black sticks). C) Accessible surface area representation of panel (B).Note that some of the residues that present strong chemical shifts, like Ile24, Gly77 and Gln92, are located on opposite faces of the protein and do not clusterin an evident way.

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ther supported by the recent discovery that PAR-3 is able tointeract on its own with different PIPs.[16]

Wu and collaborators established in an initial screeningassay that the two first PDZ domains of PAR-3 bind to bovinebrain liposome extracts (containing PIPs) in sedimentationassays. The interaction was further documented by overlayassays by using pure lipids immobilized on cellulose mem-branes (lipid strips) and by sedimentation assays by using com-position-defined liposomes. The assays suggest that PAR-3PDZ2 can interact with all of the PIPs tested with low stereo-specificity but not with other acidic membrane lipids. Yet it isdangerous to draw conclusions based on lipid strips, in whichthe results are mainly dependent on koff. Moreover, in thestudy, the authors mainly used the monophosphorylatedPtdIns-3-P (a lipid mainly concentrated on endosomes and vir-tually absent from the plasma membrane) as a model lipid,although the physiologically relevant lipid is more likely to bePtdIns-4,5-P2 due to its abundance at the plasma membrane,or possibly PtdIns-3,4,5-P3, which is produced upon cell stimu-lation at the plasma membrane. The apparent dissociation con-stant of the PDZ2–PtdIns-3-P complex was estimated by lightpolarization assays in which fluorescently labeled PAR-3 PDZ2was titrated with composite liposomes containing 10 % PtdIns-3-P. The apparent KD of PtdIns-3-P embedded in liposomes was8.0 mm, which is in the range reported for PAR-3 PDZ2–peptideinteractions. No KD was determined for other PIPs.

Structural biology of PAR-3–PtdIns-3-P interaction

The PAR-3 PDZ2 PIPs binding site was derived from NMR spec-troscopy experiments by using soluble forms of PtdIns-3-P anda radiolabeled PAR-3 PDZ2 domain. It is mentioned in thepaper that PtdIns-4,5-P2 and PtdIns-3,4,5-P3 produced the samepattern of chemical shift changes, but the data are unfortu-nately not shown. The chemical shift changes were strongly lo-calized to the aB-bF loop and bA-bB loop. The two loops forma polar pocket in which the head-group of PtdIns-3-P (IP2) wassatisfactory modeled. In the PAR-3 PDZ2-IP2 model, the phos-phate groups interact with two conserved residues, Arg532and Lys535, and one hydroxyl group of the inositol ring inter-acts with a conserved Glu469 (Figure 3 B). Mutation of any ofthese three residues lowered the KD by a factor of 10. Thedouble mutation Arg532Ala/Lys535Ala completely abolishedthe binding. Therefore, both positively and negatively chargedresidues form part of the PIP-binding site in PDZ2 domain ofPAR3. The PIP-binding site partially overlaps with the peptide-binding groove (Figure 3 C). The overlap region is importantfor peptide binding because it holds residues that form the hy-drophobic pocket for binding of the first lateral chain of thepeptide, but it is not obvious from the structure whether pep-tide and lipid binding are competitive or not. This was investi-gated by liposome sedimentation assays in which the Par-3PDZ2 was preincubated with the peptide Leu-Glu-Thr-Arg-Val(KD of 15 mm). The peptide was used in solution at a fixed con-centration of 1.0 mm, which was 30-times more concentratedthan the lipid, PtdIns-3-P incorporated in liposomes, and 1000-times more concentrated than the PDZ2 domain, and under

Figure 3. The PDZ2 domain of PAR-3 interacts with cellular membranesthrough three different mechanisms. A) Tube diagram of PDZ2 domain ofPAR-3 (PDB ID: 2OGP) highlighting the peptide-binding groove (dark gray),the hydrophobic patch for membrane penetration (residues Leu494, Pro495and Ile500 in black sticks) and the positively charged patch for electrostaticmembrane recruitment (residues Lys491, Arg496, Lys506 and Arg546 darkergray sticks). The residues Met536 (close to the PIP binding site) and Lys522(on the opposite face of the protein, used as negative control) are alsoshown. B) Tube diagram of the PDZ2 domain highlighting the residues thatdisplay the strongest chemical shifts upon PtdIns-3-P titration (black sticks).C) Accessible surface area representation of panel (B) highlighting the limit-ed overlap (C-terminal region of aB) between the peptide-binding groove(dark gray) and the PIP binding site (darker gray and black).

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these conditions a competition between lipid and peptidebinding was observed.

Additional modes of membrane recruitment

Wu et al. suggested that PAR-3 PDZ2 is not only recruited tocellular membranes by direct interaction with the PIPs but thattwo additional mechanisms are involved, namely electrostatic-mediated membrane interactions and membrane insertion.[16]

The PDZ2 domain possesses a cluster of positively charged res-idues next to the peptide-binding groove (Figure 3 A). Thecluster confers a strong and localized positive electrostatic po-tential at the surface, which is crucial for membrane binding.Mutations of these residues to alanine reduce the liposomebinding, whereas mutations to glutamic acid completely abol-ished the binding. Beside this positive cluster, the PDZ2domain of PAR-3 possesses a cluster of hydrophobic residuesexposed to the surface (Figure 3 A), which were suggested tobe inserted into the membrane leaflet. The insertion was esti-mated by mutating the candidate residue to cysteine and thenconjugating the mutant to a chemical compound that increas-es its fluorescence when inserted into a hydrophobic environ-ment. Apart from peptide binding, the PDZ2 domain of PAR-3thus presents three mechanisms for its direct interaction withcellular membranes: PIPs binding, membrane recruitmentthrough electrostatic interactions, and membrane penetration.

The PICK1 Protein

PIP interaction

PICK1 (protein interacting with C kinase 1) is a protein of about400 residues consisting of one PDZ domain and one BARdomain separated by a 40 amino acid linker region and twoacidic regions at each end of the protein. PICK1 has perinuclearlocalization in most cell types and is strongly localized at syn-apses in neuronal tissue. By regulating the subcellular localiza-tion and surface expression of its cognate partners PICK1 playsan essential role in different processes like intracellular signal-ing (regulating PKC-a), cell–cell contact and cell migration (reg-ulating the ephrin family of receptor tyrosine kinases) or long-term potentiation and long-term depression (regulating AMPA-type glutamate receptor’s subunits).[44] It was assumed that thePDZ domain of PICK1 was dedicated to protein–protein inter-actions, whereas the membrane-binding BAR domain was re-sponsible for PICK1 localization at cellular membranes.[45] Itwas demonstrated by sedimentation assays that the BARdomain directly binds liposomes from brain extracts and puremonophosphorylated PIPs spotted on lipid strips, but not tobi- or triphosphorylated PIPs. The BAR-mediated membrane in-teraction was found to be important for the biological functionof PICK1 because single point mutations in this domain causeda decrease in synaptic targeting of PICK1 and led to decreasedsynaptic targeting of the cognate partners like the GluR2 sub-unit of AMPA receptors.[46] Yet, the authors reported that thePDZ domain of PICK1 positively regulate the binding of the

BAR domain to liposomes, augmenting its binding, but noPDZ–lipid binding experiments were reported in this study.

In a later work, Pan and co-workers repeated the liposomesedimentation assays by using the isolated PDZ domain ofPICK1 fused to the C terminus of the GluR2 subunit of theAMPA receptor.[15] The PDZ–peptide construct was mixed withliposomes prepared from bovine brain extracts, and was re-trieved in the liposome fraction after centrifugation, indicatinga direct interaction between the PDZ domain and liposomes.Lipid strips analysis revealed that the PICK1 PDZ domain bindsto mono-, bi-, and triphosphorylated PIPs, with the apparentlystrongest interaction being with PtdIns-3,4,5-P3. The result wasconfirmed by sedimentation assays by using composite lipo-somes containing a bulk of phosphatidyl cholin and phospha-tidyl serine, and 10 % of the respective PIPs.

The relevance of the PICK1 PDZ lipid interaction in targetingthe protein to the membrane was shown by mutational analy-sis. It was demonstrated the PDZ-mediated membrane bindinghas the same biological relevance as the BAR-mediated mem-brane binding. In fact, single point mutations of a conservedhydrophobic Cys-Pro-Cys motif (CPC) or on a patch of positive-ly charged residues in the PDZ domain implicated in the PIPbinding, have a more severe phenotype in loss of synaptic tar-geting of PICK1 and its cognate partners than mutations in theBAR domain of PICK1.

PICK1 PDZ–lipid and –peptide interactions

The PICK1 PDZ domain is not stable and aggregates quicklyafter purification, which was the rationale behind fusing it tothe C-terminal region of the GluR2 subunit of its cognate re-ceptor, AMPA-type glutamate receptor.[15] This fusion proteinwas stable and suitable for both NMR spectroscopic structuralanalysis and sedimentation-based lipid-binding analysis. TheNMR spectroscopic analysis established that the PDZ domainadopts the canonical PDZ domain fold, and that the C-terminalregion of GluR2 binds in the canonical peptide-binding grooveby b addition (Figure 4 A). The PDZ–peptide fusion protein wasused in the lipid-binding assays discussed above in whichPICK1 PDZ was found to bind to brain liposomes and to PC/PS/PIPs reconstituted liposomes. The data thus suggest that inthe PICK1 PDZ case, peptide binding and PIP binding are notmutually exclusive but can occur simultaneously. Replacementof the C terminus of GluR2 in the PICK1 PDZ–peptide constructby the C-terminal peptide of the GB1 domain, did not alter thelipid-binding properties of the PDZ domain; this suggests thatthe relation between PIPs and peptide binding is not influ-enced by the nature of the cognate peptide.

Structural biology of the PDZ–PIPs interaction

The interactions of PICK1 PDZ domains with lipid membraneswere elucidated by biochemical experiments and mutationalanalysis. Similar to PAR-3 PDZ2, a region of membrane penetra-tion was identified, as well as a basic cluster of amino acidsproviding electrostatic membrane recruitment. The membranepenetration is mediated by a conserved hydrophobic Cys-Pro-

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Cys motif allocated in the bB-b C loop (Figure 4 A). Mutation ofthe two cysteines residues to glycines, or their chemical modi-fication with iodoacetic acid, completely abolished PICK1 PDZ-mediated liposome binding without affecting peptide binding.Upon binding to liposomes, these cysteine residues cannot bechemically modified by Ellman’s reagent; this indicates thatthey are shielded by direct interactions with the liposome,most probably by insertions into the lipidic membranes, aswas observed in the case of the Cys-Pro-Cys motif of P1B-typefamily ATPases. The PICK1 Cys-Pro-Cys motif is juxtaposed witha patch of positively charged residues (Arg76, Lys79, and Lys81at the bE-aB loop; Figure 4 A). Mutation of these basic residuesto alanine almost completely abolished the binding to PIP-con-taining liposomes, and it was therefore claimed that these resi-dues mediate a nonspecific electrostatic recruitment of thePDZ domain to PIP-enriched membranes.

The two membrane-interaction motifs are located at distinctsides of the peptide-binding grove (Figure 4 B) and do notstructurally overlap with the peptide binding site. Orientingthe PDZ domain with its membrane interaction motifs towardsa hypothetical membrane makes the PDZ-binding groove com-pletely available for C-terminal peptide binding (Figure 4 C);this allows a simultaneous binding of peptide and PIPs. Simul-taneous binding of PIPs and peptides has been suggested fora limited set of protein domains such as the disabled-1 PTBdomain and some PH domains.[47, 48] The benefit of such coinci-dence detection in vivo could be that the PDZ domain proteinwould only be efficiently recruited to the membranes whenboth cognate peptide and lipid are available.

Figure 4. Peptide-binding groove and membrane interaction motifs are located on opposite faces of the PDZ domain of PICK1 (PDB ID: 2PKU). A) Tube dia-gram of PDZ domain of PICK1 in complex with the C-terminal peptide from the GluR2 subunit (light sticks). The peptide-binding groove is highlighted indark gray, the lateral chains of the conserved Cys-Pro-Cys (CPC) motif for membrane insertion are in black sticks, and the lateral chains of the cluster of pos-itive residues for electrostatic recruitment to cellular membranes are in darker gray sticks. B) Molecular surface representation of panel (A) and its rotationthrough 908. Note that both membrane interaction motifs are on the opposite face of the peptide-binding groove. C) Orienting the membrane interactionmotifs towards a hypothetical membrane (top of panel C) makes the peptide-binding groove available for interaction with the C terminus of cognate recep-tors coming from the same membrane. The orientation is a 1808 rotation on the plane of panel (B).

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Summary and Outlook

PDZ domains are among the most abundant protein-interac-tion domains in multicellular organisms. The most distinctiverole of these domains is to act as scaffolds for the assembly ofprotein complexes that mediate all types of cellular events,from general cell trafficking to specialized synaptic long-termpotentiation. It has been assumed that the participation ofPDZ domains in these processes relies on their ability to bindthe C terminus of cognate partners, therefore acting as a unitthat holds the stability of the signaling complex. It is now be-coming clear that PDZ domains also bind to membrane lipids.They seem to use more than one mechanism for their associa-tion and binding to membrane lipids: specific PIP binding,electrostatic membrane recruitment, and membrane penetra-tion. By using these three mechanisms, PDZ domains recapitu-late what has been described for other well-documented lipid-binding domains. Wu and co-workers suggested that as manyas 20 % of the PDZ domains interact with membrane lipids.[16]

This percentage of PDZ lipid binders is similar to the percent-age of the more well-known PIP-binding PH domains.[23] Mostinvestigated PDZ domains are able to bind mono-, bis-, and tri-phosphorylated PIPs in vitro by using PIP strips. It will be cru-cial to clarify when PDZ domains are able to recognize specificPIP isoforms in vivo because specific PIPs are associated withspecific cellular processes and specific regulations. The target-ing and trafficking of PDZ–protein complexes most probablydepend on such specific recognition. Therefore, clarifying thespecific PIP interactions will help to gain insight into thepotential functional implications. So far we have learned thatsyndecan and syndecan heparan sulfate cargo recycling is con-trolled by the syntenin-1–PtdIns-4,5-P2 interaction with drasticconsequences for cell spreading.[34] The syntenin-2–PtdIns-4,5-P2 interaction appear to control subnuclear PtdIns-4,5-P2 distri-bution with potential consequence for cell division and viabili-ty.[14] Another example is the membrane localization of PAR-3,which depends on the PDZ2–PIP interaction and appears to beindispensable for cell polarization and differentiation.[16] Yet, itis not clear whether specific PIPs are biologically implicatedand whether, for instance, PIP-dependent vesicular traffickingmight play any role. Synaptic targeting of PICK1, which is es-sential for synaptic plasticity, might also be controlled by spe-cific PDZ–PIPs interactions.[15]

The available data show that there is no consensus PIP-bind-ing site on PDZ domains. Yet in several cases the PIP-bindingsite is located near the peptide-binding groove. The bindingsites share structural determinants, and both ligands might in-fluence each other. Experiments indicate that the binding ofPIPs competes with the binding of cognate protein. However,given the high number of PDZ domains and the diversity ofpeptide and lipid interactions, it would not be surprising tofind more than one way of integrating PIPs and peptide bind-ing. To correctly address whether PDZ–peptide and PDZ–PIPinteractions are mutually exclusive, concomitant, or sequentialevents, appropriate experimental approaches that recapitulatethe presentation of the ligands as in the cellular context areessential.

Interestingly, during the completion of this manuscript Meer-schaert et al. , reported on the interaction of the PDZ2 domainof zonula occludens with PtdIns-4,5-P2 in nuclear speckles.[49]

Together with the study of Mortier et al. on syntenin-2, whichis described above, this shows that PDZ domains might beeffectors of nuclear lipid signaling.[14] These findings open newavenues for the understanding of nuclear lipid signaling andPDZ protein biology.

Acknowledgements

The laboratory of P.Z. is supported by the Fund for Scientific Re-search-Flanders (FWO), the Interuniversity Attraction Poles of thePrime Ministers Services (IUAP), the Belgian Federation againstCancer (BFK), the Concerted Actions Program of the K.U.Leuvenand the EMBO Young Investigator Program. Y.I. is an EMBO long-term fellow.

Keywords: membranes · PDZ domain · phosphoinositides ·polarity · signal transduction

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Received: October 7, 2009Published online on January 20, 2010

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