Crystal structure at 2.2 Å resolution of the pleckstrin homology domain from human dynamin

Cell, Vol. 79, 199-209, October 21, 1994, Copyright 0 1994 by Cell Press

Crystal Structure at 2.2 A Resolution of the Pleckstrin Homology Domain from Human Dynamin Kathryn M. Ferguson,’ Mark A. Lemmon,t Joseph Bchlessinger,t and Paul B. Slgler* *Department of Molecular Biophysics and Biochemistry *Department of Chemistry Howard Hughes Medical Institute Yale University New Haven, Connecticut 06510 tDepartment of Pharmacology New York University Medical Center New York, New York 10016

Summary The X-ray crystal structure of the pleckstrin homology (PH) domain from human dynamln has been refined to 2.2 A resolution. A seven-stranded p sandwich of two orthogonal antiparallel p sheets is closed at one corner by a C-terminal a helix. Opposite this helix are the three loops that vary most among PH domains. The basic fold is very similar to that of two other PH domains recently determined by nuclear magnetic reaonance, confirming that PH domains are distinct structural modules. Each PH domain with known structure is electrostatically polarized, with the three varlable loops forming a positively charged surface. This surface Includes the position of the X-linked lmmunodeflclency mutation in the Btk PH domain and may serve as a ligand-binding surface.

The PH domain is a region of approximately 120 amino acids present in many proteins involved in intracellular signaling pathways. It shows homology to two regions of pleckstrin, the major protein kinase C substrateof platelets (Tyers et al., 1966). The frequent occurrence of this region of homology was originally pointed out by Mayer et al. (1993) and Haslam et al. (1993) who identified PH domains in the following: serinenhreonine kinases, regulators of small GTP-binding proteins, cytoskeletal proteins, putative signaling adapter molecules, as well as proteins involved in cellular membrane transport. Further data base searches suggested their occurrence in a large number of additional proteins, expanding these groups and including tyrosine kinasesand phospholipase C (PLC) isoforms (Musacchio et al., 1993; Shaw, 1993; Parker et al., 1994). The total number of PH domains identified now exceeds 50. Little is currently known about PH domain function, but conceptual analogy with SH2 and SH3 domains (reviewed ,by Schlessinger, 1994) has led to the argument that they may be involved in directing protein-protein interactions. Although this has yet to be established, several recent reports provide clues about possible functions of the domain. The challenge is to determine which of these are real, and what they have in common.

Supporting a role in intermolecular interactions is the finding that the N-terminal PH domain of Bruton’s tyrosine kinase (Btk) has an arginine to cysteine substitution in mice bearing the X-linked immunodeficiency (xid) mutation (Thomaset al., 1993; Rawlings et al., 1993). Btk kinase activity is not affected by this mutation, but XID 6 cells cannot respond normally to activating signals, suggesting that interactions of Btk with other components of the signaling pathway may be affected. PH domains have also been implicated in pathways of signaling mediated by the 6~ subunits of heterotrimeric G proteins (Gr,.,).The p-adrenergic receptor kinase (PARK) binds to GBrvia a C-terminal region of the kinase that includes a PH domain (Pitcher et al., 1992; Koch et al., 1993). PH domains from several other proteins have also been reported to associate with Gpr (Touhara et al., 1994) suggesting that such interactions could recruit effector molecules in GB7mediated signaling (Clapham and Neer, 1993). However, the work of Touhara et al. (1994) indicates that the affinity of isolated PH domains for GBr is very low compared with that for binding of intact PARK, and little specificity is evident. Furthermore, the association of PARK with GBr is inhibited by a 26-residue peptide that contains only the C-terminal few residues of the BARK PH domain (Koch et al., 1993). Most PH domain-containing proteins do not appear to interact with GB~.The 6 and 5 isoforms of PLC both have a PH domain at their N-terminus (Parker et al., 1994) but while PLC-3 is regulated by GBr, PLC-5 is not. The PH domain of PLC-51 appears important for binding of the enzyme to lipid bilayers containing its substrate, phosphatidylinositol 4,bbisphosphate (PIP*) (Cifuentes et al., 1993) as well as to its product, inositol 1,4,5-trisphosphate (IPa) (Cifuentes et al., 1994; Yagisawa et al., 1994). In addition, a role in dimerization of the enzyme has been suggested (Ellis et al., 1993). Finally, a portion of 5~ spectrin that contains its PH domain binds to specific sites in bovine brain membranes (Davis and Bennett, 1994) indicating a membrane localization function for the domain. Indeed, the functional importance of membrane localization is a characteristic shared by all PH domaincontaining proteins. As part of an effort to understand the role of this common domain, we have determined the crystal structure at 2.2 A resolution of the PH domain from human dynamin (DynPH). Dynamin is a GTPase of 100 kDa (Obar et al., 1990) with significant homology to the Drosophila gene shibire (Chen et al., 1991). Like the shibire gene product, dynamin appears to be involved in the initial stages of endocytosis (reviewed by Vallee et al., 1993). DynPH encompasses residues 510-633 of the 664 amino acid protein and is upstream of a C-terminal region that interacts with microtubules and SH3domains(Shpetner and Vallee, 1992; Herskovitset al., 1993; Gout et al., 1993). Our efforts to identify a binding partner for the domain have not yet been successful, but consideration of the structure presented here and comparison with other known structures may facilitate identification of PH ligands.

Cdl 200

Table I. Summary of Data Collection Statistics Crystal Detector Resolution limit Unique reflections” Completeness Rmb
Native 1 R-Axis II 2.2 A 12204 96% 9.1% 25.9 -

Native 2 CHESS Al 2.2 A 6790 73% 7.7% 33.1 -

CHsHgCl CHESS Al 2.2 A 15754 70% 7.7% 23.0 24.0% 7.2%

PCMB Xentronics 2.8 A 8978 79% 7.4% 7.4 17.4% 7.6%

Se-Met R-Axis II 2.5 A 8828 95% 8.3% 27.2 8.8%

PtClz (NH& Xentronics 3.1 A 3333 71% 6.4% 9.6 17.6% -

a For the two mercury data sets, in which anomalous data were collected, the number of unique reflections counts the Freidel equivalents, F+ and F-, separately. bR,, = & <(I,, - lh. I> I Cn. lh,, where is the average of the absolute deviation of a reflection Ih. from the average Ih of its symmetry and Freidel equivalents (except for the mercury data where the Freidel pairs were not merged). c lsomorphous difference = C 1 JF,J - JFpJ J/CJF,(, in which JFphJand JFpJare the observed amplitude for the heavy-atom derivatives and protein, respectively. “Anomalous difference = C( IF+/ -IF-J f/C ((IF+I+JF-1)/2).

Reeuite and Discussion DynPH was expressed at high levels in Escherichia coli, using the T7 expression system (Studier et al., 1990). Up to 80 mg of purified protein could be obtained from 1 liter of culture, as described in Experimental Procedures. Tetragonai crystals (a = 85.2 A, c = 132.8 A, space group 1422) of the DynPH were grown in sitting drops at 18% using Na/K phosphate (pH 8.5) as the precipitant. The crystals diffracted X-rays to at least 2.0 A. Two molecules were found in the asymmetric unit, and the solvent content of the crystals was 41%. To solve the structure, phases were determined by the method of multiple isomorphous replacement (MIR), using two mercury derivatives, a platinum derivative, as well as selenomethionine-containing protein. Further phase information was also obtained from anomalous scattering from the mercury derivatives. These data are summarized in Tables 1 and 2. The initial MIR map permitted a clear tracing of the secondary structural elements of the domains. These segments were used to determine the relationship between the two molecules in the asymmetric unit. Following solvent flattening and noncrystallographic symmetry averaging, mo8t of the connecting loops could also be traced. Model building for each of the two molecules in the asymmetric unit was performed independently, allowing a view of the domain in two differ-

ent crystal-packing environments. The current model, refined to 2.2 A, consists of 113 residues (equivalent to residues 518-830 of dynamin) in each of the two molecules in the asymmetric unit. A total of 185 water molecules is also included. The refinement statistics are summarized in Table 3. A Ramachandran plot Show8 no violators of accepted backbone torsion angles. Overview of the DynPH Structure For the purposes of describing the structure, amino acids will be numbered from 1-125, according to the sequence of the portion of dynamin that was expressed. Position 2 corresponds to residue 510 of whole dynamin (Figure 1). As depicted in Figure 2, the overall fold of DynPH is best described as an antiparallel f3 sandwich, with two nearly orthogonal sheets in which the strands are arranged as in a 9 meander (Richardson, 1977). Residues 14-58 form a four-stranded antiparallel 3 sheet that packs almost orthogonally against a second 3 sheet of three strands (residues 82-99). It is not, as previously described (Yoon et al., 1994; Wagner, 1994), a f3 barrel, since the hydrogenbonding pattern does not continue all around the structure (Woolfson et al., 1993). As is typical for orthogonal P-sheet packing (Chothia, 1984), the right-handed twist of the two sheets results in close contact only at two (close) corners. The other corners are splayed apart as seen in Figure 28.

Table 2. Summary of MIR Statistics Resolution (A) Mean figure of merit CHsHgCl lsomorphous phasing power Anomalous phasing power PCMB lsomorphous phasing power Anomalous phasing power PtCk(NH& lsomorphous phasing power Se-Met lsomorphous phasing power

15-8.7 0.86

6.1 0.88

4.7 0.82

3.8 0.73

3.2 0.64

2.8 0.54

2.5 0.46

2.2 0.40

Total 0.57

3.48 0.67

5.41 0.84

3.66 0.81

2.57 0.88

2.16 0.61

2.06 0.64

2.17 0.52

1.61 0.35

2.33 0.58

3.32 0.51

4.01 0.45

2.90 0.37

1.97 0.23

1.78 0.18

1.75 0.15

-

-

2.23 0.23

0.93

1.16

1.11

0.84

0.72

0.65

-

-

0.90

1.33

1.49

1.40

0.89

0.80

0.70

0.52

-

0.84

Phasing power = C JFb’( / C 1 J IFpOJexp(iQ,) + Fhcl - JFph”l 1, IFhcl = heavy-atom structure amplitude, and JF,0Jand JFphOlare observed amplitudes for the protein and heavy-atom derivatives, respectively: 0, is the calculated phase. Figure of merit = J P(@)exp(i@)dWJP(Q)@ in which P is the probability distribution of @, the phase angle.

EOTtal Structure of a Pleckstrin Homology Domain

Table 3. Refinement

Statistics

Resolution shell (A) Number of Unique Reflections Number of Predicted Reflections ~ Rmwb R factof R factor >2.0 o cfatac

6.00-4.21 1639 1644 15.7 0.04 0.16 0.16

rms deviations Bond lengths Angles

O.OOQA 1.602~

3.42 1574 1576 15.7 0.05 0.16 0.15

3.02 1564 1564 14.9 0.07 0.21 0.19

2.75 1555 1555 13.3 0.09 0.26 0.23

2.56 1536 1543 11.7 0.11 0.29 0.26

2.42 1534 1536 9.07 0.14 0.34 0.26

2.30 1523 1537 7.06 0.17 0.35 0.29

2.20 1213 1540 3.6 0.34 0.36 0.26

Total 12140 12467 14.0 0.06 0.22 (0.34) 0.20 (0.32)

’ is the combined value for the Native 1 and Native 2, which were merged for the refinement. bR-= C W<(In - &.I>, I Ch. IM, in which <(In - IM (> is the average of the absolute deviation of a reflection lh. from the average Ih of the two native data sets. ’ Number in parentheses is the free R value (Briinger, 1992b).

Strand 61 contributes to both sheets at one of the close corners by forming a 6 bend that results from a strong right-handed twist at W17 (4 = -62”, w = 1459 in the middle of the strand. The other close corner (between strands 64 and 65) is completed by a type II 6 turn. One of the splayed corners of the sandwich is closed off by the C-terminal amphipathic a helix (al ; residues 102-l 14).

The other splayed corner is closed off largely by the f36/ P7 connecting loop. The C-terminal a helix interacts with strand 61 and packs against f35, to which it is parallel. Hydrophobic side chains from the a helix project into the interior of the sandwich that is formed by the two 6 sheets and contribute to the well-packed hydrophobic core of the protein. Side chains from the 6 strands that participate in

60 .

nymmin

. . . . . . . ..K FY. . . . . . . . ..........

spectrin Pleckatrin

N

Pleckstrin

C

YYDPAGAE..

..........

.......... ..........

pAIlK-

RasGAP hSos WV Grb7 PLCB-1 PLCS-1 Btk

. . ..GS..KK

70

80

90

100

110

120

oynamin spectrin

Pleckstrin

N

Plecketrin $ARK-2 RBBQAF hSos

C

VaV

Grbl PL@-1 pm-1 Btk

Figure 1. Primary Sequence

Alignment

PH domains of known structure were aligned on the basis of the positions of their conserved secondary structural elements. These elements are colored green for 5 strands (61 to 57) and blue for the C-terminal a helix (al). Selected other PH domain sequences were then aligned as described in Experimental Procedures. The vertical dotted lines encompass the secondary structure elements and serve to delineate likely boundaries for these element8 in the PH domains for which the structure has not yet been determined. The observed secondary structural elements are indicated below the sequence, as are the locations of the three variable loops. The point mutation (R to C) in Btk found in XID mice (Rawlings et al., 1993) is indicated by a yellow block, with an asterisk marking the position in the sequence alignment. A number sign marks an insertion of 20 amino acids in the Btk PH domain. Numbers along the top of the alignment refer to those used for DynPH.

Figure 2. The Three-Dimensional

Structure of DynPH

The overall fold of amino acids IO-122 of DynPH is depicted. (A) View perpendicular to the plane of the two g sheets. Residue numbers corresponding to the beginning and end of each element of secondary structure are shown. See text for relationship between these sequence numbers and those in full-length dynamin. (6) View between the p sheets, toward the C-terminal a helix. The molecule has been rotated t?tY about a vertical axis with respect to the view in (A). (C) Stereo pair of an alpha carbon trace in which the relationship between the two molecules in the asymmetric unit is shown. Residue numbers in molecule 2 (green) are obtained by adding 200 to the sequence number in DynPH. Side chains of the conserved tryptophan (Wlr38, WSCJ8) and the arginine with which it interacts (R14, R214) are shown in blue.

the hydrophobic core are typical in their composition for residues most commonly found in the contact regions between 6 sheets (Chothia, 1964). W108 is the only amino acid that is invariant in all PH domain sequences (Musacchio et al., 1993). It is located in the middle of the C-terminal a helix and is quite buried, largely by van der Waals contact with the side chain of R14 in strand 61, a residue that is relatively well-conserved among PH domains. As shown in Figure 3, the interaction between strand 61 and the C-terminal a helix is stabilized by hydrogen bonds formed between the guanidino group of R14 and two residues in the C-terminal a helix, as well asseveral water-mediated interactions. G16, another well-

conserved amino acid in PH domains, is also located in this region. DynPH crystallized with two molecules per asymmetric unit. The packing between these two molecules is significantly more intimate than that seen for other contacts in the crystal and buries approximately 696 A2 on each molecule. The resulting total buried surface of 1600 A2 is comparable to that buried in the interface between several strongly interacting proteins (Janin and Chothia, 1990) leading us to suspect that the interaction may be of physiological relevance. Indeed, dynamin purified from rat brains has been reported to occur in an oligomeric state (Maeda et al., 1992; Vallee et al., 1993). Furthermore, in studies

;Oytal

Structure of a Pleckstrin Homology

Figure 3. Representative

Domain

Electron Density

Stereo pair of a (2F,-FC) map contoured at 1.5 o. The view is into the hydrophobic core of the protein, between strand 31 and al and shows the interaction between R14 on 31 and Sill on al. R14 packs against the conserved W103. and the interaction of bl and al is further stabilized by a water-mediated hydrogen bond between W103 and the carbonyl oxygen of Y17.

of recombinant PLC-51, it has been reported that the N-terminal PH domain may play a role in dimerization of the enzyme (Ellis et al., 1993). However, a series of experiments suggests that isolated DynPH does not dimerize in solution. It migrates in size exclusion chromatography as a 14 kDa protein, in good agreement with the actual molecular mass. Furthermore, a GST-fusion protein of DynPH that was immobilized on glutathione-agarose beads did not retain free DynPH. Finally, small-angle X-ray scattering (SAXS) studies show that DynPH exists as a monomer (Rc = 16.5 A) in solution under conditions similar to those used for crystallization (Z. Bu and D. M. Engelman, personal communication). Nonetheless, interactions between PH domains could stabilize the reported oligomeric structure of dynamin through a cooperative set of interactions involving additional regions of the intact molecule or a complex involving another molecular partner. The interface between the two molecules observed here involves residues that are relatively well conserved between PH domains and is largely defined by strand 61 and the C-terminal a helix (Figure 2C). In the dimer interface of the crystal, the C-terminal a helices are associated across the interface as an antiparallel coiled coil, and the apolar portion of the R14 side chain is packed against the equivalent portion of its counterpart in the neighboring molecule. In addition, several direct and water-mediated hydrogen bonds are seen between the two molecules. The N- and C-termini of DynPH are 30 A apart (10 C. to 122 C.). The portion of the protein from residues 116 122, which follows the C-terminal a helix, interacts with strand 55 forming a short parallel 6 strand, then continues in an extended conformation. The positions of the two termini appear to be consistent with the ability of PH domains to be included in a number of different protein contexts (Musacchio et al., 1993). Both termini are located toward the same side of the molecule that contains the C-terminal helix, as depicted in Figures 2A and 28.

Conservation of PH Domain Structure The overall fold of DynPH is very similar to the recently reported nuclear magnetic resonance (NMR)determined structures of PH domains from spectrin (Macias et al., 1994) and pleckstrin itself (Yoon et al., 1994; Wagner, 1994). This correspondence of the architecture of the domains provides a very persuasive argument for the existence of PH domains as structurally distinct protein modules like SH2 and SH3 domains. The argument is perhaps more important in the case of PH domains, since the sequence identity that they share is more tenuous than in the other cases. DynPH shows just 25% and 20% identity to the pleckstrin N-terminal (pleckstrin-N) and spectrin PH domains, respectively (Figure I), at which levels structural homology cannot be assumed (Chothia and Lesk, 1966). Since these levels of identity are typical for pairwise comparisons of proposed PH domains, it is remarkable that Musacchio et al. (1993) closely predicted the sequence correspondence of secondary structural elements observed in these structural studies. In each of the three PH domains, short turns connect strands 62 and 53, 64 and 65, as well as 57 and the C-terminal a helix. This is predicted to be true for all PH domains except for an insertion between the 62 and 63 strands of the IRS-l PH domain (Musacchioet al., 1993; Shaw, 1993). The remaining loops are significantly more variable in sequence and structure. For example, the loop connecting strands 53 and 54 in the spectrin PH domain is significantly longer than that in pleckstrin-N or DynPH, and includes a two-turn a helix (Macias et al., 1994) that is not present in the other PH domain structures. Figure 1 presents a sequence comparison of selected PH domains in which the alignment is based upon the secondary structural elements in those of known structure. The predicted location of secondary structural elements in other PH domains is bounded by vertical dashed lines. The p1/62,63/64, and j36/57connecting loops are the least

Figure 4. Potential Functional Features (A) Variable loops cluster on one surface. A stereo view of the C, trace of DynPH is shown in an orientation rotated by approximately 90° about a horizontal axis compared with that in Figure 2B. The variable loops are colored yellow, while the remainder of the molecule is green. The variable loops shown (see Figure 1) comprise residues 21-30 (variable loop l), 46-52 (variable loop 2) and 83-94 (variable loop 3). The side chain of Y33, which is in a position equivalent to that of R28 in the Btk PH domain, is also shown and is colored blue. (B) Polarized electrostatic potential of DynPH. The electrostatic potential of DynPH was calculated using the program GRASP (Nicholls and Honig, 1991) and is contoured at +1.5 kT (blue) and -1.5 kT, (red). The orientation of the molecule is the same as in (A) and corresponds to that presented in Figure 3 of Macias et al. (1994) highlighting the similarity in electrostatic polarization of the two different PH domains (see text).

well conserved between PH domains in sequence and length. These three variable loops are all positioned on one side of the molecule, as shown in Figure 4A. The opposite side of the molecule, surrounding the C-terminal a helix, represents a more structurally conserved surface. PH domains therefore appear to have a surface that is particularly variable, which could define the proposed specific ligand-binding characteristics of particular PH domains. This arrangement of variable loops on a scaffold of f3 sheets is reminiscent of antigen recognition sites in immunoglobulin variable domains (Mariuzza et al., 1987).

Polarized Charge Potential of PH Domains Figure 48 shows that the electrostatic potential of DynPH is also polarized. The more conserved side of the domain, including the C-terminal a helix, is a region of negative potential, while the surface that includes the variable loops has a positive electrostatic potential. Electrostatic polarization in the same direction was reported for the spectrin PH domain (Macias et al., 1994) and a cluster of basic residues close to the variable loops of the pleckstrin-N PH domain suggests a similar polarization in this case (Yoon et al., 1994). Thus, a polarization of electrostatic potential

Crystal Structure of a Pleckstrin Homology 205

Domain

that is coincident with the location of the variable loops appears to be a conserved characteristic of PH domains. This characteristic suggests that PH domain ligands may be negatively charged. Indeed, the PH domain of PLC-Sl is involved in interaction of the enzyme with the anions PIP2 and IPs. We have been unable to detect association of DynPH with these or similar molecules. As mentioned in the Introduction, substitution of R28 with cysteine in the PH domain of Btk correlates with the X-linked immunodeficiency observed in xidmice. R28 corresponds in sequence alignments to the middle of strand 82. In DynPH, a tyrosine is found at this position, while methionine and valine occupy this position in the pleckstrin-N and spectrin PH domains, respectively. In DynPH, the equivalently located tyrosine (Y33) is solvent exposed and is present on the surface of the molecule that has positive electrostatic potential and includes the variable loops (Figure 4A). Assuming that it does not have deleterious effects upon the integrity of the structure, the effect of the R28C mutation in Btk adds further weight to our suggestion that this region of PH domain is important for ligand binding. Similarity to Other Structures As pointed out by Yoon et al. (1994) the overall PH domain structure shows some similarity to other molecules that have known ligands. The Brookhaven data base contains relatively few proteins with a topology similar to that of the PH domains. The most similar actually form up-and-down 8 barrels (Branden and Tooze, 1991) rather than the sandwich of orthogonal 8 meanders observed in DynPH. Most proteins with this topologically similar P-barrel architecture have functions that are closely related, and this could provide a clue as to PH domain function. Members of the lipocalin family, such as plasma retinol-binding protein (RBP) (Zanotti et al., 1993), as well as the fatty acid-binding protein (FABP) family, such as adipocyte lipid-binding protein (Xu et al., 1993), form a structural superfamily termed the calycins (Flower et al., 1993). All members of this superfamily form up-and-down 8 barrels that bear some resemblance to the PH domain architecture, and all bind small hydrophobic molecules. This led to the suggestion that the ligand for PH domains might be similar in nature (Yoon et al., 1994). While this is an attractive hypothesis, particularly given the failure of many studies to identify a ligand (see below), one argument makes it less convincing. Figure 5 contrasts DynPH with apo-bovine RBP (Zanotti et al., 1993), a typical calycin. An obvious cavity, lined with largely hydrophobic side chains, can be seen in the center of the 8 barrel of RBP, in common with structures available for other apo-calycins. In the case of the fatty acid-binding proteins, this cavity contains several water molecules (Xu et al., 1993). The fatty acid or retinollike molecule occupies this central cavity in the holocalycins, with little conformational change in the protein upon ligand binding. By contrast, DynPH has a very wellpacked hydrophobic interior between its two 8 sheets and shows no evidence at all for the existence of a cavity (Figure 5). No obvious hydrophobic binding site can be seen, suggesting that, in the absence of major conformational

Figure 5. View of the Hydrophobic

Core

(A) A view of the cavity in the hydrophobic core of apo-REP (Zanotti et al., 1993). A van der Waals surface is represented with dots. The view is into the center of the 5 barrel and shows residues from the 5 strands that line the inside of the cavity. Coordinates were obtained from the Brookhaven Protein Data Bank. (B) The hydrophobic core of DynPH. The orientation is the same as in Figure 28. The van der Waals surface around the side chains between the 5 sheets of the sandwich illustrates the close packing. By contrast with the RBP and the other calycins, no cavity can be detected in the hydrophobic core of DynPH, and there is no other obvious hy drophobic pocket for binding of a lipophilic molecule.

changes, the ligand-binding characteristics of DynPH are unlikely to be similar to those of the calycins. We therefore suggest, on the basis of the crystal structure, that a PH domain ligand is likely to be different in nature from the ligands for the calycins. It should be noted, however, that it is not clear from the NMR studies whether or not the spectrin and pleckstrin-N PH domains have a central cavity. Another protein that is topologically similar to the PH domains is streptavidin, which also consists of an up-anddown 8 barrel (Weber et al., 1989) rather than a 8 sandwich. Streptavidin has been suggested to be related to the calycins (Flower, 1993; Holm and Sander, 1994). However, unlike the calycins, but in common with DynPH, streptavidin has a well-packed hydrophobic core in the

Cell 205

center of its 8 barrel, with no detectable cavity. Biotin binds in a surface pocket at one end of the barrel, resulting in the ordering of two loops that connect 8 strands (Weber et al., 1989). If we are to draw analogies based upon such comparisons, then more hydrophilic small molecules such as biotin might also be considered as potential PH domain ligands. Alternatively, the topological similarity between the structures discussed here may have no functional implications: this topology may represent a particularly stable arrangement. Indeed, this has been suggested by Woolfson et al. (1 Q93), based upon considerations of topological constraints for B-sheet structures as well as considerations of orthogonal sheet packings (reviewed by Chothia, 1984). Furthermore, the same basic architecture is also observed in a domain of beef liver catalase (Reid et al., 1981) where its function is not related to that of the calycins or streptavidin. Finally, it is interesting to note that the spectrin SH3 domain has a broadly similar topology that was previously compared with RBP (Musacchio et al., 1992). Functional Implications The main challenge with PH domains at present is to determine whether they have a specific binding function and, if so, the nature of the ligand to which they bind. As described in the Introduction, PH domains were originally suggested to participate in protein-protein interactions involved in intracellular signaling pathways, in a manner analogous to that seen with SH2 or SH3 domains. We have undertaken extensive screening of 17 promoterbased bacterial expression libraries with GST-fusion proteins of several PH domains, in order to identify proteins with which they might interact. We have also utilized the “interaction trap” two-hybrid system to screen libraries for protein-protein interactions in yeast cells (Gyuris et al., 1993). Neither approach yielded cDNA clones that encoded proteins with which we could demonstrate association. These negative results suggest either that the PH domain ligand is not a protein, or that the PH domain and/ or its presumed interaction partner are not expressed appropriately in the systems that we have used to search for interactions. A requirement for posttranslational modification would provide one explanation. However, GST fusion proteinsof several PH domains, linked to glutathioneagarose beads, failed to precipitate detectable quantities of proteins from [35S]methioninslabeled cell lysates that were not precipitated by GST alone. We have also been unable to detect association of these PH domains with GBr. As mentioned in the Introduction, membrane bcalization is functionally important for all PH domain-containing proteins. The variable loops of the PH domains are presented on one surface of the molecule, such that they could create a ligand-binding site. The residue corresponding to R28 of the Btk PH domain is also present in this region, supporting the role of the variable loop surface in ligand binding. The apparent conservation of the electrostatic polarization of the domains and the presence of the proposed binding site in a region of positive potential suggest binding to negatively charged molecules. If the comparison with the calycins is valid, negatively charged

lipid molecules might be considered. Indeed, an N-terminal portion of PLC-81, which includes its PH domain, has been reported to form part of a binding site for PIP2 (Cifuentes et al., 1993). The conservation of electrostatic polarization among PH domains of known structure suggests the possibility that binding to a negatively charged surface could serve to orient PH domain-containing proteins with respect to the membrane. We have performed gel-filtration studies of the binding of DynPH to phospholipid vesicles containing both neutral and anionic phospholipids, including PIP2, phosphatidylinositol Cphosphate, phosphatidylinositol, and phosphatidylserine, as well as several other membrane components. No binding to any of these vesicles could bs detected. A recent report describes centrifugation studies that indicate binding of several other PH domains to negatively charged phospholipids in vesicles (Harlan et al., 1994) although no stereo-specificity is evident. Analysis of NMR chemical shift differences upon binding to PIPn in detergent micelles indicates that this interaction involves the positively charged surface of the PH domains. It remains to be shown whether such interactions of the positively charged surface are general and important for all PH domains. Where such interactions are significant, it is important to know whether they reflect nonspecific electrostatic interactions or stereochemically defined interactions with specific negatively charged components, such as lipid-derived second messengers, many of which have yet to be identified. Experimental

Procedures

Productlon and Purification of DynPH The polymerase chain reaction was utilized to amplify from the cDNA of human dynamin a fragment corresponding to the PH domain (residues 510-533). An Ndel site was incorporated at the 5’ end of the coding sequence, adding an initiator methionine to the native coding sequence and a SamHI site at the 3’ end. The resulting fragment was digested with Ndel and BamHl and was ligated into appropriately digested pET11 a (Studier et al., 1990) to produce pETDynPH for expression directed by the phage T7 promoter. The PH domain was expressed from this construct as described (Lemmon and Ladbury, 1994). Selenomethionine-containing protein was produced from the methionine auxotrophic E. coli strain 8834 (DE3) (Novagen), transformed with pETDynPH and grown in a MOPS-based minimal medium (Neidhardt et al., 1974) containing 50 mg/liter DL-selenomethionine (Sigma) and other unlabeled amino acids at the suggested concentrations The expressed protein had the sequence 1MKTSGNQDEI LVIRKGWLTI NNIGIMKGGS KEYWFVLTAE NLSWYKDDEE KEKKYMLSVD NLKLRDVEKG FMSSKHIFAL FNTEQRNWK DYRQLELACE TQEEVDSWKA SFLRAGVYPE RVGDK-125, the predicted molecular mass for which was confirmed by mass spectrometry. Quantitative amino acid analysisof the selenomethfoninecontaining protein confirmed that more than 95% of the methionines had been substituted with selenomethionine. After IPTG induction (3 hr), cells were lysed by sonication in 100 m M NaCI, 50 m M glucose, 25 m M MES (pH 6.0) containing 5 m M DTT and 1 m M PMSF. After centrifugation, the clarified lysate, containing greater than 90% of the protein, was passed through a column of DEAE-cellulose (Pierce) equilibrated in the lysis buffer. The flowthrough was then diluted to 30 m M NaCl with a solution of 25 m M MES (PH 5.0) and was loaded onto a EMD55OS (Merck) cation-exchange column and eluted with a gradient in NaCl from O-250 m M NaCI. The PH domain eluted at approximately 150 m M NaCI. Fractions containing the protein were identified by absorbance at 250 nm, and protein was precipitated by addition of solid (NH)&O, (Ultrapure) to 75% saturation. The precipitate was collected by centrifugation at 12.090 rpm in an SS-34 rotor (Sorvall), dissolved in the minimum volume of

0#al

Structure of a Pleckstrin Homology Domain

50 m M MES (pH 6.0) 100 m M NaCI, 10% glycerol, 1 m M DTT, and subjected to gel filtration using an FPLC (Pharmacia) Superose 12 or Superdex75column. Theproteinelutedasa 14 kDamonomer, and the resulting material was at least 99% pure, as assessed on overloaded silver-stained SDS gels. Glycerol was added to the pooled fractions to a final concentration of 50%, and the protein was stored at -2OOC.

Clystellfzation and Deta Collectlon DynPH was concentrated to 16-20 mglml and buffer exchanged into 10 m M HEPES (pH 6.5) with 25 m M NaCI, using an Amicon Centriprep 10. Diffraction quality crystals were grown in sitting drops using the vapor diffusion method. Sitting drops (20 ul) containing 9 mg/ml protein, 0.45 M NalK phosphate (pH 6.5) were equilibrated against a reservoir of 1.6 M NalK phosphate (pH 6.5). Crystals typically grew to 0.3 x 0.2 x 0.15 m m in 3-4 weeks at 16OC. Crystals were stabilized for at least 6 hr in 2.0 M Na/K phosphate (pH 6.5; with or without the addition of heavy atom). These crystals were briefly (less than 15 min) exposed to a cryostabilizer of 40 (w/v) PEG 400,25 m M MES (pH 6.5)and werefrozen in liquid nitrogencooled liquid propane. The crystals diffracted to beyond 2.0 A an-d were found* to be tetragonal (unit cell dimensions a = b = 65.2 A, c = 132.6 A), belonging to the space group 1422. Data were collected either on a Siemens Xentronics multiwire area detector or on an R-Axis II image plate detector, in both cases using double mirror-monochromated/focused Cu K. from a 0.2 m m focus of a Rigaku generator operating at 3 kW (50 kV x 60 mA), or on a CCD detector at CHESS (Cornell High Energy Synchrotron Source) station Al operating at a wavelength of 0.91 A (Table I). Xentronics data were processed using the program XDS (Kabsch, 1966a, 1966b); all other data were processed using DENZO (Z. Ctwinowski, 1993). All data were scaled and merged using SCALEPACK (Z. Otwinowski).

Structure Determlnetlon and Refinement Useful heavy atom derivatives were obtained by soaking crystals in stabilizing solution containing the following heavy metal salts: 1 m M CH3HgCI for 12 hr, 1 m M PCMB (pthloromercuribenzoate) for 12 hr. and 1 m M PtCI,(NH& for 70 hr. Two heavy atom positions for the mercury (CHIHgCI) derivative were determined from an isomorphous difference Patterson map. An additional two minor sites were located for this derivative from difference Fourier maps. The PCMB derivative contained only the two major mercury sites. Also, a single unique heavy atom site was located in the PtCMNH& derivative. Finally, an isomorphous difference Fourier map using data obtained from selenomethioninecontaining crystals produced two peaks, accounting for two of the eight methionine residues in the asymmetric unit. The positions and occupancies of these sites were refined and phases were computed using ML-PHARE (Z. Ctwinowski, Daresbury Laboratory). Additional phase information was obtained from anomalous scattering by the mercury in !he CHJHgCl derivative at 0.91 A and in the PCMB derivative at 1.54 A. Table 2 presents a summary of the phasing statistics. The initial experimental map obtained using these phases permitted clear interpretation of the secondary structural elements and many of the buried side chains. A partial alpha carbon trace was built into the initial MIR map using the program 0 (Jones et al., 1991). The coordinates of the resulting model were then used to define the noncrystallographic twofold relationship between the two molecules in the asymmetric unit. The phases were improved by noncrystallographic symmetry averaging, solvent flattening (Wang, 1965) and histogram matching (Zhang and Main, 1990) using a program developed by G. Van Duyne. The solvent mask was calculated assuming asolvent content of 36%. An almost complete model was built into the final map using 0 and was refined by simulated annealing, with initial temperatures of 4000 K, using XPLOR 3.1 (Briinger. 1992a) with no local symmetry restraints. The model was refined against a combined data set in which the two native data sets in Table 2 were merged. Ten percent of the data was removed for calculating the free R factor (Briinger, 1992b). The statistics for this merged data set are shown in Table b The model was improved using the original experimentally phased sigma-weighted (Reed, 1966) and (F.-Fd maps, calculated using XPLOR 3.1. Water molecules were added in the (F,F,) map during the later stages of the refinement and accepted if they reap peared with strong density in the (2F.-F,) maps, participated in at least one hydrogen-bonding interaction and refined to have a temperature

factor of no more than 70 A* with unit occupancy. Water molecules were harmonically restrained during simulated annealing cycles. The free R factor consistently decreased throughout the refinement. Two loop regions in each molecule (25-27,70-73,225-226, and 270-272) are less well defined, the B values for main chain atoms refining to significantly higher values than the average (30 A2). The program PROCHECK (Laskowski et al., 1993) was used to assess the quality of the final structure. Figures 2A and 28 were prepared using the program RIBBONS (Carson, 1991). Figures 2C, 3, and 4A were generated using 0. FRODO (Jones, 1965) was used to generate Figure 5.

Sequence Comparfeons PH domains for which the structures have been determined were aligned on the basis of the positions of their conserved secondary structural elements. Selected other PH domain sequences were then aligned against a profile generated from this sequence alignment, using the GCG package (version 7, Genetics Computer Group, University of Wisconsin) PH domain sequences and the residues to which they correspond in their respective proteins are as follows: human dynamin, 513-629 (van der Bliek et al., 1993); mouse brain spectrin, 2199-2304 (Ma et al., 1992); human pleckstrin, 7-103 (N) and 247349 (C) (Tyers et al., 1966). human P-adrenergic receptor kinase-2 (PARK-P), 560654 (Parruti et al., 1993); human rasGAP, 300-402 (Traheyet al., 1966); rat PLC-51,23-141 (Suhet al.,lQ66); rat PLC-61, 23-132 (Suh et al., 1966); human SOS. 446-546 (Chardin et al., 1993); human vav 356-457 (Katzav et al., 1969); mouse Grb7 231-343 (Margolis et al., 1992) and human Btk, 6-136, (Vetrie et al., 1993).

Acknowledgments Correspondence should be addressed to P. B. S. We would like to thank Greg Van Duyne, Jim Geiger, and Youngchang Kim for their generous advice and invaluable assistance in solving the crystal structure, as well as other members of the Sigler lab for valuable advice and discussion. We are also grateful to Zimei Bu and Donald Engelman for SAXS analysis of DynPH; Kiki Nelson for oligonucleotide synthesis; Hiroshi Nagasaki for his efforts in searching for PH domain ligands; and Roger Brent and colleagues for providing the “interaction trap” yeast two-hybrid system. Amino acid analysis was performed in the W. M. Keck Foundation Biotechnology Research Lab at Yale University. We thank David Cowburn and Benjamin Margolis, as well as members of their respective laboratories, for many valuable discussions. This work was supported by grants from the National Institutes of Health to P. B. S. (GM22324 and GM15225) and from Sugen to J. S.; M. A. L. is the Marion Abbe Fellow of the Damon Runyon-Walter Winchell Cancer Research Fund (DRG-1243). The coordinates of DynPH will be submitted to the Brookhaven Protein Data Bank. Received August 31, 1994; revised September

15, 1994.

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