Solution structure and ligand–binding site of the carboxy–terminal SH3 domain of GRB2

Solution structure and ligand-binding site of the carboxy-terminal SH3 domain of GRB2 Daisuke Kohda', Hiroaki Terasawa 1' 2, Saori Ichikawa1 , Kenji Ogura l , Hideki Hatanaka 1, Valsan Mandiyan 3, Axel Ullrich4, Joseph Schlessinger 3 and Fuyuhiko Inagaki 1 * Department of Molecular Physiology, Tokyo Metropolitan Institute of Medical Science, 18-22, Honkomagome 3-chome, Bunkyo-ku, Tokyo 113, Japan, 2 Molecular Biology Research Laboratory, Daiichi Pharmaceutical Co., Ltd., 16-13, Kitakasai 1-chome, Edogawa-ku, Tokyo 134, Japan, 3Department of Pharmacology, New York University Medical Center, New York, NY 10016, USA and 4Max-Planck Institut fur Biochemie, 8033 Am Klopfrspitz, 18A Martinsried, Germany Background: Growth factor receptor-bound protein 2 (GRB2) is an adaptor protein with three Src homology (SH) domains in the order SH3-SH2-SH3. Both SH3 domains of GRB2 are necessary for interaction with the protein Son of sevenless (Sos), which acts as a Ras activator. Thus, GRB2 mediates signal transduction from growth factor receptors to Ras and is thought to be a key molecule in signal transduction. Results: The three-dimensional structure of the carboxy-terminal SH3 domain of GRB2 (GRB2 C-SH3) was determined by NMR spectroscopy. The SH3 structure consists of six [-strands arranged in two P-sheets that are packed together perpendicularly with two additional -strands forming the third -sheet.

GRB2 C-SH3 is very similar to SH3 domains from other proteins. The binding site of the ligand peptide (VPPPVPPRRR) derived from the Sos protein was mapped on the GRB2 C-SH3 domain indirectly using H and 5 1 N chemical shift changes, and directly using several intermolecular nuclear Overhauser effects. Conclusions: Despite the structural similarity among the known SH3 domains, the sequence alignment and the secondary structure assignments differ. We therefore propose a standard description of the SH3 structures to facilitate comparison of individual SH3 domains, based on their three-dimensional structures. The binding site of the ligand peptide on GRB2 C-SH3 is in good agreement with those found in other SH3 domains.

Structure 15 November 1994, 2:1029-1040 Key words: GRB2, ligand-binding site, NMR, SH3, solution structure

Introduction Growth factor receptor-bound protein 2 (GRB2) is a 25 kDa 'adaptor' protein that links receptor tyrosine kinases to Ras signaling [1,2]. GRB2 is also known as ASH (abundant Src homology) [3]. It is the human homologue of the Sem-5 protein of Caenorhabditis elegans [4] and the drk protein of Drosophila melanogaster [5,6]. In the cytoplasm of unstimulated cells, GRB2 exists as a complex with Son of sevenless (Sos) protein, binding by use of its two Src homology 3 (SH3) domains. In the presence of growth factors, growth factor receptors are autophosphorlylated by a dimerization mechanism [7]. GRB2 associates with the phosphotyrosine sites of the activated receptors via its single Src homology 2 (SH2) domain, so that the GRB2-Sos complex is recruited to the plasma membrane. Sos, a guanine-nucleotidereleasing factor, converts membrane-bound Ras from the inactive GDP-bound form to the active GTP-bound form [8,9]. Knowledge of the three-dimensional structure of the GRB2 protein is a prerequisite to understanding the function of GRB2 on a molecular basis. We report here the solution structure of the carboxy-terminal SH3

domain of GRB2, determined by NMR. SH3 domains are protein motifs of about 60 amino acid residues found in various proteins involved in signal transduction [10,11]. SH3 domains are also found in cytoskeletal proteins such as spectrin and myosin 1. We reported previously the NMR structure of the SH3 domain of human phospholipase C-,y [12]. Other groups have reported the crystal structures of the SH3 domains of chicken ot-spectrin [13], human Fyn [14], human Csk [15] and human Lck [16], and the NMR structures of the SH3 domains from chicken c-Src [17], p8 5 subunit of human and bovine phosphatidylinositol 3-kinase (PI3K) [18,19], and human GTPase activating protein (GAP) [20]. SH3 domains are known to participate in protein-protein interactions. It has been found that the conserved residues form a hydrophobic patch on one side of the molecular surface [13]. This patch overlaps with residues which produce NMR signals sensitive to the addition of their target peptide [17,19]. The first identified target peptide sequence was a 10-amino acid proline-rich sequence of the 3BP-1 protein [21,22]. Both the amino-terminal SH3 domain of GRB2

*Corresponding author. © Current Biology Ltd ISSN 0969-2126

1029

1030

Structure 1994, Vol 2 No 11 (N-SH3) and the carboxy-terminal SH3 domain (C-SH3) of GRB2 were found to bind to proline-rich sequences in the carboxy-terminal portions of Sos and dynamin, and very weakly to the proline-rich sequence in 3BP-1 [23]. We investigated the interaction of GRB2 C-SH3 with a Sos-derived peptide, VPPPVPPRRR 0 (Kd= .1 mM). The low affinity of the peptide, however, made it difficult to determine the structure of the SH3-peptide complex directly using the nuclear Overhauser effect (NOE), although we found several intermolecular NOEs. Thus, the binding site for the substrate peptide was identified indirectly by mapping the amino acid residues with 1H and 15N resonances that were perturbed upon the addition of the peptide. Recently, Yu et al. have determined the structure of the PI3K SH3 domain in a complex with a high affinity peptide (Kd <10 pM) [24]. In the present paper, we describe the structural details of the peptide-binding surface of the GRB2 C-SH3 domain and compare it with that of the PI3K SH3 domain.

Results and discussion NMR measurement conditions The carboxy-terminal SH3 domain of GRB2 that we analyzed by NMR has 59 residues consisting of residues 159-215 of human GRB2 and two additional residues (Gly-Ser) at the amino terminus. 1'H NMR and 15 N HSQC spectra of GRB2 C-SH3 were recorded at pH 7.5 (direct pH meter reading) and 37 0C in the presence of 10% dimethyl sulfoxide (DMSO) and 1 mM dithiothreitol (DTT). The protein is stable for several months when stored at pH 8.0 at 4C and in the presence of a protease inhibitor, but when the pH is decreased below 7.5 in order to observe exchangeable amide proton resonances during NMR measurements [25], the protein precipitates. We found that high temperature (>300 C) and the addition of DMSO were critical to avoid protein precipitation at lower pH. The presence of DMSO did not alter the structure of GRB2 C-SH3 because the one-dimensional spectra were almost identical, but it did induce gradual unfolding of the protein with a half-life of about a week. Thus, a set of NMR spectra were collected within 3-4 days after NMR sample preparation. The slightly alkaline pH (and probably the presence of DMSO) made it very difficult to record NMR spectra in 1H20 with usual water presaturation. Thus, water suppression using a combination of a shaped pulse with a pulsed field gradient as a read pulse was essential to obtain good quality spectra in 1H 20 [26]. Fig. 1 shows an example of a nuclear Overhauser effect spectroscopy (NOESY) experiment using this read pulse. Note that the spectrum is asymmetric, since the resonances around the water signal are not excited by the read pulse. Structure of the carboxy-terminal SH3 domain of GRB2 The solution structure of the GRB2 C-SH3 has been calculated using the program X-PLOR with 828 upperlimit distance constraints and 46 torsion angle constraints. The final 20 structures of the C-SH3 are

L

I

. . i::.....I

I, M.

'0 0

3

?-;;

O

.··

-

I I

8

4 F1 (ppm)

0

Fig. 1. NOESY (150 ms mixing time) spectrum in 90% 'H 20/10% DMSO-d 6 with water suppression using a combination of shaped pulses (SS and S) and pulsed field gradient. Projections along F1 and F2 axes are shown. shown superimposed on the mean structure for the backbone atoms of residues 4-56 (Fig. 2). The atomic root mean square difference (rmsd) of these 20 individual structures around the mean structure is 0.38+0.08 A for the backbone atoms (N, Coa, C') of residues 4-56 and 0.83±0.09 A for the non-hydrogen atoms of the same residue range. Fig. 3 shows the number of NOE distance constraints and the averaged rmsd values around the mean structure plotted as functions of the residue number. The terminal residues, 1-3 and 57-59, were considered to be disordered and excluded from the rmsd calculation. The structure is less defined at segment 14-18 (Fig. 3b). If these residues are excluded, the rmsd decreases to 0.28±0.05 A for the backbone and 0.72+0.07 A for all heavy atoms. Structural statistics for the 20 simulated annealing structures and their mean structure are summarized in Table 1. Four or five distance constraint violations greater than 0.5 A (the largest is 0.68 A) remain in the final structures. These violations are permissible because we used a set of distance constraints tighter than that produced by the usual three-level classification (e.g. strong < 2.8 A, medium < 3.5 A, weak < 5.0 A). In fact, we found that the value of the NOE potential, FNOE' was reduced to about 30 kcal mol-1 with no residual violations greater than 0.3 A when such a three-level classification of NOEs was used. Two or three torsion angle constraints greater than 50 also remain. Those greater than 100 are found exclusively around 4)of Tyr4 and X1 of Phe9 and Glu18. Note that Tyr4 is located near the amino terminus, and these three residues are all solventexposed. As we did not find any reasons to omit these torsion angle constraints, these three constraints were retained. The final structures are well-defined, enabling

Structure of the carboxy-terminal SH3 domain of GRB2 Kohda et al.

Fig. 2. Superposition of final structures of the carboxy-terminal SH3 domain of GRB2. (a) Stereoview of a superposition of the backbone (N, Ca, C') atoms of the 20 final structures of the GRB2 C-SH3 domain best-fitted to their mean structure for the backbone atoms of residues 4-56. The 31-sheet is drawn in red, the 311-sheet is in blue, the 111lsheet is in green, the 310 helix is in purple, and the rest is in white. The amino and carboxyl termini are labelled with N and C. (b) Certain well-defined side chains are shown on the superposition of the backbone atoms of the 20 final structures. A slightly different view direction was used. The side chains of residues Val5, Ala7, Leu8, Phe9, Phel 1, Pro13, Leu19, Phe21, lle27, Val29, Pro35, Trp37, Trp38, Met48, Phe49, Pro50, Tyr53, Val54 and Pro56 are shown in red and the backbones, in green. The structures were displayed on an Iris workstation with the program QUANTA (Molecular Simulations). (c) Same view as (b), showing residue numbers.

us to discuss the backbone and side-chain conformations (Fig. 2b). Deviations from idealized geometry are small, and a negative Lennard-Jones energy term shows good non-bonded contacts. A Ramachandran plot also shows the good quality of the final 20 structures (Fig. 4). The secondary structure of the C-SH3 domain was identified using the inter-strand NOEs between backbone protons (Fig. 5). Alternative NMR information for assigning secondary structure is derived from slowly exchanging amide protons. However, we could

not use this information due to the absence of slowly exchanging amide protons in GRB2 C-SH3. The six 13-strands are named 13a, P3bl, 3b2, 3c, 13d and 3e according to the first description of the SH3 structure [13]. These strands form two triple-stranded antiparallel 13-sheets, 3PIand II. 13I is composed of ,a, 3bl and 13e, and II is composed of Pb2, 3c and 3d. An additional two 3-strands named 13f and P3g form a double stranded antiparallel 3-sheet, which is absent from the original description [13]. The inset in Fig. 5 shows a schematic structure of the GRB2 C-SH3 domain, which delineates

1031

1032

Structure 1994, Vol 2 No 11

(

-o

0 ,3

a -180

Fig. 3. (a) The number of the inter-residue distance constraints (closed bars) and intra-residue distance constraints (open bars), (b) the averaged values of the backbone rmsds around the mean structure, and (c) the rms changes of chemical shift of proton resonances per residue (rms 1H change=(Y(A8) 2 /N)1/2; closed bars) and absolute value of changes of chemical shift of amide nitrogen resonances per residue (ABS 5 N change; open bars) were plotted as a function of residue number. The rmsds per residue were computed after the individual structures were fitted to the mean structure for the backbone atoms of residues 4-56.

Table 1. Structural statistics.a

Rms deviations from experimental distance constraints (A) (828) No. of distance constraint violations >0.5 A Rms deviations from experimental dihedrel angle constraints () (46) No. of torsion angle constraint violations > 5' FNOE(kcalmol -1)b Ftor(kcalmol - 1)b Frepel(kcalmol-)b EL(kcal-mol- )c Rms deviations from idealized geometry Bonds (A) (925) Angles (°) (1643) Impropers (°) (478)

< SA>

(SA),r

0.079 ± 0.001

0.078

4.55

5

3.0

0.8

2.8 259.1 6.7 109.2 -277.7

I 6.3 + 3.7

i 5.1 i 11.0

0.008 0.0004 2.45 ± 0.01 1.07 0.04

2.0 3 254.8 2.7 105.1 -265.9 0.008 2.43 0.98

aThe refers to the final set of the simulated annealing structures and (SA)r is the mean structure. The number of terms is given in parentheses. bThe values of the square-well NOE potential, FNOE, and torsional angle potential, Fto,, are calculated with a force constant of 50kcal-mo-l 1A- 2 and 50 kcalmol - 1rad- 2, respectively. The value of the repel term, Frepel, is calculated with a force constant of 4kcal mol- 1 A- 4 with the van der Waals radii scaled by a factor of 0.8 of the standard values used in the CHARMm empirical function. CELJ is the Lennard-Jones van der Waals energy calculated with the CHARMm empirical energy function, which was not included in the simulated annealing calculations.

-120

-60

0

60

120

180

Fig. 4. Ramachandran plot of the backbone (, xp)angles of residues 4-56 of the 20 final structures of GRB2 C-SH3. ('+' and '.' represent glycine and non-glycine residues, respectively). the architecture of the secondary structure. PI1 and P3II associate with each other almost perpendicularly to form a B-sandwich. 3III bends over the P3-sandwich forming an eight-stranded antiparallel 13-barrel structure. Inspection of our structure indicates that the interior of the molecule is packed with hydrophobic side chains of the residues Val5, Ala7*, Leul9*, Phe21, Ile27, Val29, Trp38*, Phe49, Arg51 and Val54* ('*' indicates conserved residues). The side chains of the conserved amino acids Leu8*, Phe9*, Phell*, Trp37*, Pro50* and Tyr53*, and that of the non-conserved amino acid Met48 make a hydrophobic patch on the surface of the SH3 domain (Fig. 2). We classified loop/turn residues based on sequential NOE patterns, 3JHN, values, hydrogen bonds (found by modelling), and the q4, values of residues. Richardson has suggested that tight turns fall into one of four major classes [27]: type I, type I', type II and type II', while other minor classes contain type VIa, VIb and IV (miscellaneous). One possible addition is type Ib: this is not a true 'tight turn', but a 'half turn' described by Wagner et al. [28]. At a half turn, a polypeptide chain changes its direction at approximately right angles. The experimental data indicate that the types of turns at residues Ala7-Leu8, Arg23-Gly24 and Val29-Met30 are Ib, II and Ib, respectively. The 3-hairpin belongs to a subset of tight turns: it connects two adjacent strands of an antiparallel 3-sheet. 13-Hairpins can be described by the loop length and are grouped into classes 1-4 (for a precise definition, see [29,30]). In the GRB2 C-SH3 domain there is a nine-residue 3-hairpin of class 3 at Asp12-Gly20, a five-residue, class 3 13-hairpin at residues Ser33-Trp37, and a two-residue, class 2 (type I') 3-hairpin at residues His43-Gly44. Identification of successive dN(i,i+2) NOE connectivities indicates the

Structure of the carboxy-terminal SH3 domain of GRB2 Kohda et al.

Fig. 5. Secondary structure of GRB2 C-SH3. Inter-strand NOEs observed in 1 H O NOESY and 2 H20 NOESY are indicated by solid arrows. The thickness of the arrows indicates the approximate intensity of the NOESY crosspeaks. A broken arrow indicates an expected but ambiguous NOE due to accidental overlap with another intra-residue NOE. The residues that project their side chains above the sheet are shown in shadowed font, and the residues that project their side chains below the sheet are in normal font. The inset shows a schematic structure of GRB2 C-SH3. existence of one turn (Arg51-Asn52-Tyr53) of a 310 helix that connects the last two P-strands, Ed and 3e. Comparison with other SH3 domains and structure-based sequence alignment Ten structures of the SH3 domains from nine different proteins have been published so far. The coordinates of spectrin with the entry name 1SHG, Fyn (1SHF, molecule A) and bovine PI3K (1PNJ, 2PNI) are now available from the Protein Data Bank (PDB). The coordinates of the PLC-y SH3 domain determined by us are also available from the PDB, but we used a revised set of coordinates, which has been deposited at the PDB (1HSQ, 2HSP). The coordinates of the SH3 domains from Src and human PI3K were obtained from S Koyama, H Yu and SL Schreiber, Harvard University, USA. Here we compare the five SH3 structures mentioned above and the carboxy-terminal SH3 domain of GRB2 (Fig. 6). The topographical similarities make it possible to construct a better sequence alignment of the

SH3 domains than that based solely on the primary structures. Such structure-based sequence alignments have been reported previously [14,15,18], but our alignment includes more SH3 domains. We collected information on the secondary structures of the SH3 domains from the literature, and found that they were not identical. This is not surprising, since some irregularity of secondary and tertiary structures may lead to different descriptions of the SH3 structures. Of course, the possibility that there are true structural differences due to sequence variations involving insertion and deletion must also be considered. Thus, we think it useful to propose a general description of the SH3 structures, based on the structural-based sequence alignment. The nomenclature of the P-strands is based on the first SH3 structure by Musacchio et al [13] and is consistent with other papers, with slight modifications. Fig. 7 shows structure-based alignment of the amino acid sequences. The standard SH3 structure consists of eight

1033

1034

Structure 1994, Vol 2 No 11

Fig. 6. Schematic representation of GRB2 C-SH3 and other SH3 domains.

The 31-sheet is drawn in red, the 311sheet is in blue, the III1-sheet is in green, the 310-helix is in magenta, and the rest is in white. P13K SH3 was

drawn using the coordinates of bovine P13K. The ribbon diagrams in Figs 6 and 8 were generated using the program MOLSCRIPT [521.

3-strands, 3a, 3f, [3g, 13bl, [b2, [3c, [3d and 3e from the amino terminus to the carboxyl terminus. Strand 3a is four or five residue long, strand 3e is three or four residues long. The lengths of both strands vary because they are flanked by the amino-terminal or carboxyterminal non-SH3 segment. In particular, the length of the strand 3e seems to be determined by a proline residue at the third position, where strand 3e is three residues long. Strand 3e of PI3K SH3 contains a classical 3-bulge [19], thus it actually consists of five residues. The assignment of strand 13c and 13d, each consisting of six residues, and one turn of 310 helix between [3d and 3e is very straightforward since all the SH3 structures contain these secondary structure elements. Strand 13d of PLCCy SH3 has one additional residue that makes a classical 3-bulge [12]. A 3-hairpin between [3c and 3d (distal loop) is a two-residue loop of class 2 with the type I' or II' conformation in the GRB2 C-SH3, spectrin and PLCy SH3 domains, or a four-residue loop of class 4 with type I conformation in Src, Fyn and PI3K SH3s [30]. The tip of the four-residue loop of type I hairpin is bent over like the raised head of a cobra (Fig. 6). The existence of strands 13f and 13g may be controversial. In the previous description, these strands are involved in a RT-Src loop [14]. They were found in Src, human PI3K, PLC-y and GRB2C-SH3. As for Src and human PI3K, we consider that the strand S1 in the literature corresponds to 13a and [3f, and that the 3-bulge on strand S1 should be reinterpreted as a type lb turn [31,32]. Strand S2 corresponds to [3g, but S2 should be shortened by two residues at the amino-terminal side and one residue at the carboxy-terminal side. By contrast, 3f and 13g were not described in spectrin, Fyn and bovine PI3K. However, the authors stated the presence of "an irregular antiparallel 3-sheet" [19] and "a hairpin-like structure"

[13]. As for the GRB2 C-SH3 and PLCy SH3 domains, we have no reason to state that 3III is more irregular than PI and 13II on the basis of the inter-strand NOE patterns. Thus, it is reasonable to include 3f and 13g into our standard description of the SH3 structure if we admit that the 3-sheet formed by 3f and 3g varies in regularity. Such irregularity found in 3III suggests flexibility of this 13-sheet, which may be important for the binding of ligand peptides. A loop between 3f and 13g is a seven-residue loop of class 1. This loop has a high content of acidic residues. The region around 3bl and b2 shows the largest variation in the SH3 structures. Originally, two 3-strands, 3bl and [b2, were described as one 13-strand, (13b) with a kink, but we prefer to divide the 13b into two strands because the kink forms a type lb turn and changes the direction of the polypeptide chain sharply. It is to be noted that a splicing site exists at the last residue of strand 3bl in the two SH3 domains of the Sem-5 protein [4]. In PLCy SH3, the position of the type lb turn shifts by one residue, which was clearly shown by a strong dNN NOE connectivity [12]. This results in a one-residue shortening of 3bl and a one-residue extension of [b2 in PLC-y SH3. Finally, a 15-residue insertion in PI3K SH3s extends 3bl to six residues, and deletes strand b2. The long insertion sequence, called the n-Src loop in the previous description [14], forms several helical segments [18,19]. The loop connecting [b2 and [3c is a five-residue or seven-residue 3-hairpin of class 3. In addition to the three-dimensional structures of the SH3 domains drawn as a ribbon diagram whose secondary structure is defined according to the standard SH3 structure (Fig. 6), we also show the overlays of the

Structure of the carboxy-terminal SH3 domain of GRB2 Kohda et a.

N-ternnm

a

Of

g

bl

b2

** *****

H GRB2C

H PLIg

*

** *

gsPTFK CAVKA L FDYK AREDEL TFI KS AIIQ

H PI3K

R-sheet turn

**

I

PIII lb

EL DI

II

**

QEMG WhDY G G KLWFP SNY VEEM VNPegihrd 0 KGSLVAUGFSOXAP EEIG WLNGYN ETTG ERGDFP GTY VEYG RKKISPP I

KGSLVALGFWSXEAKP EEIG WLGYN ETI

E D

I

*

N VEK

GD E PIII

***

TEGD WWLAHS LTIG QCIYIP SNY VAP S SEGD WEARS LTIG ETGYIP SNY VAP VD

B PI3a gsfiAEGS YQYRA L YDYK KEREEDI DLH LG DILTVN

A L YDY I FNF

C-tern

L NS

MSAEG YQYRA L YDYK KEREEDI DLH LG DILTVN

consensus

pfe

** * *

TNKD WVEV N D RGFVP AAY VKKL D

*

TFVA L YDYE SRTETDL SFK KG ERLII V NN

mVr LFVA L YDYE ARTEDDL SFH KG EKFQI

H fyn

pd

SDPN WWXGAC H G QlCMEP RNY VTP VNR

C speca mDGKE LVIA L YDYQ EKSPREV TMK KG DILTL L NS C csrc gsHMGVr

PC ** * **

gsT YVOX L FDFD PQfGEL GFR RG DFIHV M DN

*

insert

PII

i}D

WW G YA

pII

ERDFP GTY VEYG RKKISP I

G D

G

YV FI

P

1'

II I'/II'(2) I(4)

3

10

Fig. 7. Alignment of SH3 sequences and definition of the secondary structure. Abbreviations: H GRB2C, human GRB2 carboxyterminal SH3; C speca, chicken a-spectrin; C csrc, chicken c-src; H fyn, human Fyn; H PLCg, human phospholipase Cy; H P13K, p85 subunit of human phosphatidylinositol-3'-kinase; B P3Ka, P85d subunit of bovine phosphatidylinositol-3'-kinase. The sequences are aligned on the basis of the structural data, in addition to the conserved residues noted previously 113]. The notation of the S-strands is shown at the top. The assignment of the 13-strand to the three p-sheets, and the types of the loop/turns are shown at the bottom.. Residues of GRB2C, Src and P13K SH3 domains with 1H and/or 15N resonances affected by complex formation with substrate peptides (in the case of GRB2C, rms 1H change >0.04 ppm or 5NH change >0.2 ppm) are marked with an asterisk [17,191.

SH3 structures superimposed for the backbone atoms of the 38 residues found in regions of conserved secondary structures (Fig. 8). The rmsd values range between 0.83 A and 1.64 A (Table 2). It is interesting to compare the human and bovine structures of the PI3K SH3 domains, since they are identical except for one residue

Fig. 8. The overlay of the backbone atoms of the 38 residues found in regions of the conserved secondary structures, The SH3 structures superimposed are GRB2 C-SH3 (white), PLCy (yellow), spectrin (red), Fyn (purple), Src (green) and bovine P13K (blue). The structures are in the same orientation as in Fig. 2a.

in the insert region. The comparison of the Src and Fyn SH3s is also of interest because of their high sequence identity. These rmsds (0.83 A and 1.19A) are regarded as an estimate of the error level in structure determination, and are comparable to the rmsd values of 0.5-2 A between four independently determined interleukin-4

1035

1036

Structure 1994, Vol 2 No 11

Table 2. Sequence identities and rms deviations between various SH3 domains.a GRB2C spectrin GRB2C spectrin Src Fyn PLCy hPI3K bPI3K

26 34 32 37 26 26

0.86 34 34 27 28 28

Src

Fyn

PLCy

hPI3K

bPI3K

1.57 1.19 78 37 26 26

1.17 0.94 1.19 40 35 35

1.29 b

1.18 0.97 1.28 0.88 1 .5 3b 99

1.24 1.04 1.33 0.83 1. 46b 0.83 -

1 .1 8b 1.64 b

1.61 b 35 35

aSequence identities (%) are shown in the lower triangle. Rmsds (A) in the upper triangle were computed for the backbone atoms (N, Ca, C') of 38 residues of a (last 4 residues), af (1 res), 3f(4 res), Og (3 res), gbl (2 res), pb1 (first 4 res), c (6 res), cd (peripheral 2 res), d (6 res), de (3 res) and e (first 3 res), where 'af' means the loop connecting pa and f, etc. bThe values reduce by 0.13-0.31 A if three residues around the -bulge structure in strand [3d of PLCy SH 3 are omitted during comparison.

structures [33]. The other rmsd values between SH3 structures with lower sequence identities in Table 2 do not exceed the error level, indicating highly conservative three-dimensional structure of the SH3 domains. Ligand binding Each of the two SH3 domains of the GRB2 protein is known to bind to the carboxy-terminal portion of the Sos protein [9,22]. Point mutations of GRB2 suggests that both SH3 domains are necessary for full binding to Sos although the carboxy-terminal SH3 domain seems to be less important than the amino-terminal SH3 ([1,8] and references therein). The target sequence of the amino-terminal SH3 domain was suggested as the sequence containing VPPPVPPRRR (residues 1152-1161 of mSosl). The dissociation constant of the complex of the amino-terminal SH3 and the decapeptide, VPPPVPPRRR, was 5.6 ,uM [34]. The carboxy-terminal SH3 domain also binds to this peptide, but with lower affinity (Kd= 3 9 RIM). In order to find a peptide sequence with higher affinity, we synthesized two other candidate peptides deduced from the Sos sequence, and determined the dissociation constants of

the complexes using fluorescence spectroscopy. A dodecapeptide, HSIAGPPVPPRQ (residues 1288-1299), bound to the carboxy-terminal SH3 with a Kd of 0.7 mM, while another dodecamer, PPESPPLLPPRE (residues 1210-1221) showed no binding (data not shown). The strongest interaction we found was the binding of the decamer, VPPPVPPRRR, with a Kd of 0.1 M, which is comparable to the reported value of 39 mM. The consensus of the binding peptide is PPVPPR. The NMR experiments were carried out on a 1:1 complex of the GRB2 C-SH3 domain (unlabelled or '5 N-labelled) and VPPPVPPRRR (VPP peptide). The 1 H and 15N chemical shifts of the SH3 domain in the complex were determined using the sequential assignment method. The 1 H chemical shifts of the bound peptide were also obtained. One complete set of the proton resonances for the peptide was observed, but we do not exclude the existence of minor conformations. The ligand-binding site was deduced from the identification of amino acid residues whose chemical shifts were perturbed by the addition of the VPP peptide. We found changes in the chemical shifts of 36 proton resonances (>0.05 ppm) of 18 residues, and 9 amide nitrogen resonances (>0.2 ppm). The chemical shift perturbations are summarized for each residue (Figs 3c and 7), and the residues affected by the complex formation are mapped on the backbone structure of the SH3 domain (Fig. 9). These residues form a hydrophobic patch on the molecular surface, with anionic charged residues at the periphery. Note that this patch contains conserved residues, Phe9, Phell, Trp37, Pro50 and Tyr53. Resonances in residues at similar positions were found to be shifted upon addition of the peptides for Src and bovine PI3K SH3 domains (Fig. 7) [17,19]. We searched for NOEs between the SH3 domain and the VPP peptide in the two-dimensional 1H NOESY spectra, and found some intermolecular NOEs. The number was, however, unexpectedly small, either due to overlap of the proton resonances or due to the weak binding of the VPP peptide to the carboxy-terminal

Fig. 9. The proposed binding surface of the VPP peptide. The residues with peptide-sensitive 1H (rms shift per reside >0.04 ppm) and 15NH (>0.2 ppm) chemical shifts are shown superimposed on the backbone structure of the GRB2 C-SH3 domain. Phe9 is also shown because it is conserved.

Structure of the carboxy-terminal SH3 domain of GRB2 Kohda et al. SH3 domain. All the unambiguous and strong NOEs were observed between the methyl protons of Val5' of the peptide and aromatic resonances of Phel 1 and Tyr53 of the SH3 domain, demonstrating that a hydrophobic pocket formed by these conserved aromatic residues recognized the side chain of Val5'. Though less unambiguous, weak NOEs between Val5' and Pro50, and between Val5' and Arg51 were also detected. Note that these residues of C-SH3 were also found to contain 1H or 15 N resonances (or both) sensitive to the VPP peptide binding (Fig. 7). The total number of the intermolecular NOEs was eight. We then collected 57 intra-peptide NOEs consisting of 39 intra-residue, 18 sequential and no longer range NOEs. Strong daN(i,i+l) and das(i,i+l) NOE connectivities were seen over the Vall'-Pro7' sequence of the VPP peptide, suggesting that the peptide adopted an extended conformation and its peptide bonds were trans in the bound state. Recently, the structure of the complex of the human PI3K SH3 domain and its high affinity peptide, RKLPPRPSK-methyl ester (RLP1 peptide), has been reported by Yu et al. [24]. The key to these authors' success was to find peptides with high affinity (Kd <10 IM). The high affinity resulted in observing four out of five expected amide protons of RLP1; in contrast, we observed only one (Val5') out of four expected amide protons of the VPP peptide. Yu et al. used 34 intermolecular distance constraints between the PI3K SH3 and the RLP1 peptide. Their calculated structures revealed that the RLP1 peptide took the left-handed type II polyproline helix and bound to the cleft formed between II and 3III. Considering the PI3K complex structure, we can deduce two major sites of contact with the VPP peptide on the GRB2 C-SH3 domain. The first site is a pocket formed by the side chains of Phe9 and Tyr53, and the second site is formed by those of Phell, Trp37, Pro50O and Tyr53. The intermolecular NOEs we observed clearly show that ValS5' of the ligand peptide binds to the second pocket on the C-SH3 domain. However, our data are insufficient to constrain the peptide on the SH3 domain. If we assume that the VPP peptide takes the same orientation as in PI3K, then the first pocket on GRB2 C-SH3 is occupied by Pro7' and Arg8'. Yu et al. also pointed out that another class of ligand having no RXL motif (for example, the VPP peptide) may bind in the opposite orientation to RLP1 [24]. This is plausible because an electrostatic interaction between basic residues of the VPP peptide and acidic residues on the loop between f and g may be formed when the VPP peptide binds in the opposite orientation. In this case, the first pocket is occupied by Pro2' and Pro3'. A model building study of the complex of the carboxy-terminal domain of Sem-5 and a polyproline peptide also concluded that the orientation differed from the case of the PI3K-RLP1 [35]. At the present stage, we can not conclude which orientation is correct. In parallel to the C-SH3 study, we have determined the complex structure of the GRB2 N-SH3 domain with the VPP peptide. As the VPP peptide binds strongly to

N-SH3, we collected a sufficient number of intermolecular NOEs to obtain a high-resolution structure of the complex (H Terasawa et al., unpublished data). The VPP peptide binds to GRB2 N-SH3 in opposite direction to the orientation in PI3K-RLP1. Slowly exchanging amide protons in SH3 domains Slowly exchanging amide protons are defined by their observation in an NMR spectrum several hours after changing the solvent from H 2 0 to 2 H 20. Slowly exchanging amide protons are involved in hydrogen bonds or buried in the interior of proteins. Considering the high content of the p-structure in the SH3 domains, it is expected that the SH3 domains would contain many slowly exchanging amide protons. The human and bovine PI3K SH3 domains have 38 and 23 slowly exchanging amide protons, respectively [18,19,32]. In contrast, the Src SH3 contains only three slowly exchanging amide protons and PLCy SH3 contains only one [12,17,31]. No slowly exchanging amide protons were detected in GRB2 C-SH3. These findings suggest that SH3 domains possess structural flexibility that could accelerate amide proton exchange rates. The long insertion sequence might restrain the flexibility, reducing the exchange rates of the amide protons in the PI3K SH3s. A more detailed study is necessary to understand the dynamical aspects of the SH3 domains. In conclusion, we have determined the solution structure of the carboxy-terminal domain of the human GRB2 protein using NMR, and then mapped the ligandbinding site on the SH3 domain by using chemical shift perturbation and intermolecular NOEs to study the formation of a complex with a Sos-derived peptide, VPPPVPPRRR. These results are the basis for understanding the functions of the GRB2 protein, a key molecule in signal transduction.

Biological implications Growth factor receptor bound protein 2 (GRB2) plays an important role in the transduction of signals from receptor tyrosine kinases. GRB2 has two Src homology (SH) type 3 domains, which are connected by a SH2 domain. The SH3 domains interact with proline-rich regions of the son of sevenless (Sos) protein. In response to EGF-receptor activation and autophosphorylation, the GRB2-Sos complex binds to a phosphotyrosine residue at the carboxy-terminal tail of the EGF receptor through the SH2 domain of GRB2, so that GRB2 functions as an 'adaptor', relocating Sos to the plasma membrane. The interaction between the guanine nucleotide releasing activity of Sos and membrane-bound Ras leads to the formation of GTP-bound Ras, which activates a kinase cascade and other pleiotropic responses essential for cell growth and differentiation. In this sense, GRB2 is a key molecule in the signal transduction process and

1037

1038

Structure 1994, Vol 2 No 11

knowledge of the three-dimensional structure of GRB2 is essential for understanding the detailed mechanism of signal transduction.

vector. Thus the recombinant SH3 domain for NMR analysis contains 59 amino acid residues and two amino-terminal residues derived from the expression vector.

We have determined the three-dimensional structure of the GRB2 carboxy-terminal SH3 domain (GRB2 C-SH3) and investigated the binding mode with a proline-rich peptide from Sos (VPPPVPPRRR). The present structure of C-SH3 is the first step in determining the threedimensional structure of intact GRB2. The structure consists of eight -strands arranged in three -sheets. We have also determined the site of interaction between the proline-rich peptide of Sos and the C-SH3 structure, a hydrophobic patch comprising Phe9, Phell, Trp37, Pro50 and Tyr53 of GRB2. The Sos peptide forms an extended structure, possibly adopting the conformation of a type II polyproline helix [24], where Val5' of the peptide binds to the pocket formed by Phell, Trp37 and Tyr53.

Fluorescence of tryptophan residues of GRB2 C-SH3 was recorded with a Shimadzu RF-5000 fluorescence spectrophotometer. The temperature of the sample solution (2.0 ml, 25 mM potassium phosphate buffer, pH 7.1) containing -1 ,uM C-SH3 was controlled at 37+0.1°C.

One problem encountered when comparing SH3 domains is an apparent discrepancy in the assignment of the secondary structures. We propose here a general description of the sequence alignment and the secondary structure of SH3 domains on the basis of the three-dimensional structures.

Materials and methods Purification of GRB2 C-SH3 domain The carboxy-terminal SH3 domain of GRB2 (residues 159-217) was expressed as a glutathione S-transferase (GST) fusion protein using, pGEX-2T (Pharmacia). Transformed Escherichia coli BL21 cells were grown in LB medium, or M9 minimal medium containing 15 NH 4 C1 and 1 g 1-1 CELTONE-N9 (Martek Corp., USA) and induced at OD600 =1.8 (LB) or 0.5 (M9) with 0.4 mM isopropyl-thio-[D-galactoside. The cells were collected after 3 h induction, suspended in phosphate-buffered saline (PBS) containing 1% Triton X-100, and disrupted by sonication. The GST-SH3 fusion protein was recovered by affinity chromatography using glutathione Sepharose 4B (Pharmacia) and eluted with 15 mM reduced glutathione followed by concentration with an Amicon Centriprep-10 and addition of 5 mM dithiothreitol (DTT). The fusion protein was cleaved using 0.01% w/w trypsin (Sigma) for 20 min at room temperature in 20 mM Tris buffer, pH 7.8 containing 5mM DTT and 2.5 mM CaC12 . The SH3 domain was further purified using a Mono-Q column HR 10/10 (Pharmacia) with a 70-170 mM NaCl gradient in 20mM Tris buffer, pH 7.8, and a Superose 12 column (Pharmacia) that had been equilibrated with 20 mM Tris, pH 8.0 containing mM DTT and 0.15 M NaCl. The fractions containing SH3 were combined and stored at 4°C in the presence of Pefabloc SC (Merck) at the final concentration of 0.2 mM. The yield of purified protein was 15 mg 1-1 LB culture and 4 mg 1-1 M9 culture. Mass spectroscopy showed that the carboxy-terminal two residues are removed by trypsin digestion: the measured mass was 6775, compared with 6778.3 calculated from the GRB2 sequence (residues 159-215) and including the amino-terminal glycine and serine from the

NMR measurements The buffer of the stock solution of C-SH3 was changed to an NMR buffer with a Centricon-3 (Amicon): 2 H2 0/dimethyl sulfoxide-d 6 (DMSO), from CEA Commissariat a L'Energie Automatique, (9:1) or H 2 0/DMSO-d 6 (9:1) in 25 mM potassium phosphate buffer containing mM DL-1,4-dithiothreitol-d, 0 (DTT), from ICON Service Inc. The buffer change must be done at 25-37°C to avoid (reversible) precipitation of the protein. DMSO is necessary to keep the protein soluble at millimolar concentration below pH 7.5, otherwise the protein irreversibly precipitates. The final concentration of the C-SH3 domain was 1.5-2 mM. The buffer used for preparation of the NMR sample was pH 7.1, but an addition of DMSO slightly increased the pH meter reading to 7.5. NMR spectra were recorded on a 500 MHz Varian Unity at 37°C with spectral widths of 6500 Hz (1H) and 1800 Hz ( 5 N) in the TPPI-States method [36]. The deuterium signal of DMSO-d 6 was used as an internal lock signal for 1 H2 O0 samples. For H NMR spectra in 2 H2 0, the residual HDO signal was suppressed by presaturation with a transmitter channel. DQF-COSY [37], TOCSY (75 ms mixing time with the clean MLEV17) [38], PE-COSY [39] and NOESY (75 nms and 150 ms mixing time) [40] were recorded using Varian's standard pulse library. For H NMR spectra in H2 0, the water resonance was suppressed by the combination of the shifted laminar pulse [41] and the pulsed field gradient. Water suppression was achieved by using SS90°-G-S180°-G'-acquisi tion scheme in place of the last 90 ° read pulse for NOESY and TOCSY, where SS is a shifted laminar pulse whose amplitude profile is a complete cosine cycle, while S is a half cycle of cosine [26], G and G' are delays with a pulsed field gradient; SS90°=0.36 ms; S180°=0.6 ms; G=l.lms and G'=1.4 mis with 1.0 ms-pulsed filed gradient at 8 G cm - l. For DQF-COSY, hard 90°-G-S180°-G'-acquisition scheme was used. In TOCSY, a DIPSI-2 spin lock sequence (80 ms) [42] bracketed by 1.0 ms-trim pulses was used followed by a hard 90° flipback pulse and a delay for clean TOCSY before the SS-S read pulse. Another pulse-field gradient (1.0 ms, 8 G cm 1 ) was inserted at the centre of the mixing time of NOESY and the delay after the flip-back pulse of TOCSY. Two-dimensional tSN/iH HSQC [43] and three-dimensional 5 N/1 H TOCSYHSQC [44] were obtained using the sensitivity enhanced scheme [45], with an extra pulse-field gradient to suppress the water signal. Eight transients were averaged for each increment. All FID data were processed with FELIX (Biosym Technologies). Baseline correction and peak picking were performed with our own C programs. The resonances were assigned to individual protons in sequence-specific manner using the conventional sequential assignment method [25]. Interproton distance constraints were derived from NOE cross peak intensities (peak height) in the two-dimensional NOESY spectra recorded with a mixing time of 150 mns. The peak intensities were transformed into distances on the basis of known distances: sequential dN in -strand=2.2 A in H 2 0 NOESY and inter-strand da,~ in antiparallel 3-sheet=2.3 A in

Structure of the carboxy-terminal SH3 domain of GRB2 Kohda et al. 2

H2 0 NOESY [25] and a relation of (NOE intensity) oc -a (distance) . An empirical adjustable parameter 'a' is used for taking into account spin diffusion effects [46]. We used a=5 instead of the theoretical value, a=6, considering the molecular mass of GRB2 C-SH3 and the mixing time used. The upper bound distance constraints were the calculated distance plus 0.2 A. The lower bound constraints were all set to 1.8 A. The distances involving methylene and methyl protons and ring protons of tyrosine and phenylalanine were referred to single () -1/ 6 average distances so that no corrections for centre 1 averaging were made [47]. 3JHNa values were read from H 20 2 3 DQF-COSY and Jab values from H20 PE-COSY. Torsion and X and stereospecific assignments angle constraints for were obtained according to [48]. Several 4 constraints were obtained from the shape analysis of NH-CaoH cross peaks of 1 DQF-COSY in H 20 [49]. Slowly exchanging amide protons 5 were identified in two-dimensional ' N-1H HSQC spectrum 2 1 5 h after changing the solvent from H2 0 to H2 0. Calculations of three-dimensional structures A total of 828 upper bound distance constraints including 56 constraints between multiple protons were derived from 1172 assigned NOE crosspeaks. These restraints include 291 intraresidue, 147 sequential (I i-j =1), 63 short-range (2< l i-j <5) and 327 long-range (i-j 1>5) constraints. An additional 82 intra-residue upper bound distance constraints were obtained, but not incorporated in the constraint set because they were not structurally meaningful. 19 4) and 27 X torsion angle constraints were included. All peptide bonds were trans, including those preceding the proline residues. The three-dimensional structures were calculated with the program X-PLOR (Molecular Simulations) using YASAP [50]. Initially, structures were built using unambiguous distance constraints. Additional constraints were incorporated in successive stages as described previously [51]. A final set of 20 converged structures was selected from 200 calculations on the basis of agreement with the experimental data and van der Waals energy, with the cut383 kcal mo- 1 . A mean off taken at FNOE+Ftor+Frepel< structure was obtained by averaging the coordinates of the structures that are superposed in advance to the best converged structure, and then minimizing under the constraints with reduced van der Waals radii [47]. We previously reported the solution structure of the SH3 domain of phospholipase C-y (PLCy) using 532 upper bound constraints [12]. We have further refined the structure of the PLCy SH3 using 821 upper bound constraints. rmsd of the final 20 structures around the mean structure is 0.54±0.13 A for the backbone atoms of residues 8-62 and 1.17±0.11 A for non-hydrogen atoms of the same residue range. Coordinates have been deposited with the Brookhaven protein data bank (GRB2 CSH3, 1GFC and 1GFD; PLCy SH3, 1HSQ and 2HSP). Acknowledgements: We thank Professor SL Schreiber, Drs H Yu and S Koyama for the coordinates of the Src and PI3-kinase SH3 structures. This work was supported by a grant from the Human Frontier Science Program and a Grant-in-Aid from the Ministry of Education, Science and Culture ofJapan.

References

1. Lowenstein, E.J., et al., & Schlessinger, J. (1992). The SH2 and SH3 domain-containing protein GRB2 links receptor tyrosine kinases to Ras signaling. Cell 70, 431-442. 2. Schlessinger, J. (1993). How receptor tyrosine kinases activate Ras. Trends Biochem. Sci. 18, 273-275.

3. Matsuoka, K., Shibata, M., Yamakawa, A. & Takenawa, T. (1992). Cloning of ASH, a ubiquitous protein composed of one Src homology region (SH) 2 and two SH3 domains, from human and rat cDNA libraries. Proc. Natl. Acad. Sci. USA 89, 9015-9019. 4. Clark, S.G., Stern, M.J. & Horvitz, H.R. (1992). C. elegans cell-signalling gene sem-5 encodes a protein with SH2 and SH3 domains. Nature 356, 340-344. 5. Simon, M.A., Dodson, G.S. & Rubin, G.M. (1993). An SH3-SH2SH3 protein is required for p21 Raslactivation and binds to sevenless and Sos protein in vitro. Cell 73, 169-177. 6. Olivier, J.P., et al., & Pawson, T. (1993). A Drosophila SH2-SH3 adaptor protein implicated in coupling the sevenless tyrosine kinase to an activator of Ras guanine nucleotide exchange, Sos. Cell 73, 179-191. 7. Ullrich, A. & Schlessinger, J. (1990). Signal transduction by receptors with tyrosine kinase activity. Cell 61, 203-212. 8. Downward, J. (1994). The GRB2/Sem-5 adaptor protein. FEBS Lett. 338,113-117. 9. Pawson, T. & Schlessinger, J. (1993). SH2 and SH3 domains. Curr. Biol. 3, 434-442. 10. Koch, C.A., Anderson, D., Moran, M.F., Ellis, C. & Pawson, T. (1991). SH2 and SH3 domains: elements that control interactions of cytoplasmic signaling proteins. Science 252, 668-674. 11. Musacchio, A., Gibson, T., Lehto, V.-P. & Saraste, M. (1992). SH3an abundant protein domain in search of a function. FEBS Lett. 307, 55-61. 12. Kohda, D., et al., & Inagaki, F. (1993). Solution structure of the SH3 domain of phospholipase C-y. Cell 72, 953-960. 13. Musacchio, A., Noble, M., Pauptit, R., Wierenga, R. and Saraste, M. (1992). Crystal structure of a Src-homology 3 (SH3) domain. Nature 359, 851-855. 14. Noble, M.E.M., Musacchio, A., Saraste, M., Courtneidge, S.A. & Wierenga, R.K. (1993). Crystal structure of the SH3 domain in human Fyn; comparison of the three-dimensional structures of SH3 domains in tyrosine kinases and spectrin. EMBOJ. 12, 2617-2624. 15. Borchert, T.V., Mathieu, M., Zeelen, J.P., Courtneidge, S.A. & Wierenga, R.K. (1994). The crystal structure of human CskSH3: structural diversity near the RT-Src and n-Src loop. FEBS Lett. 341, 79-85. 16. Eck, M.J., Atwell, S.K., Shoelson, S.E. & Harrison, S.C. (1994). Structure of the regulatory domains of the Src-family tyrosine kinase Lck. Nature 368, 764-769. 17 Yu, H., Rosen, M.K., Shin, T.B., Seidel-Dugan, C., Brugge, J.S. & Schreiber, S.L. (1992). Solution structure of the SH3 domain of Src and identification of its ligand-binding site. Science 258, 1665-1668. 18. Koyama, S., Yu, H., Dalgarno, D.C., Shin, T.B., Zydowsky, L.D. & Schreiber, S.L. (1993). Structure of the P13K SH3 domain and analysis of the SH3 family. Cell 72, 945-952. 19. Booker, G.W., et al., & Campbell, I.D. (1993). Solution structure and ligand-binding site of the SH3 domain of the p85a subunit of phosphatidylinositol 3-kinase. Cell 73, 813-822. 20. Yang, Y.S., et al., & Roques, B.P. (1994). Solution structure of GAP SH3 domain by H NMR and spatial arrangement of essential Ras signaling-involved sequence. EMBO . 13, 1270-1279. 21. Ren, R., Mayer, B.J., Cicchetti, P. & Baltimore, D. (1993). Identification of a ten-amino acid proline-rich SH3 binding site. Science 259, 1157-1161. 22. Li, N., et al., & Schlessinger, J. (1993). Guanine-nucleotide-releasing factor hSosl binds to GRB2 and links receptor tyrosine kinases to Ras signaling. Nature 363, 85-88. 23. Gout, I., et al., & Waterfield, M.D. (1993). The GTPase dynamin binds to and is activated by a subset of SH3 domains. Cell 75, 25-36. 24. Yu, H., Chen, J.K., Feng, S., Dalgarno, D.C., Brauer, A.W. & Schreiber, S.L. (1994). Structural basis for the binding of proline-rich peptides to SH3 domains. Cell 76, 933-945. 25. Wuthrich, K. (1986). NMR of Proteins and Nucleic Acids. John Wiley and Sons, New York. 26. Smallcombe, S.H. (1993). Solvent suppression with symmetricallyshifted pulses. J.Am. Chem. Soc. 115, 4776-4785. 27. Richardson, J.S. (1981). The anatomy and taxonomy of protein structure. Adv. Protein Chem. 34, 167-337. 28. Wagner, G., Neuhaus, D., Worgotter, E., VasAk, M., Kagi, J.H.R. & Wuthrich, K. (1986). Nuclear magnetic resonance identification of 'half-turn' and 310-helix secondary structure in rabbit liver metallothionein-2. J. Mol. Biol. 187, 131-135. 29. Sibanda, B.L. & Thornton, J.M. (1985). 3-hairpin families in globular proteins. Nature 316, 170-174. 30. Milner-White, E.J.& Poet, R. (1986). Four classes of 3-hairpins in proteins. Biochem J. 240, 289-292.

1039

1040

Structure 1994, Vol 2 No 11 31. Yu, H., Rosen, M.K.& Schreiber, S.L. (1993). 'H and 5 N assignments and secondary structure of the Src SH3 domain. FEBS Lett. 324, 87-92. 32. Koyama, S., Yu, H., Dalgarno, D.C., Shin, T.B., Zydowsky, L.D. & Schreiber, S.L. (1993). 'H and 5N assignments and secondary structure of the P13K SH3 domain. FEBS Lett. 324, 93-98. 33. Smith, L.J., et a., & Wlodawer, A. (1994). Comparison of four independently determined structures of human recombinant interleukin-4. Sruct. Biol.1, 301-310. 34. Chen, J.K., Lane, W.S., Brauer, A.W., Tanaka, A. & Schreiber, S.L. (1993). Biased combinatorial libraries: novel ligands for the SH3 domain of phosphatidylinisitol 3-kinase. J. Am. Chem. Soc. 115, 12591-12592. 35. Lim, W.A. & Richards, F.M. (1994). Critical residues in an SH3 domain from Sem-5 suggest a mechanism for proline-rich peptide recognition. Sruct. Biol. 1, 221-225. 36. Marion, D., Ikura, M., Tschudin, R. & Bax, A. (1989). Rapid recording of 2D NMR spectra without phase cycling. Application to the study of hydrogen exchange in proteins. J. Magn. Reson. 85, 393-399. 37. Rance, M., Sorensen, O.W., Bodenhausen, G., Wagner, G., Ernst, R.R. & Wuthrich, K. (1983). Improved spectral resolution in COSY proton NMR spectra of proteins via double quantum filtering. Biochem. Biophys. Res. Commun. 117, 479-485. 38. Bax, A. & Davis, D.G. (1985). MLEV-17 based two-dimensional homonuclear magnetization transfer spectroscopy. J. Magn. Reson. 65, 355-360. 39. MOller, L. (1987). P. E. COSY, a simple alternative to E. COSY. J. Magn. Reson. 72, 191-196. 40. Macura, S., Huang, Y., Suter, D. & Ernst, R.R. (1981). Two-dimensional chemical exchange and cross-relaxation spectroscopy of coupled nuclear spins. J. Magn. Reson. 43, 259-281. 41. Patt, S.L. (1992). Single- and multiple-frequency-shifted laminar pulses. . Magn. Reson. 96, 94-102. 42. Shaka, A.J., Lee, C.J. & Pines, A. (1988). Iterative schemes for bilinear operators; application to spin decoupling. J. Magn. Reson. 77, 274-293.

43. Bodenhausen, G. & Ruben, D. (1980). Natural abundance nitrogen15 NMR by enhanced heteronuclear spectroscopy. Chem. Phys. Lett. 69, 185-189. 44. Marion, D. et al., & Clore, G.M. (1989). Overcoming the overlap problem in the assignment of IH NMR spectra of larger proteins by use of three-dimensional heteronuclear 1H-1 5N Hartmann-Hahnmultiple quantum coherence and nuclear Overhauser-multiple quantum coherence spectroscopy: application to interleukin 1 . Biochemistry 28, 6150-6156. 45. Kay, L.E., Keifer, P. & Saarinen, T. (1992). Pure absorption gradient enhanced heteronuclear single quantum correlation spectroscopy. J. Am. Chem. Soc. 114, 10663-10665. 46. Suri, A.K. & Levy, R.M. (1993). Estimation of interatomic distances in proteins from NOE spectra at longer mixing times using an empirical two-spin equation. J. Magn. Reson. B 101, 320-324. 47. Clore, G.M., Brunger, A.T., Karplus, M. & Gronenborn, A.M. (1986). Application of molecular dynamics with interproton distance restraints to three-dimensional protein structure determination: a model study of crambin. J. Mol. Biol. 191, 523-551. 48. Wagner, G., Braun, W., Havel, T.F., Schaumann, T., Go, N. & Wuthrich, K. (1987). Protein structures in solution by nuclear magnetic resonance and distance geometry; the polypeptide fold of the basic pancreatic trypsin inhibitor determined using two different algorithms, DISGEO and DISMAN. . Mol. Biol. 196, 611-639. 49. Bartik, K. & Redfield, C. (1993). A method for the estimation of X1 torsion angles in proteins. J. Biomol. NMR 3, 415-428. 50. Brunger, A.T. (1990). X-PLOR Version 2.1 Manual. Yale University, New Haven, CT. 51. Hatanaka, H., et al., & Inagaki, F. (1994). Tertiary structure of erabutoxin b in aqueous solution as elucidated by two-dimensional nuclear magnetic resonance. J. Mol. Biol. 240, 155-166. 52. Kraulis, P.1. (1991). MOLSCRIPT: a program to produce both detailed and schematic plots of protein structure. J. Appl. Crystallogr. 24, 946-950. Received: 2 Aug 1994; revisions requested: 22 Aug 1994; revisions received: 5 Sep 1994. Accepted: 15 Sep 1994.