The Solution Structure and Dynamics of the Pleckstrin Homology ...

The Solution Structure and Dynamics of the Pleckstrin HomologyDomain of G Protein-coupled Receptor Kinase 2 (b-AdrenergicReceptor Kinase 1)A BINDING PARTNER OF Gbg SUBUNITS*

(Received for publication, August 8, 1997, and in revised form, October 13, 1997)

David Fushman‡, Taraneh Najmabadi-Haske§, Sean Cahill‡, Jie Zheng‡, Harry LeVine III§,and David Cowburn‡¶

From the ‡Laboratory of Physical Biochemistry, The Rockefeller University, New York, New York 10021-6399and §Parke-Davis Laboratories, Warner-Lambert Inc., Ann Arbor, Michigan 48105

The solution structure of an extended pleckstrin ho-mology (PH) domain from the b-adrenergic receptor ki-nase is obtained by high resolution NMR. The structureestablishes that the b-adrenergic receptor kinase ex-tended PH domain has the same fold and topology asother PH domains, and there are several unique fea-tures, most notably an extended C-terminal a-helix thatbehaves as a molten helix, and a surface charge polaritythat is extensively modified by positive residues in theextended a-helix and the C terminus. These observa-tions complement biochemical evidence that the C-ter-minal portion of this PH domain participates in protein-protein interactions with Gbg subunits. This suggeststhat the C-terminal segment of the PH domain may func-tion to mediate protein-protein interactions with thetargets of PH domains.

G protein-coupled receptor kinases (GRKs)1 are a uniquefamily of serine-threonine kinases, which are responsible foractivator-dependent phosphorylation of G protein receptorsand provide rapid desensitization of the agonist occupied re-ceptors (1). The GRKs are recognized to have three functionalcomponents: an N-terminal section believed to interact directlywith the seven-trans-membrane helical receptor protein and/or

other membrane targets, a central section, which is the cata-lytic domain, and a C-terminal section containing a generallyconserved autophosphorylation region and a variable regionthat mediates membrane association by various means. InGRK2 (also known as b-adrenergic receptor kinase-1) or GRK3(b-adrenergic receptor kinase-2), the C-terminal variable re-gion contains a pleckstrin homology (PH) domain (2, 3), confer-ring binding specificity to Gbg proteins (reviewed in Ref. 1).

The PH domain family (reviewed in Refs. 4–6) appears to bea very large family of structurally homologous protein domainsof moderate to low sequence similarity. The PH domain isbelieved to play a role in intracellular signal transduction, andthe functional role of the PH domain has been characterized forseveral systems. In phospholipase Cd, the PH domain has ahigh affinity (Kd , 1 mM) site for phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and inositol 1,4,5-trisphosphate(Ins(1,4,5)P3) (7), which forms a crystallographically observed,well defined structural interaction (8). Other PH domains havedifferent lipid specificities, and a well defined set of bindingmotifs does not readily emerge (9–15). One hypothesis is thatPH domains present a framework with a polymorphic surfaceused for specific recognition, analogous to immunoglobulins (5,9). In addition, the overall fold of the PH domain was observedto be common with that of the PTB (phosphotyrosine binding)domain (16, 17), a protein domain that shows little sequencehomology to PH domains. In light of these developments, it is ofsignificance to establish whether the nominal PH domain ofGRK-2 (b-adrenergic receptor kinase (bARK-1)), which clearlybinds (both in vivo and in vitro) to a protein partner, Gbg

subunits of the heterotrimeric G protein family (18), truly hasthe common structural motif of the PH/PTB domains, and whatthe relationship of putative lipid and protein binding sitesmight be in such a structure.

In this paper, we present the solution structure of an ex-tended PH domain from human bARK1, determined at 0.4 År.m.s.d. by high resolution NMR using heteronuclear tripleresonance methods. Although the overall fold and topologyclearly establishes that the bARK1 extended PH domain is ofthe same family as other PH domains, there are several signif-icant alterations (most notably an extension of the C-terminala-helix, which in solution presents as a “molten helix” having aclear gradient of mobility) on the subnanosecond, as well asmillisecond to microsecond, time scales, increasing toward thefree C terminus. The polarity of the surface charge observed inother PH domains is altered by positively charged residues inthe extended a-helix. This unusual clustering may be comple-mented by a highly negatively charged area on Gbg subunits.Although a direct study of the Gbg/PH domain complex is

* This work was supported by American Cancer Society Grant NP-922 and by National Institutes of Health Grants GM-47021 and RR-00862 (mass spectral resources). The costs of publication of this articlewere defrayed in part by the payment of page charges. This article musttherefore be hereby marked “advertisement” in accordance with 18U.S.C. Section 1734 solely to indicate this fact.

The atomic coordinates and structure factors (code 1BAK) have beendeposited in the Protein Data Bank, Brookhaven National Laboratory,Upton, NY.

¶ To whom correspondence should be addressed: The RockefellerUniversity, 1230 York Ave., New York, NY 10021-6399. Fax:212-327-7566.

1 The abbreviations used are: GRK, G protein-coupled receptor ki-nase; PH, pleckstrin homology; PI(4,5)P2, phosphatidylinositol 4,5-bisphosphate; Ins(1,4,5)P3, myo-inositol 1,4,5-trisphosphate; PTB,phosphotyrosine binding domain; bARK, b-adrenergic receptor kinase;GST, glutathione S-transferase; r.m.s.d., root mean square deviation;NOE, nuclear Overhauser effect; PLC, phospholipase C; HCCH, 1H-13C-13C-1H multidimensional correlation spectroscopy; TOCSY, total corre-lation spectroscopy; NOESY, nuclear Overhauser spectroscopy;ROESY, rotating frame NOESY; HMQC, heteronuclear multiquantumcoherence spectroscopy; HTQC, heteronuclear triple quantum coher-ence spectroscopy; HSQC, heteronuclear single quantum coherencespectroscopy; HOHAHA, homonuclear Hartmann-Hahn (correlation)spectroscopy (The acronyms HNCA, HNCO, CBCANH, CBCA(CO)NH,and WATERGATE refer to pulse sequence selection programs and arereferred to in the text. DIANA, DYANA, REDAC, and ECEPP arecomputer analysis programs.)

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 273, No. 5, Issue of January 30, pp. 2835–2843, 1998© 1998 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

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beyond the range of current NMR technology, the structurepresented here supports a model in which the C-terminal por-tion of bARK PH domain in particular, and PH domains ingeneral, participate in protein-protein interactions.

MATERIALS AND METHODS

Sample Preparation—Recombinant human bARK PH domain(hbARK1-(556–670)) was obtained by GST fusion expression frompGEX-2T (Pharmacia Biotech Inc.) in BL21 (DE3) Escherichia coli cells(Novagen, Madison, WI) and subsequent bacterial expression and pro-tein purification as described previously (19) on a larger scale. Thefull-length hbARK1 cDNA clone was provided by Dr. Antonio DeBlasi(Mario Negri Sud, Santa Maria Imbaro, Italy). The sequence of the119-residue construct used in the present study is shown in Fig. 1. Itcontains both the PH domain and the Gbg-binding motif (20). The firstfour residues are not from the natural sequence. Uniform 15N and15N,13C labeling was achieved by growing the cells in M9 minimalmedium using standard procedures.

Solutions used for NMR studies contained 1–2 mM protein in 10 mM

acetate buffer at pH 4.5 (uncorrected for isotope effects), 0.02% sodiumazide, 1 mM [U-2H]EDTA, 5 mM [U-2H]dithiothreitol, and 10% 2H2O inthe H2O samples. These low salt and low pH conditions were necessaryto prevent protein aggregation. CD data indicated no changes in theprotein secondary structure, between the buffer used for NMR studiesand phosphate-buffered saline, pH 7.2. The external 1H chemical shiftreference used was sodium 2,2-dimethyl-2-silapentane-5-sulfonate, andindirect referencing was used for 15N (21) and 13C. Spectra were essen-tially identical among several preparations of the PH domain.

NMR Spectroscopy—NMR experiments were run on Bruker DMX-500 and DMX-600 spectrometers. Quadrature detection was achievedby the States or States-time proportional phase incrementation meth-ods. Some of the pulse schemes implemented pulse field gradients forcoherence selection (HCCH-TOCSY, 13C-separated NOESY-HMQC),and some used the sensitivity enhancement method (HSQC, hetero-nuclear NOE) (22). The water signal was suppressed either by theWATERGATE method (23) or by using selective on-resonance irradia-tion during a relaxation delay of ;1.3 s. Experiments were run at 35 °Cwith sweep widths of 8000 and 2000 Hz for 1H and 15N (at 600 MHz),respectively, unless indicated otherwise.

The homonuclear experiments, HOHAHA and NOESY, were run inboth H2O and 2H2O using standard pulse sequences and phase cycling.A range of t1 increments from 200 to 512, each consisting of 2048complex points, was typically acquired with 32–128 scans/increment.

The heteronuclear experiments consisted of two-dimensional HSQC(1H-15N and 1H-13C), HSQC-J, and HTQC, three-dimensional CBCA-(CO)NH, CBCANH, HNCA, HN(CO)CA, HNCO, HCCH-TOCSY, and13C-separated NOESY-HMQC, and two- and three-dimensional 15N-separated NOESY-HMQC (in H2O and in 2H2O) and HOHAHA-HMQC.The mixing time in the NOESY-HMQC experiments was 100 and 150ms, and the spin lock duration was 30 ms in the HOHAHA-HMQC and19 ms and 6 ms in the HCCH-TOCSY. The three-dimensional spectrawere recorded with a 32 3 100 3 1000 hypercomplex matrix, with 32

scans/increment. The degree of amide hydrogen protection was as-sessed (a) by measuring hydrogen-deuterium exchange rates by follow-ing the intensity of cross-peaks in HMQC experiments after exchanginga fully protonated, lyophilized sample with 99.996% 2H2O, and (b) bycomparison of cross-peak intensities in two HSQC experiments, withand without water presaturation (24). The three-bond H9-Ha couplingwas assessed by the method of (25). Heteronuclear 15N{1H} NOEs weremeasured using standard methods as described elsewhere (26). Two-dimensional H2O-selective heteronuclear 15N-edited ROESY experi-ments (27) were performed to map those amide hydrogens in the bARKPH domain that are exposed to and interacting with water molecules.Signal processing and assignment were done as discussed previously(26, 28).

Structure Calculation—Structure calculations used DIANA with RE-DAC strategy (29) or DYANA (30) with ECEPP stereochemistry, withstructurally significant constraints of 1956 upper and 76 lower distancebounds (from ;3000 NOEs), 38 hydrogen bonds chosen within strandsor helix with slowed exchange, 99 f-angle constraints derived from3JHNHa coupling constants, and 99 c-angle constraints (derived from Cachemical shift data) corresponding to conservative ranges of allowedtorsion angles, in those regions of strand or helix that were well defined(Fig. 2). All peptide bonds were assumed to be trans. A final selection of20 structures from 400 starting structures was done by using the lowesttarget functions (the ensemble statistics are shown in Table I). DIANAand DYANA use no assumptions about protein energetics, other thanvan der Waals repulsion; structures are unrefined and only adjusted byrotation/translation for comparison purposes. Structures were alignedusing XPLOR or in-house software written in MATLAB (MathWorks)and displayed and analyzed with the INSIGHTII package (Biosym) orwith MOLMOL (31).

FIG. 1. The sequence of the proteinconstruct used in the present workand its relation to the nominal PHdomain and to the Gbg-binding regionof bARK1. At the top is the nominallength PH domain section; below is thedomain demonstrated previously (50) tobe sufficient and optimal for Gbg binding,below which is the construct used here,which has the same Gbg binding. The low-ercase “gshm” residues are from the GSTconstruct, and are not further referred to.At the bottom, the complete sequence ofhbARK1 PH domain, and the similarhbARK2 are compared, with the second-ary structural elements of hbARK1 su-perimposed in color. The more flexible re-gion of the C-terminal a-helix is shown inlight blue.

TABLE IRoot-mean-square deviation from the mean structure calculated for

the ensemble of 20 structures of the bARK1 PH domain

Selected residues Selected atoms r.m.s.d.

Å

All 119 residues Backbone heavy atoms 3.80 6 0.65Residues 560–658, floppy

tails clipped offBackbone heavy atoms 1.08 6 0.40

Residues 560–658, floppytails clipped off

All heavy atoms 1.38 6 0.41

All elements of secondarystructurea

Backbone heavy atoms 0.38 6 0.09

All elements of secondarystructure

All heavy atoms 0.82 6 0.16

All elements of secondarystructure

All atoms 1.20 6 0.17

All b-strands Backbone heavy atoms 0.44 6 0.11a-Helix Backbone heavy atoms 0.31 6 0.10

a Strands b1 through b7 and the a-helix. There were no NOE viola-tions of upper limits .1 Å, and eight in the ensemble of 20 structures.0.5 Å, and eight dihedral angle violations .5 °.

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Alignment of the PH/PTB Domains—Pairwise comparison of thePH/PTB domain structures was performed by superposition of the back-bone heavy atoms (N, Ca, C9, and O) of the residues from regions ofregular secondary structure, as indicated by boxes in Table II. Thea-helical insertions in the loop regions of the PH domains were nottaken into account. The alignment was done by direct calculation ofr.m.s.d. values and optimized by relative shift of the protein sequenceswithin each secondary structure element (b1–b7 strands, a-helix), aswell as by adding or removing individual residues. The resulting align-ment and r.m.s.d. values are presented in Tables II and III, respectively.

Protein Backbone Dynamics—The backbone dynamics were assessedvia 15N spin relaxation studies comprising T1, T2, and heteronuclearsteady state NOE measurements using previously described protocols(26). Fifteen two-dimensional spectra with the relaxation delays of 4(32), 200, 400 (32), 600, 900 (32), and 1200 ms (positive initial 15Nmagnetization), and 4, 200 (32), 400, 600 (32), and 900 ms (negativeinitial 15N magnetization) were acquired in the alternate-sign T1 ex-periment (duplicate experiments are indicated by 32) (26). Eleventwo-dimensional spectra were collected for the T2 measurements, withthe relaxation delays of 8 (32), 16, 32 (32), 48, 64 (32), 80, 96 (32), 112,128 (32), and 160 ms. The heteronuclear {1H}15N steady state NOEswere assessed as a ratio of cross-peak intensities in the experimentswith and without proton presaturation. The relaxation data analysiswas performed using programs RELAXFIT and DYNAMICS (26), ex-

tended to include anisotropic character of the overall motion of theprotein (32).

RESULTS

hbARK-PH domain corresponding to residues 556–670 ofhuman bARK1 (Fig. 1) was produced in E. coli and isolated asa GST fusion protein, cleaved, and purified. Solubility andstability limited observation to a narrow range of conditions,and the majority of studies were conducted in 20 mM acetatebuffer at pH 4.5, 35 °C. Under these conditions, binding of theconstruct to Gbg is maintained (data not shown). The 546–670construct was also produced, and NMR spectra indicated thatthe additional N-terminal residues did not belong to the do-main fold, and were apparently unstructured. It was concludedthat the first construct contained the essential domain. Assign-ment used standard triple resonance methods, complementedby study of the [U 13C, 15N; 12C, 14N-Met]PH domain to helpidentify methionine residues that underwent partial oxidationduring sample preparation. Assignment and NOE data aresummarized in Fig. 2.

In Figs. 3 and 4, the overall fold and the electrostatic poten-

FIG. 2. Summary of NMR data used for identification of the secondary structure. From top to bottom, deviations from standard chemicalshift (in random coil peptides) (53, 54), of a-hydrogens (DHa), a- and b-carbons (DCa, DCb, DCa-DCb), and carbonyl carbons (DC9) (54); 3JHNHa, themagnitude (in Hz) of the three-bond scalar (intraresidue) spin-spin coupling between the a- and the amide hydrogens; intensities of the NOEcross-peaks (on an arbitrary log scale) between the a- and amide hydrogens, da,N(i,i11), and between amide hydrogens, dN,N(i,i11), of the adjacentresidues; horizontal bars indicate NOEs observed between the a- and amide hydrogens three (da,N(i,i13)) or four (da,N(i,i14)) residues apart, andbetween the a- and b-hydrogens three residues apart (da,b(i,i13)) characteristic for the a-helix; heteronuclear 15N{1H} steady state NOE; circlesindicate amides protected from exchange with (solid circles) or exposed to (open circles) solvent. a-Helices are typically characterized by DHa , 0,DCa . 0, DCb , 0, and DC9 . 0, whereas chemical shift deviations of the opposite sign are expected for the b-strands (54). 3JHNHa is directly relatedto the intervening torsion angle f, so that 3JHNHa , 6 Hz is characteristic of the a-helix. Based on all the data, seven b-strands (b1, 561–568, b2,578–584; b3, 587–591; b4, 600–602; b5, 606–613; b6, 618–624; b7, 628–634) and a C-terminal a-helix (residues 638–655) were identified. Arepresentation of the secondary structure of the bARK PH construct is shown on the bottom. Shaded in gray is the C-terminal extension of thea-helix (656–658) where some of the helical features are still preserved (see text), characteristic of a “molten” helix.

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tial of the hbARK PH domain are shown, along with the PHdomain of PLCd (8) for comparison. The topology of the fold istypical for PH domains, and consists of seven b-strands form-ing a b-sandwich flanked on one end by a C-terminal a-helix.The termini of the construct are disordered and highly flexible,with large amplitudes of backbone motion on a nanosecondtime scale (Fig. 3). The b1/b2 and b3/b4 loops are disorderedand display increased amplitudes of backbone dynamics on asubnanosecond to nanosecond time scale, as well as motions ona millisecond to microsecond time range (data not shown).

Of specific note, the C-terminal a-helix is clearly extended bymore than one turn compared with C-terminal a-helices ofmost previously determined PH domain structures. The posi-tion/orientation of the helix appears to be fixed by interactionswith the protein core, namely by a hydrophobic strip formed byLeu-640, Trp-643, Leu-647, Ala-650, Tyr-651, and Ala-654,which are located on the side of the helix facing the b-sandwichand are involved in contacts with several residues in the firsttwo b-strands. The aromatic ring of Trp-643, the only con-served residue among PH domains, is buried in the protein coreand exhibits numerous NOE contacts to residues in b1 and b2.Another aromatic residue in the helix, Tyr-651, is also orientedtoward the interior of the b-sandwich. The NOESY data indi-cate several close contacts between the aromatic ring of Tyr-651 and the residues in the b4/b5 loop and in the strand b5.Both the structure of this loop and the orientation of the Tyr-651 ring are well defined, as indicated by low r.m.s.d. values in

these parts of the structure, and by chemical shift non-equiv-alence of all four ring hydrogens of Tyr-651.

DISCUSSION

Relation to Other PH/PTB Domain Structures—ThehbARK1 PH domain has very low sequence similarity to otherPH domains of known three-dimensional structure, and there-fore cannot be satisfactorily homology-modeled from knownstructures. The structure-based alignment of the hbARK1 PHwith these PH domains (Tables II and III) demonstrates thesame overall topology of the protein fold. The expected range ofr.m.s.d. values between sequences of the same structural class,but with varying degrees of homology, has been derived previ-ously (33). The r.m.s.d. values between different members ofthe PH/PTB domain family (Fig. 7) are within the range ex-pected for such homologous sequences of low identity. Thecharge distribution is, however, different among the PH do-mains, and the large positive charge associated with the C-terminal helix of bARK is unusual.

Validity of the Derived Structure of the bARK PH Domain—The low pH (4.5) required for this study is close to the pKa

values for both glutamate and aspartate, so variations in theside chain charges of these residues compared with physiolog-ical conditions are expected. Since this might result in a per-turbed structure in those regions containing negativelycharged residues, a question arises of whether the NMR struc-ture derived under these conditions represents the protein

FIG. 3. Three-dimensional structure of the bARK1 PH domain. A, view of the backbone (N, Ca, C9) of 20 superimposed NMR-derivedstructures of the bARK1 PH domain. Parts of the backbone belonging to the elements of secondary structure are colored (b-strands, yellow; a-helix,blue) and labeled. The termini of the construct and some residues in the loops b1/b2 and b3/b4 are disordered (see also D and E). B, a ribbonrepresentation of the tertiary solution structure of the bARK1 PH domain, the same orientation as in A. C, the structure of PLCd H domain fromx-ray diffraction (8). D and E, ribbon representation of the backbone of the bARK1 PH domain. The ribbon width in D represents local backboner.m.s.d. values in the ensemble of 20 calculated structures and, in E, the amplitude of nanosecond-subnanosecond backbone motion (as inferredfrom 1-S2, S is the order parameter). The 15N relaxation data indicate a dynamic character of structural disorder in the N and C termini (S2 , 0.5for the backbone NH groups in residues 552–557 and 659–670) and in the loops b1/b2 and b3/b4. The backbone mobility in the elements ofsecondary structure is restricted (S2 . 0.85, local correlation time in a subnanosecond time range). The ratio of the principal components of therotational diffusion tensor of the molecule is Dz/Dx 5 1.30; the z axis of the diffusion tensor is tilted by ;20° angle from the C-terminal helix axis.Residues 556–670 (bARK1 PH domain) are represented in A, 556–664 in B and D. Unanalyzed residues (unassigned or with insufficiently resolved1H-15N correlations) are colored gray in E. The drawings were performed with INSIGHTII (Biosym) (A, D, and E) and with SETOR (55) (B and C).

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structure under physiological conditions. As mentioned above,the circular dichroism data indicate no changes in the proteinstructure as compared with the more physiological conditionsin a phosphate-buffered saline pH 7.2. To address this issue ingreater detail, the 1H-15N correlation maps (HSQC) were alsorecorded for the PH domain dissolved in the phosphate-buff-ered saline (pH 6.0, temperature 25 °C) or in 0.1 M Tris buffer(pH 7.9, 35 °C). The minor chemical shift changes (up to 0.06ppm in 1H and 0.6 ppm in 15N) are consistent with expectationsof variations in pH, temperature, and buffer content. The ab-sence of significant chemical shift perturbations in this finger-print region suggests no significant changes in the proteinstructure. The bARK PH domain tertiary structure here is alsogenerally similar to other PH domain structures measured inthe range pH 6.0–9.0 (8, 11, 34–39). The high flexibility of theC terminus in the extended bARK PH domain construct re-ported here is also preserved at the more physiological condi-tions, as indicated by negative steady-state heteronuclearNOEs observed for the C-terminal residues (663–670) in phos-phate-buffered saline (pH 6.0, 25 °C). The binding to Gbg sub-units is also retained at pH 4.5, with c. 100 nM affinity of theGST fusion protein at pH 4.5 and 7.5, from an immunoblottedWestern assay (19).

Structure of the C-terminal Extension: Molten Helix—TheC-terminal segment shows an unusual structural feature that,to our knowledge, has not been reported previously in proteins.NOEs characteristic for an a-helix are preserved for residuestoward the C terminus despite the gradual loss of other NMRcharacteristics of helical structure (deviations from standardchemical shifts, heteronuclear 15N{1H} NOEs, 3JHNHa cou-pling) (Fig. 2). Increased mobility is indicated by both relax-ation and solvent exchange/accessibility data, suggesting thatthe C-terminal part of the a-helix is present as a “molten helix”in solution. Molecular dynamics calculations (40) of a-helicalmelting appear to be qualitatively consistent with our NMRobservations.

The hydrophobic residues C-terminal to Gln-656 (Leu-657,Val-658, and Val-661) are located at proper sequence positionsto extend the already existing hydrophobic strip on the helix

surface. However, being extended beyond possible interactionwith the protein core’s b-sandwich and therefore exposed tosolvent, these residues lack proper hydrophobic contacts toother residues that might stabilize the helix formation. It ispossible, however, that the C-terminal extension of the helixbecomes structured under certain conditions, e.g. in the pres-ence of a binding partner such as Gbg subunits. It is possiblealso, that the apparent “molten helix” is a consequence of theexpression of the C-terminal region of bARK in the absence ofits native protein context, and therefore might be stabilizedthrough tertiary contacts with residues N-terminal to the PHdomain. It is unlikely, however, that the thermodynamic sta-bility of the protein is a result of special conditions of this study(low salt, pH4.5), since both the CD and NMR data (see above)indicate no significant perturbations in the bARK PH domainstructure upon exchange into phosphate buffer.

In the human SOS PH domain, residues N-terminal to thenormal PH domain (in the Dbl homology domain) make welldefined structural contacts with the PH domain, (41) and in theBtk PH domain, the “Btk motif” C-terminal to its nominal PHdomain packs against the C-terminal b-sheet (39).

Binding of Phosphatidylinositides and Inositol Phos-phates—A general hypothesis that has been advanced in theliterature is that the PH domain recognizes specifically andwith high affinity highly anionic phospholipids, especiallyphosphatidylinositol (4, 5) bisphosphate (42). This has beenillustrated for the PLCd PH domain, in which the binding ofPI(4,5)P2 and Ins(1,4,5)P3 is submicromolar, and a well definedstructural interaction with the PH domain has been character-ized (8). However, other PH domains appear to have signifi-cantly weaker affinity (11, 12, 42). There is also sensitivity tothe isomer identity of the inositide, since the PH domain of Akt(13, 14) binds to phosphatidylinositol 3,4-bisphosphate, and anewly identified GRP1 binds to phosphatidylinositol 3,4,5-triphosphate (43). It is evident that the basic residues in theN-terminal section involved in the PLCd PH domain/ligandcomplex (8) are not generally present in the structure-basedsequential alignment of Table II. The physiological relevance ofphosphatidylinositol phosphates and phosphoinositides bind-

FIG. 4. The effect of the C terminuson the electrostatic potential of thehbARK1 PH domain, and comparisonwith the PLCd PH domain. Surfacesare contoured at 22 kT/e (red) and 2 kT/e(blue) (GRASP; Ref. 56) for variouslengths of the C-terminal extension: a,full-length construct, 556–670; b, resi-dues 556–666; c, residues 556–661; d,residues 556–656; e, nominal PH domain,residues 556–651. The bARK1 PH do-main constructs in b–d correspond to C-terminal deletion studies of Gbg binding(b and c (50) and d (19)). The most C-terminal residues, upon truncation, areindicated. For comparison, the electro-static potential of the PH domain fromPLCd (Protein Data Bank entry 1MAI) isshown in f; the arrow indicates a posi-tively charged area at the opening of theb-barrel, which is involved in the phos-pholipid binding (8). The molecular orien-tations are similar, as indicated by thebackbone tube diagrams.

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TABLE IIStructure-based alignment of the PH/PTB domains

Domains are as follows (top to bottom): GRK-2 (this work); human dynamin PH domain (35); PH domain of human SOS (41); PH domain of mouseb-spectrin (36); PH domain of Drosophila b-spectrin (37); N-terminal PH domain from pleckstrin (38); PLC-d PH domain (8); PTB domain of Shc(16); PTB domain of IRS (17). The alignment was done by pair-wise superposition of the structures and direct calculation of aligned RMSD (TableIII), based on the elements of regular secondary structure, as described in the text. Residues belonging to the elements of secondary structure usedfor the alignment are enclosed in boxes, labeled on the top. Numbers indicate residue positions within the corresponding secondary structureelement. Conserved hydrophobic residues are colored green. The only conserved residue in PH domains, Trp a1–7, is colored blue and underlined.In the Shc PTB domain, residues 58–114, belonging to an insertion in the b1/b2 loop are omitted.

TABLE IIIPairwise root-mean-square deviations (in Å) between the PH/PTB domain structures

The elements of regular secondary structure of the proteins used for the comparison and their alignment are indicated in Table II. Numbersshown above the diagonal were obtained with only Ca atoms selected for the alignment and r.m.s.d. evaluation, whereas numbers below thediagonal correspond to all heavy backbone atoms (N, Ca, C9, and O) in the selected core elements taken into account. The percent of sequenceidentity between core elements of the compared proteins is indicated in the parentheses. PH/PTB domain notation is the same as in Table 2. Proteinatom coordinates were obtained from the Protein Data Bank, PDB entries 1DYN (dynamin), 1AWE (hSOS), 1PLS (pleckstrin), 1BTN (b-spectrin,mouse), 1DRO (b-spectrin, Drosophila), 1MAI (PLCd), 1SHC (Shc PTB), and 1IRS (IRS PTB).

a Dyn (A) and (B) refer to the crystal structures of the two monomers in the dynamin PH domain dimer observed in the crystallographic studies (35).

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ing to PH domains remains unresolved for the PH domains ofb-spectrin, N-pleckstrin, dynamin, and bARK.

Possible PI(4,5)P2 Binding Site on bARK-1 PH Domain andIts Significance—Using Ins(1,4,5)P3 as a model compound forPI(4,5)P2, the 15N and 1H spectral perturbations upon titrationwere mapped for hbARK-1 PH domain using a previously pub-lished procedure (12). Under the experimental conditions, thebARK PH domain binds Ins(1,4,5)P3 with Kd of 207 mM, accord-ing to our protein fluorescence titration measurements usingthe protocol described in Ref. 12. The data (not shown) indi-cated maximal shift perturbations at residues Gly-569, Trp-576, Arg-578e, Tyr-580, and Ala-596, located in the N-terminalsegment of the domain, a pattern seen similar to, but differentin detail from, other PH domains. The amide 1H chemical shiftof Asp-635 is also perturbed (0.02 ppm), that is probably causedby variation in the distance between this site and the closelylocated (in the unbound state) positively charged side chain ofArg-578.

The association of bARK with membranes is complex. It hasbeen suggested (44) that the high affinity binding of bARK tomicrosomal membranes depends on a segment of the N termi-

nus of bARK, distinct from the putative PH domain. However,other investigators have shown that the bARK PH domainbinds with moderate specificity to PI(4,5)P2 and suggest thatsynergistic interactions of binding to both PI(4,5)P2 and to Gbg

proteins via the PH domain are required for activation ofbARK. (45). This synergism was not observed in a model sys-tem of higher turnover, where PI(4,5)P2 was inhibitory (46).

Residues perturbed by the Ins(1,4,5)P3 binding are locatedmostly in the b1/b2 loop and in the N-terminal part of theb2-strand. This rather flexible loop is relatively distant fromboth the putative Gbg binding site and the area of hydrophobiccontacts between the a-helix and the protein core. The presentdata provide no direct structural evidence for a possible rela-tionship between the phospholipid and Gbg binding regions.

Protein Interaction—The clustering of positive residues inthe extended C-terminal helix creates a positively charged siteon the bARK PH domain surface (Fig. 4, a–e), in addition to acluster of positive charges at the opening of the b-barrel, a siteimplicated for phospholipid binding (11) in other PH domains(Fig. 4f). This causes a different polarity of the surface chargein the case of bARK PH domain, so that in the fully extendedconstruct, the dipole moment of the molecule is aligned at ;30°angle relative to the helical axis (Fig. 5). Truncation of a fewC-terminal residues causes a 2.5-fold reduction in the proteindipole moment and alters the orientation of the dipole vector toclose to perpendicular to the helix axis. Further truncationcauses only minor variations of the dipole vector. This reduc-tion could explain the observed differences of Gbg binding totruncated bARK1 PH domains (19).

A depression on the bARK PH domain surface between thea-helix and the b5-strand, flanked by positively charged sidechains of Lys-644, Lys-645, Arg-648, and Arg-652 (helix); Lys-623 (b6); and Arg-625 (b6/b7) (Fig. 4) resembles the site in thePTB domains involved in the phosphopeptide binding of thatprotein. This topological similarity suggests that these residuesmight be involved in electrostatic interaction with a negativelycharged cluster on the Gb surface formed by Glu-226, Asp-228,Asp-246, and Asp-247 of the WD5 and Asp-267, Asp-290, andAsp-291 of the WD6 subunits (Fig. 6). With the exception of theAsp-247 and Asp-291, which are highly conserved among theWD40 subunits and are involved in the formation of an inter-and intra-blade hydrogen bonds (47, 48), other negativelycharged residues in this cluster are unique for WD5 and WD6,and highly conserved in eukaryotes. Asp-228 and Asp-246 aredirectly involved in ionic interactions with the switch-II region(a-Lys-210, Fig. 6) of Ga, which plays a critical role in G proteinheterotrimer formation (47). It is possible that in the absence ofGa the PH domain of bARK interacts (via its positively chargedC terminus) with the same residues on the top of Gb surfaceand therefore either directly or indirectly interferes with Ga

binding to Gbg.The bARK2 (GRK-3) PH domain has a different receptor and

Gbg selectivity, but possesses a similar pattern of basic residues(Lys-644, Lys-645, Lys instead of Arg at 625 and 652, and Arginstead of Lys at 623) (Fig. 1). The difference of sequence in theC-terminal segment of the bARK2 PH domain as comparedwith bARK1 (Arg/Asn-648, Gln/Arg-656, and Gln/Arg-659) re-sults in an increased total positive charge and thus a largerpolarization of the PH domain. Consistent with the above elec-trostatic model of the bARK - Gbg interaction, a recent Gbg

binding assay (49) involving the C-terminal-peptide sequences(corresponding to the segments 643–670 and 648–665) indi-cates a higher potency of the PH domain of bARK2 comparedwith that of bARK1.

The structure of the bARK PH domain allows rationalizationof the results of bARK truncation studies (50). The modifica-

FIG. 5. The dipole moment as a function of C-terminal trunca-tion. The magnitude of the dipole moment (A) and its orientation (B)relative to the a-helix (the polar angle between the dipole vector and thea-helix axis), for various lengths of the C-terminal extension in bARK1PH domain. Upon truncation, the C terminus was capped with the COO-group. The following partial charges were assigned to the side chainatoms: 11 (NZ in Lys), 10.5 (NH2, NH3 in Arg), 20.5 (OG1, OG2 in Aspand OD1, OD2 in Glu) (56), and the protein mass centroid was used as theorigin (57). Each peptide bond was assigned a dipole moment of 3.5 Debyealigned along the CO bond (57, 58). To account for the observed flexibilityin the loops and in the termini, the results are the average of the valuesfrom the ensemble of 20 bARK PH domain structures (Fig. 3A). Similarresults were obtained using a much larger ensemble of 100 NMR-derivedlowest-target-function structures from the same distance-geometry calcu-lation. Positions corresponding to the charged residues are labeled in A.Insets show the orientation and relative strength (in arbitrary units) ofthe effective electric dipole vector, colored green, in the full-length (B) andin the residue 556–656 construct (A).

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tions in bARK that affect the Gbg binding in that assay can beexplained either by deletion of one or more core elements,hence causing a disruption of the overall fold of the PH domain,or by deletion of positively charged residues on the C terminus(see below).

Several fusion proteins, containing sequences encompassinga PH domain (from Ras-GRF, Ras-GAP, OSBP, b-spectrin,IRS-1, and others), were shown to bind Gbg in vitro with vary-ing affinities, and some of these PH domains were able tocompete with the bARK PH domain and Ga for binding to Gbg

(18). The C-terminal extended PH domains of Btk (51), IRS-1,and Dbl (19) were also demonstrated to interact with the Gbg

heterodimer in vitro. The results of indirect assays (51, 52)suggest that some of these PH domain constructs may alsointeract with Gbg in vivo. The C-terminal parts of all theseproteins, in particular region corresponding to residues 644–670 in bARK (Figs. 1 and 4), are rich in basic residues, and thissupports the proposed bARK-Gbg interaction model. Other lit-erature data are also consistent with this model. Mutation ofthe last four of the five basic residues in the highly charged Cterminus (Arg-660, Lys-663, Lys-665, Lys-667, and Arg-669) toacidic ones leads to almost complete loss of the Gbg binding(20), as does a deletion of the last nine residues (662–670) (50).A truncated fragment, ending with Asn-666, does retain an

FIG. 6. Electrostatic polarization ofthe Gbg heterodimer. A, molecular sur-face of the Gbg complex colored by theelectrostatic potential, from red (210kT/e) to blue (110 kT/e). A fragment ofGa (green) contains the switch II region(a2 helix), which is intimately involved ina direct contact with the top of the Gb

propeller in the heterotrimer (47, 48).Those positively charged residues of Ga

(Lys-209, Lys-210) in the Gabg complexless than ;3 Å between side chain heavyatoms from Asp-228 and Asp-246 of Gb

are labeled. The highly negativelycharged area around this position mayserve as a complementary charged sur-face to the bARK1 PH domain. B, a dia-grammatic representation of the locationof negative charges on the Gb surface. Thesurface corresponding to the highly nega-tively charged area in A is indicated by abroken line. The general sequence consen-sus of a WD motif and location of nega-tively charged residues in the sequencesof WD5 and WD6 subunits are also shown(B, top). The residues in the WD repeatsequence are marked: x, a non-conservedposition; h, a conserved hydrophobic posi-tion; r, a conserved aromatic; p, a con-served polar position; t, a tight turn con-taining Gly, Pro, Asp, or Asn. Thesuperscripts indicate the range of resi-dues observed in the various known Gb

subunits. Those acidic residues that areunique for the WD5 and WD6 subunitsare shaded in gray. A was generated us-ing GRASP (56); atom coordinates wereextracted from PDB entry 1GG2; theschematic representation of Gb and thegeneral sequence consensus of WD motifswere adopted from Ref. 59.

FIG. 7. Backbone r.m.s.d. values between the PH/PTB domainsversus percent of sequence identity in the superimposed sec-ondary structure elements. Data are taken from Table III. The solidline represents the relation: r.m.s.d. 5 0.4 e1.87 (1 2 h), where H is afraction of identical residues (33), obtained for homologous proteins.Data corresponding to bARK1 PH domain are indicated by solid sym-bols. The leftmost data point (open square) corresponds to r.m.s.d.between DynA and DynB (see Table III).

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intermediate level of Gbg activation, whereas further trunca-tion up to Val-661 leads to a significantly lower amounts of Gbg

binding compared with the full-length construct (50). On theother hand, the results of deletion analysis (19) indicate thatthe C-terminal extension beyond Gln-656 is not absolutelyrequired for binding to Gbg; the full-length C-terminal exten-sion (553–689 construct) dramatically increases the maximalextent of Gbg binding although does not significantly alter thebinding affinity. A 18-residue peptide comprising the C-termi-nal amino acids 648–665 was recently shown to bind Gbg, alsosuggesting that the critical C-terminal extension in bARK1 PHdomain required for Gbg binding might be shorter than initiallysuggested by (50). However, other functions may be associatedwith the extended domain.

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David CowburnDavid Fushman, Taraneh Najmabadi-Haske, Sean Cahill, Jie Zheng, Harry LeVine III and

SUBUNITS γβPARTNER OF G-Adrenergic Receptor Kinase 1): A BINDINGβProtein-coupled Receptor Kinase 2 (

The Solution Structure and Dynamics of the Pleckstrin Homology Domain of G

doi: 10.1074/jbc.273.5.28351998, 273:2835-2843.J. Biol. Chem. 

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