The Crystal Structures of the SH2 Domain of p56lckComplexed with Two Phosphonopeptides Suggest a Gated Peptide Binding Site

JMB—MS 328 Cust. Ref. No. RH 81/94

[SGML] J. Mol. Biol. (1995) 246, 344–355

The Crystal Structures of the SH2 Domain of p56lck Complexed with Two Phosphonopeptides Suggest a Gated Peptide Binding Site Vincent Mikol, Go¨tz Baumann, Thomas H. Keller, Ute Manning and Mauro G. M. Zurini Preclinical Research, Sandoz Pharma AG, CH-4002 Basel Switzerland

Src homology-2 (SH2) domains are protein modules found within a wide variety of cytoplasmic signalling molecules that bind with high affinity to phosphotyrosyl-containing protein sequences. In order to develop SH2 inhibitors that contain phosphotyrosyl analogues resistant to cellular phosphatases, we have solved the crystal structures of the SH2 domain of p56lck in separate complexes with two high-affinity p-(phosphonomethyl)phenylalanine-containing peptides. The structures have been determined at 2.3 Å and 2.25 Å, and refined to crystallographic R-factors of 19.2% and 18.5%, respectively. The conformation of the SH2 domain of p56lck is essentially similar to that observed in Src and Lck complexed with a phosphotyrosine-containing peptide except in some loops and especially in the loop that connects the second and third b-strands. This loop, which was involved in hydrogen-bond interactions with the phosphotyrosine moiety, has moved away in the phosphonopeptide complexes as a rigid body by about 7 Å on two hinges leaving the tyrosine phosphate mimetic moiety accessible to the solvent. Some intramolecular hydrogen bonds with other residues of the third and fourth b-strands stabilize an open conformation of the lid, suggesting a flap mechanism for peptide binding. Keywords: SH2 domains; phosphonopeptides; protein-peptide interactions; protein crystallography; flexible loop

Introduction The Src homology-2 (SH2) domains are modules of about 100 amino acid residues that are found in many intracellular signal-transduction proteins (for a review, see Pawson & Schlessinger, 1993). The SH2 domains are thought to mediate specific protein-protein interaction via binding with high affinity to specific, phosphotyrosine-containing peptide sequences in their targets. The crucial role of tyrosine phosphorylation in intracellular signaling cascades and the specificity of binding of different SH2 domains for different phosphorylated intracellular proteins has made the development of specific inhibitors for SH2 domains an attractive target for potential intervention in a number of human diseases. Studies using small phosphotyrosine Abbreviations used: SH2, Src homology-2 domain; N-Gap, N terminus Src homology-2 domain of the GTPase activating protein; Lck, Src homology-2 domain of p56lck; PEG, polyethylene glycol; Pmp, p-(phosphonomethyl)phenylalanine; pY, phosphotyrosine; r.m.s.d., root-meansquare difference. 0022–2836/95/070344–12 $08.00/0

(pY)-containing peptides have shown that molecules containing only five residues can effectively block SH2-mediated binding to activated receptors (Fantl et al., 1992). The combination of the small size of the pY-containing peptide required for inhibition, the dominant role of the phosphotyrosine moiety, the fact that recognition occurs via a variable contiguous peptide sequence, and the cell-type restricted expression of some SH2 domains provides a good basis for the design of selective inhibitors of signal transduction (Brugge, 1993). The protein p56lck is a protein tyrosine kinase of the p60src family and is predominantly expressed in T lymphocytes. The three-dimensional structure of its SH2 domain has already been solved complexed with a pY-containing peptide (Eck et al., 1993, 1994) as have the structures of other SH2 domains, by protein crystallography and by nuclear magnetic resonance (for a review, see Kuriyan & Cowburn, 1993). They all share a common fold characterized by a central antiparallel b-sheet, flanked by two helices. Crystal structure analysis of the SH2 domains of p56lck (Lck) and of p60src (Src) complexed with a pY-containing peptide (Eck et al., 1993, 1994; 7 1995 Academic Press Limited

JMB—MS 328 345

Crystal Structures of Lck with Phosphonopeptides

Waksman et al., 1992, 1993) have revealed that the peptide binds in an extended conformation roughly perpendicular to the plane of the b-sheet, resembling a two-pronged plug (the prongs being pY and the third residue after pY). The hydrolytic lability of tyrosine phosphate in vivo has led several groups to examine the potential of p-(phosphonomethyl)phenylalanine (Pmp) as a stable amino acid analogue of pY (Burke et al., 1993; Garbay-Jaureguiberry et al., 1992a; Lee & Cushman, 1992). It has been shown that a Pmp-containing peptide with the sequence corresponding to Y315 of the polyoma virus middle T antigen blocks the in vitro association between the c-Src/mT complex and phosphatidyl inositol 3kinase. However a five to sixfold higher concentration of the Pmp-peptide was required to inhibit this interaction when compared with the corresponding phosphopeptide (Domcheck et al., 1992; Burke et al., 1994). It has been suggested, that the lower affinity may either be due to the higher pKa of the phosphonate group (Domchek et al., 1992) compared with the phosphate group or to the loss of a hydrogen bond between the phenolic oxygen atom and the SH2 domain. In order to examine the structural basis for the reduced affinity of Pmp-containing peptides we have performed the crystal structure analysis of Lck complexed with two different phosphonopeptides. One amino acid sequence is derived from the p75HS1 protein, the expression of which is limited to cells of the hematopoietic lineage (Yamanashi et al., 1993). In activated Ramos B cells the p75HS1 protein is the major ligand for Src-like tyrosine kinases (Baumann et al., 1994). The other phosphonopeptide originates from the amino acid sequence of the hamster polyomavirus middle-sized tumor antigen (Songyang et al., 1993).

Results The Lck structure Lck has the same folding as Src (Waksman et al., 1992, 1993) (Figure 1) and the main differences occur in three loops, residues Asp21 to Gly27 (AB loop), Asp49 to Gly53 (CD loop) and especially in the loop Ser34 to Ser40 (BC loop) of Lck. The r.m.s.d. for backbone atoms N, Ca, C, O of the Asp21 to Gly27 loop ˚ after superposibetween Src and Lck2A† is 1.17 A tion of the corresponding atoms, and the maximum ˚ ). The shift for a Ca atom is observed for Pro22 (3.95 A difference in conformation of the AB loop is a likely result of the presence of Arg46 in the central b-sheet (Ser in Src), which by steric hindrance favours a more extended conformation of the loop. Furthermore, with one of its peripheral Nh atoms this Arg makes two hydrogen bonds with the carbonyl oxygen atoms of Ala21 and of Asn24, inducing a cis-conformation for the proline of the b-turn (Pro22 ). The r.m.s.d. between † The conventions for the notations are given in the Materials and Methods section.

Figure 1. Schematic diagram of the superposition of Lck(grey)/peptide2A on Src (black). Labels for the N and C termini and for the 3 loops of Lck that have different conformation in both structures are given. The phosphonopeptide is represented as a ball-and-stick model. All pictures representing molecules were made with the program MOLSCRIPT (Kraulis, 1991).

Lck2A and Src for backbone atoms N, Ca, C, O of the ˚ after superposition of Asp49-Gly53 (CD loop) is 0.86 A the corresponding atoms and the maximum shift for ˚ ). The CD loop a Ca atom is observed for Gln50 (3.23 A 49 50 in Src (sequence Asp -Asn -Ala51-Lys52 ) corresponds to a b-hairpin with a distorted type II b-turn structure. In Lck (sequence Asp49-Gln50-Asn51-Gln52 ), this turn has twisted to give a slightly distorted type I b-turn, presumably because Asn occurs in position (i + 2) of the turn (Wilmot & Thornton, 1988). The major difference between the structures of Lck presented here and other available SH2 domain X-ray crystal structures can be found in one of the loops that delineates the pY binding site. The BC loop (Ser34-Ser40 ), which connects the second and third b-strand, lies in well-defined electron density and adopts in Lck1, Lck2A and Lck2B a completely Table 1 ˚ ) of backbone atoms (N, Ca, Root-mean square difference (A C, O) between Src, Lck1, Lck2A and Lck2B for all residues and for all atoms without the three loops (Asp21-Gly27, Ser34-Ser40 and Asp49-Gly53 ) Lck1 No loops

All

—

1.76

—

1.71

—

0.87

—

1.07

—

1.10

—

—

0.62

—

0.63

—

—

—

—

0.64

—

0.56

—

—

—

—

0.47

—

—

—

—

—

—

0.41

All All 1.69 Src No loops — All Lck1 No loops All Lck2A No loops

Lck2A No loops

All

Lck2B No loops

JMB—MS 328 346

Crystal Structures of Lck with Phosphonopeptides

Figure 2. Stereoview of the superposition of the Ser34-Ser40 loop of Lck (grey) as reported in this paper on that of Src (black: Waksman et al., 1993). Some residues of Lck are labelled. The corresponding sequence in Src is SETTKGA.

different orientation when compared with Src (Figure 1). There is no significant difference in the conformation of this loop between Lck1, Lck2A and Lck2B despite different crystal packing environments (Table 1). Only in Lck1 is a residue of the loop involved in crystal packing contacts: Glu35 makes a salt-bridge with Arg62 of neighbouring molecule, whereas in Lck2A and Lck2B the side-chain of Glu35 is disordered. All other residues of the loop are exposed to the solvent. Interestingly, this loop has an internal conformation that does not differ from that observed in the Src complex (Waksman et al., 1992, 1993) or in the complex between a pY-containing peptide and Lck (Eck et al., 1993, 1994). However, the r.m.s.d. for the backbone atoms L Ser34 to L Ser40 ˚ (Table 2, and the corresponding atoms in Src is 0.39 A Figure 2). Indeed there are three direct intramolecular hydrogen-bonds within the loop that stabilize its overall conformation. These result in this loop just being canted at a different angle (about 40° difference when compared with Src), thus revealing the existence of a rigid body movement on two hinges (Glu33 and Phe41 ). The maximum shift for a Ca atom ˚ for Lck1, 8.4 A ˚ for Lck2A is observed for Ser36 (7.1 A ˚ for Lck2b, respectively). The orientation of and 6.8 A the loop is stabilized by two direct hydrogen bonds with the rest of the molecule (Table 3).

Table 3 Intra- and intermolecular hydrogen bonds of the loop Ser34-Ser40 in Lck1 A. Intramolecular hydrogen bonds Loop Dist Water ˚) Atoms (A molecule

The structures of the bound phosphonopeptide molecules Peptide1 binds in an extended conformation almost perpendicular to the central b-sheet. Gly−2 and Glu−3 induce a 90° bend in the peptide chain. However, residues at the N terminus lie in relatively weak electron density and, apart from the carbonyl oxygen atom of Asp−1, do not appear to interact with Table 2 ˚ ) of the backbone atoms of Root-mean-square difference (A the loop Ser34-Ser40 between Src, Lck1, Lck2A and Lck2B Src Lck1 Lck2A

the protein, whereas the four residues from Pmp0 to Val+3 are particularly well defined in the density and seem tightly anchored. The phenyl ring of Pmp has tilted by about 37° towards the third b-strand (Gly53 to Asn63 ) when compared with that of pY, which lies nearly perpendicular to it (Waksman et al., 1992, 1993; Figure 3). The presence of two hydrogen bonds between the main chain of Val+3 and a crystallographic neighbouring molecule (Val+3-N–Od1-Asn89, Val+3-O–Nd2-Asn89 ) might explain why Val+3 appears to cover more than to lie inside the +3 pocket. However, the side-chain of Val+3 seems rather inaccessible as 92% of the side-chain surface area are protected by the pocket from exposure to the solvent. The distance between the Ca and Cb of Val+3 and the corresponding atoms of Ile+3 in the Lck2A structure ˚ and 0.6 A ˚ , respectively. Interestingly, a are 1.1 A water molecule is buried in the so-called ‘‘hydrophobic’’ pocket bridging the hydroxyl group of Tyr87 with the carbonyl atom of Asp92, thus raising doubt about the strong hydrophobic character of the pocket. There is one close contact between one oxygen atom

Lck1

Lck2A

Lck2B

0.39 — —

0.39 0.39 —

0.49 0.44 0.28

Ser34-N Ser34-Og Thr37-O Ser34-Og Ser36-O

2.87 3.00 3.00 3.27 2.63

W54 W22

Dist ˚) (A

Loop atoms

2.86 2.85

O-Ser40 N-Thr37 N-Ser40 Og-Ser36 N-Ala38

Dist ˚) (A

Atoms of the rest of the molecule

2.53 2.83 2.90 2.78 3.24 3.11

Oe1-Glu33 Nd2-Asn63 Ne-Lys60 Od1-Asn63 O-Ile61 N-Phe41 Og-Ser42 N-Ser42 O-Arg32

B. Intermolecular hydrogen bonds Loop atoms

Dist ˚) (A

Gly39-N Gly39-O Ser40-Og Gly39-O Gly39-O Gly39-O Ser40-O Ser40-O Ser40-O

2.74 2.75 3.02 3.16 3.16 3.16 3.40 3.40 3.40

Water molecule

W29 W29 W29 W4 W4 W4

JMB—MS 328 347

Crystal Structures of Lck with Phosphonopeptides

of the phosphonate group of Pmp and the side-chain of Asp65 from the adjacent molecule in the crystal lattice. Since the side-chain of Asp65 does not lie in well-defined density, it is probably an artefact of the crystallographic refinement. There are ten water ˚) molecules hydrogen-bonded (distance cutoff 3.4 A −3 −2 0 to the peptide atoms (Glu -N, Gly -N, Pmp -OP1, Pmp0-OP3, Pmp0-O, Glu+1-O, Glu+1-Oe1, Glu+1-Oe2, Glu+2-Oe1, Glu+2-Oe2, Leu+4-O). The two peptide2 structures (A and B) have rather similar extended conformations (r.m.s.d. for back˚ and 1.37 A ˚ for all atoms; bone atoms is 0.34 A Figure 3). Their conformation is closer to that of ˚ ) than to peptide1 (r.m.s.d. for backbone atoms 0.7 A that of the pY-containing peptide complexed to Src ˚ and 1.1 A ˚ for (Waksman et al., 1992; 1993; 1.2 A peptide2A and peptide2B, respectively) despite

nearly identical sequence. In both structures, the phenyl ring of Pmp has tilted by 37° for peptide2A and by 40° for peptide2B towards the third strand of the central sheet. The fact that B Ile+3 adopts alternative conformations with relatively higher ˚ 2, Figure 4) temperature-factor (average value 39.7 A 0 2 +1 ˚ ˚ 2) than the residues P (B = 34.7 A ), P (B = 34.6 A suggests that the interaction in the +3 pocket is not as strong as previously thought and that the pocket could easily accommodate larger amino acid groups. There are four water molecules hydrogen˚ ) to the peptide2A bonded (distance cutoff 3.4 A atoms (AcQ−1-O, Pmp0-OP3, Glu+1-O, Glu+1-Oe1, Glu+1-Oe2, Ile+3-O) and 6 waters hydrogen-bonded to peptide2B atoms (AcQ−1-Oe1, AcQ−1-O, Pmp0OP3, Pmp0-O, Glu+1-O, Glu+1-Oe2, Glu+2-Oe1 ), respectively.

(a)

(b)

(c) Figure 3. Comparison of the conformation of bound phospho- and phosphonopeptides. Oxygen and nitrogen atoms are represented in grey. (a) Stereoview of the superposition of the pY-containing peptide from Src (carbon atoms are displayed in white: Waksman et al., 1993) on peptide1 (carbon atoms are displayed in black). (b) Stereoview of the superposition of peptide1 (carbon atoms are displayed in black) on peptide2A (carbon atoms are displayed in white). (c) Stereoview of the superposition of peptide2A (carbon atoms are displayed in white) on peptide2B (carbon atoms are displayed in black).

JMB—MS 328 348

Crystal Structures of Lck with Phosphonopeptides

Table 4 ˚) Peptide1/Lck interatomic distances (distance cutoff 3.4 A Peptide1 atoms Asp−1-O Pmp0-Ch Pmp0-OP2 Pmp0-OP2 Pmp0-OP2 Pmp0-OP2 Pmp0-OP3 Glu+1-N Glu+1-Cb Glu+1-Cb Glu+1-Cg Leu+4-C Leu+4-O' Leu+4-O' Leu+4-O"

Distance ˚) (A

Lck1 atoms

2.78 3.32 2.83 3.20 3.36 2.73 3.02 2.77 3.24 3.32 3.37 2.78 3.40 2.55 2.50

Nh2-Arg12 Og-Ser42 Ne-Arg12 Nh2-Arg12 Nh1-Arg32 Nh2-Arg32 Nh1-Arg32 O-His58 O-His58 Ce2-Tyr59 Ce2-Tyr59 Nh1-Arg74 Cz-Arg74 Nh1-Arg74 Nh1-Arg74

˚ of peptide1: Arg12, Arg32, Lck1 residues which are within 4.0 A Ser42, Lys57, His58, Tyr59, Lys60, Ile71, Ser72, Arg74, Gly93.

phosphate oxygen atoms, an ion-pair between the phosphate group and Arg32, and one amino-aromatic bond between Arg12 and the phenyl ring of Pmp (Figure 5). One new feature when compared with Src is that the side-chain oxygen atom of A AcQ−1 is involved in a weak hydrogen-bond with the guanidinium group of Arg12, while the NH2 group is interacting with one phosphate oxygen atom. This latter intramolecular interaction is not observed for the other molecule of the asymmetric unit Lck2B, where the side-chain of AcQ−1 faces out towards the Table 5 Peptide2A/Lck2A interatomic distances (distance cutoff ˚ ) and comparison with peptide2B/Lck2B (corre3.4 A sponding values are given in parentheses if they are less ˚) than 3.4 A

Figure 4. Temperature-factors of the phosphonopeptides. Average main-chain (N, Ca, C; continuous lines) and side-chain (broken lines) temperature factors as a function of residue number for (a) peptide1, (b) peptide2A and (c) peptide2B.

The binding pocket Because of the hinge movement of the Ser34-Ser40 loop, the pY binding pocket appears much more open than that observed in the Src complex; 34% of the surface area of Pmp remains exposed to the solvent. However, in the three complexes Lck1, Lck2A and Lck2B, the main features of the interactions between the peptide and Lck are conserved (Tables 4 and 5). They include hydrogen bonds between the main-chain carbonyl group of the residue at position −1 with Arg12, the main-chain amide group of the residue at position +1 with the carbonyl atom of His58, Arg12 with one of the

Peptide2 atoms

Distance ˚) (A

Lck atoms

AcQ−1-Ca AcQ−1-Oe1 AcQ−1-Oe1 AcQ−1-Oe1 AcQ−1-C AcQ−1-C AcQ−1-O AcQ−1-O AcQ−1-O Pmp0-Ch Pmp0-Ch Pmp0-Cd1 Pmp0-OP2 Pmp0-OP2 Pmp0-OP2 Pmp0-OP2 Pmp0-OP2 Pmp0-OP3 Glu+1-N Glu+1-Cb Glu+1-Cb Ile+3-Cg2 Ile+3-Cg2 Pro+4-C Pro+4-O' Pro+4-O' Pro+4-O"

3.08 (3.36) 3.08 — 3.33 — 3.26 — 3.18 (3.39) 3.18 (3.10) 3.29 (3.37) 2.70 (2.91) 3.05 (2.99) 3.18 (3.35) 3.29 — 3.39 (3.20) 2.91 (2.89) 3.32 (3.25) 2.88 (3.24) 3.24 (3.07) 2.73 (2.59) 2.96 (2.83) 2.69 (2.69) 3.35 — 3.36 — 3.18 — 3.25 — 3.35 — 2.87 (2.92) 2.50 (2.47) 3.28 —

Nh1-Arg12 Cz-Arg12 -Ne-Arg12 Nh2-Arg12 Nh1-Arg12 Nh2-Arg12 Cz-Arg12 Nh1-Arg12 Nh2-Arg12 Og-Ser42 Ne-Lys60 (alt2) O-His58 Ne-Arg12 Cz-Arg12 Nh2-Arg12 Nh1-Arg32 Nh2-Arg32 Nh1-Arg32 O-His58 O-His58 Cd2-Tyr59 Cg2-Ile71 O-Ile71 C-Ser72 Og-Ser72 Nh1-Arg74 Og-Ser72

JMB—MS 328 Crystal Structures of Lck with Phosphonopeptides

349

(a)

(b)

Figure 5. Direct and water-mediated hydrogen bonds (broken lines) between (a) peptide1 and Lck, (b) peptide2A and Lck. The protein is displayed with a ball-and-stick model and the peptide with a stick model. The values of the interatomic distances are given in Tables 4, 5 and 6.

JMB—MS 328 350

Crystal Structures of Lck with Phosphonopeptides

Table 6 Water-mediated contacts between peptide1 and Lck1, between peptide2 and Lck2A and between Lck2B and peptide2 Peptide1 atoms

Dist ˚) (A

Water molecule

Dist ˚) (A

Water molecule

Dist ˚) (A

Lck1 atoms

Pmp0-OP3 Glu+1-O Glu+1-Oe1 Glu+1-Oe1 Glu+1-Oe2 Glu+1-Oe2

3.25 2.97 2.63 2.63 3.08 3.08

W13 W2 W3 W3 W12 W12

3.24

W93

2.79 2.79

W16 W16

3.15

W17

3.01 2.72 3.03 3.25 2.66 3.14

N-Glu35 N-Lys60 Oh-Tyr59 O-Gly93 N-His58 O-Val56

Peptide2A atoms

Dist ˚) (A

Water molecule

Dist ˚) (A

Water molecule

Dist ˚) (A

Lck2A atoms

AcQ−1-O Glu+1-O Glu+1-O Glu+1-O Glu+1-O Glu+1-O Glu+1-Oe2 Ile+3-O

2.54 2.80 2.80 2.80 2.80 2.80 3.16 2.55

W6 W1 W1 W1 W1 W1 W6 W77

3.10 3.10 3.10 3.10

W22 W22 W22 W22

2.94 2.79 2.69 2.84 3.00 2.90 2.94 2.59

N-His58 N-Lys60 O-Lys60 Ne-Arg62 Nh2-Arg62 O-Ile71 N-His58 O-Ile71

Peptide2B atoms

Dist ˚) (A

Water molecule

Dist ˚) (A

Water molecule

Dist ˚) (A

Lck2B atoms

AcQ−1-O Pmp0-OP3 Glu+1-O Glu+1-O Glu+1-O Glu+1-Oe2 Glu+1-Oe2 Glu+1-Oe2

2.79 2.83 2.82 2.82 2.82 3.18 3.18 3.18

W206 W270 W201 W201 W201 W206 W206 W206

2.98 2.98

W222 W222

2.51 3.34

W264 W291

2.95 2.66 2.76 2.58 2.67 2.95 2.56 2.80

N-His58 N-Arg12 N-Lys60 O-Lys60 O-Ile71 N-His58 O-Val56 Ne-Arg62

solvent. The main difference is the absence of the three hydrogen-bonds between the Ser34-Ser40 loop and the Pmp when compared with Src complexed with a pY-containing peptide. In the three structures determined, the carboxyl terminus of the peptide accepts hydrogen bonds from the guanidinium group of Arg74 and from the hydroxyl group of Ser72. However, as indicated by the relatively high B-factors of the C-terminal residues of the peptide (Figure 4), these contacts are unlikely to be strong interactions. There are six water-mediated interactions between Lck1 and peptide1 and 8 for Lck2A/peptide2A and Lck2B/peptide2B. Only two water-mediated contacts are conserved in the three complexes studied (Table 6). One involves the main-chain amide group of Lys60 and the main-chain carbonyl group of position +1, and the other conserved water molecule bridges one carboxylate oxygen atom of Glu+1 and the main-chain amide group of His58. Both water molecules have low ˚ 2 ), B-factors (average for the three structures of 27.5 A which are comparable with the average value for the protein. There are six conserved (out of eight) water-mediated contacts between Lck2A/peptide2A and Lck2B/peptide2B. This suggests that most of the water-mediated interactions (except the two conserved ones) depend primarily on the sequence of the peptide and are not critical for binding. One of the two conserved water molecules in all complexes bridges one carboxylate oxygen atom of Glu+1 with the main-chain carbonyl atom of P−1, thus stabilizing the conformation of the peptide.

This water molecule makes a hydrogen bond with the amide nitrogen atom of His58 (Figure 5). The presence of this network of hydrogen bonds centered around this water molecule might explain why Glu, although not involved in a direct hydrogen bond with the protein, is determinant in position +1 for binding to the protein (Songyang et al., 1993).

Discussion In order to study the selectivity of peptide binding to SH2 domains, we have synthesized a variety of different peptides containing p-(phosphonomethyl)phenylalanine as a stable and easily prepared tyrosine phosphate mimetic. Not surprisingly we have found, as recently described (Burke et al., 1994), that peptides containing this residue are generally six to ten times less active than the corresponding peptides containing tyrosine phosphate (for an example see Table 7). The reduced

Table 7 Relative affinities of phosphopeptides and phosphonopeptides for Src, Lck and N-Gap Compound

Src (mM)

Lck (mM)

N-Gap (mM)

Peptide sequence

2 3 4 5

0.11 3.1 0.2 10

0.16 0.79 0.18 6.3

3.1 4.0 5.0 6.3

PEGD(pY)EEVL PEGD(Pmp)EEVL EPQ(pY)EEI EPQ(Pmp)EEI

JMB—MS 328 351

Crystal Structures of Lck with Phosphonopeptides

affinity for the phosphonopeptide when compared with the phosphopeptide is likely to result from the smaller contact area between the protein and the phosphonotyrosine, and from the reduced acidity of the phosphono versus phosphotyrosine derivatives and/or from the loss of the hydrogen bonds with the BC loop. In the structure of Src complexed with a pY-containing peptide, the BC loop is involved in three hydrogen bonds with the phosphotyrosine moiety: the Og of Ser34 donates a proton to the phosphate ester oxygen atom, the Og of Thr36 and the amide nitrogen atom of Glu35 are within hydrogen bond distance from phosphate oxygen atoms. None of these contacts are observed in the phosphonopeptide complex structures reported here. Interestingly, in an analogue series of peptides, when the acidity and the hydrogen-bond capabilities of the carbon atom adjacent to the phosphate group of the Pmp were restored by incorporating two fluorine substituents the affinity of the compound was similar to that of pY (Burke et al., 1994). We have also noticed that in certain cases the binding selectivity of phosphonopeptides is markedly different from the corresponding phosphopeptides. For example the seven residue phosphonopeptide EPQ(Pmp)EEI inhibits the binding of biotinylated peptides to the SH2 domains of Src, Lck and N-Gap (N terminus SH2 domain of the GTPase activating protein) with and IC50 of about 10 mM in all the three cases, while for the corresponding phosphopeptide there is a more than 25-fold difference between the IC50 ’s values of the Src-family SH2 domains and N-Gap (see Table 7). The selectivity between Src, Lck and N-Gap seems to be reduced with phosphonopeptides when compared with phosphopeptides. This is likely to be a result of the sequence and conformation variability of the BC loop within the SH2 domains (sequence of the BC loop for N-Gap and Src are SDRRPGS and SETTKGA, respectively). It suggests that the pattern of interactions between the BC loop and the pY moiety is not universally conserved among the SH2 family as already observed in the structure of the amino-terminal SH2 domain of the Syp tyrosine phosphatase (Lee et al., 1994). As a consequence, the combination of the flexibility of the BC loop that delineates an enlarged binding pocket and of its sequence variability could permit the design of SH2 sub-type specific, non-peptidic inhibitors. Comparing the crystal structure of Src free and complexed with a pY-containing peptide, Waksman et al. (1993) have noticed that some small conformational changes occur in the BC and EF loops. This led Waksman et al. (1992) to speculate that the BC loop might be in an open orientation in the absence of ligand as in the free form of Src, the BC loop is seen to become relatively disordered for two molecules (out of four) of the asymmetric unit. This is consistent with NMR results on two apo SH2 domains (Booker et al., 1992; Overduin et al., 1992), which have indicated that this loop is poorly ordered in solution. In the structures reported here with a Pmp-containing peptide, the conformation of this

Figure 6. Amino acid structures : (a) tyrosine (Y); (b) phosphotyrosine (pY); (c) p-(phosphonomethyl)phenyl-alanine (Pmp).

loop appears to be well defined as typified by an ˚ 2 for Lck1 (22.3 A ˚ 2 for the average B-factor of 25.8 A whole molecule). This loop in Src shields all of the pY moiety from the aqueous environment. The pY moiety is then confined within the protein and cannot be released without a conformational change of the SH2 domain, in contrast to the Pmp moiety, which is accessible to the solvent because of the hinge motion of the loop. The change pY to Pmp therefore appears to favour an open orientation for the BC loop. This suggests that pY-containing peptide recognition occurs in two steps; (1) binding in a solvent-exposed, highly charged pocket; (2) closure of the BC loop through a hinge motion. The fact that this loop seems rather disordered in solution (Booker et al., 1992; Overduin et al., 1992) suggests that there may be a dynamic equilibrium between the closed and open conformation with only slight difference in energy between them. Analogous loop closures upon substrate binding have been observed in several proteins (e.g. see Kempner, 1993; Gerstein et al., 1994) and illustrate a simple low-energy, induced-fit mechanism.

Materials and Methods Notations A sequential notation for the residues (Eck et al., 1993) will be employed. Table 8 shows the equivalence between the sequential numbering and that used by Eck et al. (1993) which refers to residues by their relative positions within elements of secondary structure. The structure of Lck when complexed with peptide1 will be referred to as Lck1. Since the crystal of Lck with peptide2 contains two molecules of complex per asymmetric unit, two independent structures of Lck complexed with peptide2 could be determined and will be denoted Lck2A/peptide2A and Lck2B/peptide2B. Pro22, A Pro22, B Pro22 designate Pro22 of Lck1, Lck2A and Lck2B, respectively. The central p- (phosphonomethyl)phenylalanine (Pmp) is designated as position P0 and neighbouring residues are numbered accordingly. A Glu+1 indicates Glu at the position P+1 of the phosphonopeptide complexed with Lck2A. The three oxygen atoms of the phosphate group are denoted O1P, O2P and O3P, respectively.

JMB—MS 328 352

Crystal Structures of Lck with Phosphonopeptides

Table 8 A conversion table for the Lck sequential and Eck et al. (1993) numbering systems Sequence ASMTGGQQMGR GS WFF KNL SRKDAERQLL APGNTHG SFLIRES ESTAG SFSLSVRD FDQNQGE VVKHYKIRNL DNG GFYI SPR ITF P GLHDLVRHYTNA SDGLCTRLSRPC

Sequential numbering

Secondary structure

−8:2 3:4 5:7 8 : 10 11 : 20 21 : 27 28 : 34 35 : 39 40 : 47 48 : 54 55 : 64 65 : 67 68 : 71 72 : 74 75 : 77 78 79 : 90 91 : 102

— — bA AA aA AB bB BC bC CD bD DE bE EF bF FB aB BG

The prefixes a and b designate a-helix and b-strand, respectively. The sequential numbering is used here as it appears that some residues as indicated by their torsion angles do not fit properly into the elements of secondary structures delineated by Eck et al. (1993). Residues − 8 to 0 correspond to residues of the T7 gene leader sequence required for expression.

Peptide synthesis and protein preparation The pY analogue used is p- (phosphonomethyl)phenylalanine (Figure 6), a phosphonate-based mimetic of pY in which the phosphate ester oxygen atom has been replaced by a methylene group. Peptide1 and Peptide2 are Pro-Glu-Gly-Asp-Pmp-Glu-Glu-Val-Leu and AcQ-PmpGlu-Glu-Ile-Pro, respectively (AcQ designate N-acetylglutamine). Fmoc-p (dimethylphosphonomethyl)-l-phenylalanine was prepared as described (Garbay-Jaureguiberry et al., 1992b). The peptides were synthesized using a stepwise Na-Fmoc strategy on a Milligen 9050 peptide

synthesizer. The demethylation of the Pmp residue was performed using a mixture of TFMSA/TFA/DMS/mcresol 10:50:30:10 (by vol.) for four hours at room temperature (Lee & Cushman, 1992). Peptides were purified by HPLC after which they had mass spectra and amino acid analyses in agreement with the expected structures. The SH2 domain of Lck was expressed in Escherichia coli (BL21 (DE3)pLysS) by the phage T7 expression system (NOVAGEN) essentially as described (Baumann et al., 1994). The DNA fragment corresponding to the SH2 domain of human Lck was isolated by the polymerase chain reaction using complementary oligonucleotides with suitable BamHI restriction sites at either end (Perlmutter et al., 1988). The amplified DNA fragment was isolated, BamHI digested and subsequently cloned into the BamHI restriction site of the bacterial expression vector pET3A (Novagen) by standard recombinant technology. To prove integrity the inserted DNA-fragment was entirely sequenced. The overexpressed SH2 domain of Lck was purified as follows. Typically, 30 g of recombinant E. coli pellet was resuspended in 150 ml of a buffer containing 50 mM Tris-HCl (pH 8.0), 5 mM EDTA, 5 mM DTT and 5 mM benzamidine and disrupted by two passages in a French Press at 1200 × 105 Pa. After centrifugation, the pellet was subjected to a freeze/thaw cycle, resuspended in 100 ml of the above buffer and sonicated for ten minutes at 4°C with one second pulses at 50% duty cycles. After centrifugation, the resulting pellet was subjected to two more freeze/thaw/sonication cycles yielding 3 to 4 g of highly purified inclusion bodies. The resulting inclusion bodies were dissolved in about 20 ml of a buffer consisting of 50 mM Hepes (pH 8.5), 5 mM DTT, 5 mM EDTA, 5 mM benzamidine and 6 M guanidine, and after centrifugation to remove insoluble material, the clear solution was immediately diluted with one litre of 20 mM Hepes (pH 7.0), 100 mM NaCl and 2 mM DTT. The slightly cloudy solution was clarified by centrifugation and immediately applied to a 20-ml bed volume column of phosphotyrosineSepharose 4B at a flow-rate of 2 ml/min. After washing of the column to base-line with the above dilution buffer,

Table 9 Crystals and diffraction data Crystal parameters

Lck/peptide1

Lck/peptide2

Initial drop composition

10 mM CH3 COOH/CH3 COO − Na + (pH 5.05), 10.5% (w/v) PEG8000 , 0.02% (w/v) NaN3 , [Lck/peptide1] = 2 mM

10 mM Hepes/NaOH (pH 7.1), 13.5% (w/v) PEG8000 , 0.02% (w/v) NaN3 , [Lck/peptide2] = 2 mM

Well composition

20 mM CH3 COOH/CH3 COO − Na + (pH 5.05), 21% (w/v) PEG8000 , 0.04% (w/v) NaN3

20 mM Hepes/NaOH (pH 7.1), 27.0% (w/v) PEG8000 , 0.04% (w/v) NaN3

Crystal size

0.8 mm × 0.3 mm × 0.3 mm

0.4 mm × 0.2 mm × 0.1 mm

Space group, cell dimensions

˚ , b = 46.66 A ˚, P21 21 21 , a = 44.66 A ˚ , a = b = g = 90° c = 59.30 A ˚ 3/Da 1, 50.2%, 2.46 A

˚ , b = 41.84 A ˚ , c = 45.52 A ˚, P1, a = 35.17 A a = 68.71°, b = 82.94°, g = 87.36° ˚ 3/Da 2, 50.4%, 2.47 A

Number of crystals used, measured reflections, unique reflections

1, 13232, 5815

1, 19230, 10015

Rsym (%), independent reflections, ˚) resolution (A

Rsym = 7.7% for 5072 independent ˚ reflections up to 2.25 A ˚ 95.6% up to 2.25 A

Rsym = 6.0% for 8054 independent ˚ reflections up to 2.3 A ˚ 93.9% up to 2.3 A

Number of complex per asymmetric unit, solvent fraction, VM Diffraction data

Completeness

JMB—MS 328 353

Crystal Structures of Lck with Phosphonopeptides

Table 10 Results of structure refinement Lck/peptide2 Lck/peptide1 No. of Lck atoms No. of phosphonopeptide atoms No. of solvent molecules ˚ 2) Average B-value for Lck (A ˚ 2) Average B-value for the phosphonopeptide (A ˚ 2) Average B-value for the solvent molecules (A ˚) Range of spacings (A R-value (=F = > 2s) (%) No. reflections weighted r.m.s.d. from ideality ˚) Bond length (A Bond angle (°)

elution was achieved by changing the buffer to 100 mM Na2 HPO4 (pH 7.4) yielding about 100 mg of very pure material. Competition assays to estimate relative binding of peptides to SH2 domains were performed using a modified version of the method described by Payne et al. (1993) using the BIAcore system (Pharmacia). The reference compound was a biotinylated peptide derived from the hamster polyomavirus middle-sized tumor antigen (sequence Glu-Pro-Gln-Pmp-Glu-Glu-Ile-Pro) with a 6-aminocaproic spacer between the biotin and the N terminus of the peptide. The IC50 values were determined as the concentration of peptide required to inhibit 50% of specific biotinylated reference peptide binding. Preparation of the crystals and data collection Purified Lck with a stoichiometric amount of phosphonopeptide were concentrated to 2 mM by ultrafiltration in 0.02% (w/v) NaN3 . Crystals of the Lck/peptide1 and Lck/peptide2 complexes were grown by vapour diffusion at 22°C using the hanging drop method (see Table 9). Both complexes tend to crystallize under rather similar conditions in various different crystal systems. Two crystal forms of Lck/peptide1 could be grown using PEG as the precipitant: an orthorhombic form that was used for structure solution appears at slightly acidic pH (5.0 to 6.5) and a tetragonal form that diffracted X-rays to medium ˚ ) was obtained at neutral pH. At least resolution (3.0 A three different crystal forms for Lck/peptide2 could be grown under similar conditions (Table 9) and they were distinguishable only by their X-ray diffraction patterns.

Table 11 Occupancies of residues having alternative conformations Occupancy of Occupancy of Residues conformation 1 conformation 2 A A A A B B B B B B

Leu30 Ile31 His58 Lys60 His26 Phe29 Ile31 Glu54 Cys95 Ile + 3

0.63 0.48 0.49 0.58 0.49 0.49 0.41 0.45 0.77 0.62

0.37 0.52 0.51 0.42 0.51 0.51 0.59 0.55 0.23 0.38

Averaged-B-factor of the side-chain ˚ 2) atoms (A 17.2 12.8 16.0 24.1 28.2 17.6 27.1 40.3 37.9 38.1

780 71 129 22.3 35.7 43.4 8–2.25 18.5 5525 0.015 1.85

LCK2A

LCK2B

780 59

779 59 217

26.3 41.5 40.6 8–2.3 19.2 9897

28.0 39.9

0.014 1.80

The X-ray intensity data were collected on a FAST television area detector. The X-ray source was a rotating anode generator (FR571) operating at 40 kV and 80 mA. The evaluation of the measured intensity was performed by the program MADNES (Messerschmidt & Pflugrath, 1987). Data reduction was carried out using the CCP4 package (Daresbury Laboratory, UK). The crystal parameters and diffraction data are given in Table 10. Structure solution and crystallographic refinement The structure of the complex between Lck and peptide1 was solved first. The refined structure of Src without peptide was used for molecular replacement (Waksman et al., 1992, 1993) as it was the only SH2 domain for which coordinates were available. All residues that differ in sequence from Lck were set to alanine, and were used as the model for phase determination by molecular replacement of Lck/peptide1. Rotation calculations using direct space Patterson search followed by Patterson correlation refinement as implemented in XPLOR ˚ yielded (Bru¨nger, 1992) using data between 8.0 and 4.0 A an unambiguous orientation of the search model (Patterson correlation coefficient of the first peak 0.16; of the second peak 0.098). The translation search was then performed by optimizing the standard linear correlation coefficient between the normalized observed structure factors and the normalized calculated structure factors. The position of the model computed by the translation search gave an excellent packing of the symmetry-related molecule and a crystallographic R-factor of 49.7% for data between 8.0 and ˚ . The model was then subjected to rigid body 3.5 A refinement by running 40 steps of conjugate gradient minimization. The R-factor dropped to 47% for data ˚ . Energy restrained least-squares between 8.0 and 3.5 A refinement followed by simulated annealing refinement were performed using XPLOR (Bru¨nger, 1992) with the parameter file compiled by Engh & Huber (1991). At this stage (R-factor = 29.5% for data between 8.0 and ˚ ), inspection of a (Fobs − Fcal ) difference Fourier map 3.0 A showed good density in terms of changes in the sequence of Lck relative to Src as well as for part of the phosphonopeptide. The amino acid sequence of the model was changed to that corresponding to Lck in alternating cycles of manual rebuilding using the program O (Jones et al., 1991) and least-squares refinement using the program XPLOR (Bru¨nger, 1992). The phosphonopeptide was then fitted into the map and then co-refined with the Lck model. In the final stages of refinement, individual temperature factors were also refined.

JMB—MS 328 354 Solvent molecules were included if they were on sites of difference electron density with values above 2.5s and if ˚ of the protein complex or of a water they were within 3.5 A molecule. The correctness of the final model, namely the regions that differ significantly from the Src structure, was checked using simulated annealing omit maps (Hodel et al., 1992). The final model has been refined to a ˚ (see crystallographic R-factor of 18.5% for data up to 2.25 A Table 10). All residues have f and c angles that lie in the allowed region of the Ramachandran diagram. The quality of the map was very good and has allowed confident placement of all main and side-chain atoms except 11 residues at the N terminus (from residue − 8 to residue 2, see Table 8 for the numbering of residues), two residues at the C terminus (residues 101 and 102) and the proline residue of the phosphonopeptide that were not visible in the electron density. The structure described here is for residues 3 to 100. The side-chain of the last visible residue (Arg100 ) could not be detected in the map and was set to alanine. The side-chains of the following residues display poor electron density: for the peptide Glu−3, Asp−1, Leu+4, for the protein Gln50. Three residues of the protein (Lys8, Lys13 and Asp65 ), although they do not show continuous density for their whole side-chain, have been positioned without ambiguity up to their Cg atom. The crystals of Lck complexed with peptide2 (space group P1) contain two molecules of complexes per asymmetric unit. A self-rotation calculation for data of the ˚ reveals a Lck/peptide2 complex between 8.0 and 4.0 A clear 2-fold axis at c = 63.0°, f = 54° and k = 180°. Rotation search in Patterson space followed by Patterson correlation refinement (Bru¨nger, 1992) was performed for data ˚ using the Lck1 structure from which between 8.0 and 3.5 A the peptide and the solvent molecules were deleted. This yielded two major orientations (Patterson correlation coefficient of the first peak 0.36; of the second peak 0.30; of the third peak 0.10) that are related by c = 64°, f = 52° and k = 177°, corresponding essentially to the local 2-fold axis determined by the self-rotation calculation. Since the crystals of Lck/peptide2 are triclinic, one can position arbitrarily one molecule. A translation search for the second molecule was then performed by optimizing the standard linear correlation coefficient between the normalized observed structure factors and the normalized calculated structure factors. The position of the model computed by the translation search gave a crystallographic ˚ . The R-factor of 47.1% for data between 8.0 and 3.0 A model was then subjected to rigid body refinement by running 40 steps of conjugate gradient minimization. The ˚. R-factor dropped to 34.7% for data between 8.0 and 3.0 A Energy restrained least-squares refinement using XPLOR (Bru¨nger, 1992) gave an R-factor of 23.2% for data between ˚ . Inspection of a (Fobs − Fcal ) difference Fourier 8.0 and 3.0 A map showed good density for four residues of the phosphonopeptide for both complexes in the asymmetric unit. They were fitted into the map and then co-refined with the Lck model. The two complexes of the unit cell were refined independently following the procedure described for Lck1. The final model has been refined to a ˚ (see crystallographic R-factor of 19.2% for data up to 2.3 A Table 10). All residues have f and c angles that lie in the allowed region of the Ramachandran diagram: 217 water molecules have been located in the electron density (109 for Lck2A and 108 for Lck2B). The quality of the map was very good and has allowed confident placement of all main and side-chain atoms except 11 residues at the N terminus (from residue − 8 to residue 2), two residues at the C terminus (residues 101 and 102) and the acetyl termini of A AcQ−1 and of B AcQ−1. The

Crystal Structures of Lck with Phosphonopeptides

side-chain of the last visible residue (Arg100 ) could not be detected in the map and was set to an alanine for LckA and to a glycine for LckB. The side-chains of the following residues display poor electron density : for the peptide B Glu+2, for the protein A Asn9, A Glu35, A Gln50, A Gln52, B Asp14, B Glu35 and B Ser36. One residue of the protein (B Lys8 ), although it does not show continuous density for its whole side-chain, has been positioned without ambiguity up to its Cg atom. Alternative conformations for ten doubly disordered residues could be determined (Table 11).

Acknowledgements We thank Armin Widmer and Trevor Payne for helpful discussions and Malcolm D. Walkinshaw for constant support.

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JMB—MS 328 Crystal Structures of Lck with Phosphonopeptides

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Edited by R. Huber (Received 17 August 1994; accepted 15 November 1994)