Kinetic analysis of the binding of human matrix metalloproteinase-2 and -9 to tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2

The dissociation constants (Kd) of tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 for the active and latent forms of matrix metalloproteinase (MMP)-2 and MMP-9 were evaluated using surface plasmon resonance (SPR) and enzyme inhibition studies. SPR analysis shows biphasic kinetics with high (nM) and low (microM) affinity binding sites of TIMP-2 and TIMP-1 for MMP-2 (72- and 62-kDa species) and MMP-9 (92- and 82-kDa species), respectively. In contrast, binding data of TIMP-2 to an MMP-2 45-kDa active form lacking the C-terminal domain and to an MMP-2 C-terminal domain (CTD) fragment displays monophasic kinetics with Kd values of 315 and 60 nM, respectively. This suggests that the CTD contains the high affinity binding site, whereas the catalytic domain contains the low affinity site. Also, binding of TIMP-2 to pro-MMP-2 is stronger at both the high and low affinity sites than the corresponding binding of TIMP-2 to the MMP-2 62-kDa form demonstrating the importance of the N-terminal prodomain. In addition, the Kd value of TIMP-1 for the MMP-2 62-kDa species is 28. 6 nM at the high affinity site, yet neither the MMP-2 45-kDa species nor the CTD interacts with TIMP-1. Enzyme inhibition studies demonstrate that TIMPs are slow binding inhibitors with monophasic inhibition kinetics. This suggests that a single binding event results in enzyme inhibition. The kinetic parameters for the onset of inhibition are fast (kon approximately 10(5) M-1 s-1) with slow off rates (koff approximately 10(-3) s-1). The inhibition constants (Ki) are in the 10(-7)-10(-9) M range and correlate with the values determined by SPR.     

Specific, high affinity binding of tissue inhibitor of metalloproteinases-4 (TIMP-4) to the COOH-terminal hemopexin-like domain of human gelatinase A. TIMP-4 binds progelatinase A and the COOH-terminal domain in a similar manner to TIMP-2

The binding properties of the newly described tissue inhibitor of metalloproteinases-4 (TIMP-4) to progelatinase A and to the COOH-terminal hemopexin-like domain (C domain) of the enzyme were examined. We present evidence for the first time of a specific, high affinity interaction between TIMP-4 and the C domain of human gelatinase A and show that TIMP-4 binds both progelatinase A and the C domain in a similar manner to that of TIMP-2. Saturable binding of recombinant C domain to TIMP-4 and to TIMP-2 but not to TIMP-1 was demonstrated using a microwell protein binding assay. The recombinant collagen binding domain of gelatinase A, comprised of the three fibronectin type II-like repeats, did not bind to TIMP-4, indicating that binding is mediated selectively by the C domain. Binding to TIMP-4 was of high affinity with an apparent Kd of 1.7 x 10(-7) M but slightly weaker than that to TIMP-2 (apparent Kd of 0.66 x 10(-7) M). Affinity chromatography confirmed the TIMP-4-C domain interaction and also showed that the complex could not be disrupted by 1 M NaCl or 10% dimethyl sulfoxide, thereby further demonstrating the tight binding. To verify the biological significance of this interaction, binding of full-length progelatinase A to TIMP-4 was investigated. TIMP-4 and TIMP-2 but not TIMP-1 bound specifically to purified TIMP-2-free human recombinant full-length progelatinase A and to full-length rat proenzyme from the conditioned culture medium of ROS 17/2.8 cells. Preincubation of the C domain with TIMP-2 was found to reduce subsequent binding to TIMP-4 in a concentration-dependent manner. Competition between TIMP-2 and TIMP-4 for a common or overlapping binding sites on the gelatinase A C domain may occur; alternatively TIMP-2 may prevent the binding of TIMP-4 by steric hindrance or induction of a conformational change in the C domain. We propose that the binding of progelatinase A to TIMP-4 represents a third TIMP-progelatinase interaction in addition to that of progelatinase A with TIMP-2 and progelatinase B with TIMP-1 described previously. This new phenomenon may be of important physiological significance in modulating the cell surface activation of progelatinase A.     

High affinity binding of latent matrix metalloproteinase-9 to the alpha2(IV) chain of collagen IV

Association of matrix metalloproteinases (MMPs) with the cell surface and with areas of cell-matrix contacts is critical for extracellular matrix degradation. Previously, we showed the surface association of pro-MMP-9 in human breast epithelial MCF10A cells. Here, we have characterized the binding parameters of pro-MMP-9 and show that the enzyme binds with high affinity (Kd approximately 22 nM) to MCF10A cells and other cell lines. Binding of pro-MMP-9 to MCF10A cells does not result in zymogen activation and is not followed by ligand internalization, even after complex formation with tissue inhibitor of metalloproteinase-1 (TIMP-1). A 190-kDa cell surface protein was identified by ligand blot analysis and affinity purification with immobilized pro-MMP-9. Microsequencing and immunoblot analysis revealed that the 190-kDa protein is the alpha2(IV) chain of collagen IV. Specific pro-MMP-9 surface binding was competed with purified alpha2(IV) and was significantly reduced after treatment of the cells with active MMP-9 before the binding assay since alpha2(IV) is hydrolyzed by MMP-9. A pro-MMP-9.TIMP-1 complex and MMP-9 bind to alpha2(IV), suggesting that neither the C-terminal nor the N-terminal domain of the enzyme is directly involved in alpha2(IV) binding. The closely related pro-MMP-2 exhibits a weaker affinity for alpha2(IV) compared with that of pro-MMP-9, suggesting that sites other than the gelatin-binding domain may be involved in the binding of alpha2(IV) to pro-MMP-9. Although pro-MMP-9 forms a complex with alpha2(IV), the proenzyme does not bind to triple-helical collagen IV. These studies suggest a unique interaction between pro-MMP-9 and alpha2(IV) that may play a role in targeting the zymogen to cell-matrix contacts and in the degradation of the collagen IV network.     

Plasma membrane-bound tissue inhibitor of metalloproteinases (TIMP)-2 specifically inhibits matrix metalloproteinase 2 (gelatinase A) activated on the cell surface

The cell-surface activation of pro-matrix metalloproteinase 2 (pro-MMP-2) is considered to be critical for cell migration and invasion. Treatment of human uterine cervical fibroblasts with concanavalin A activates pro-MMP-2 on the cell surface by converting it to the 65-kDa form with a minor form of 45 kDa. However, the 65-kDa MMP-2 was inactivated by tissue inhibitor of metalloproteinases (TIMP)-2 that was bound to the plasma membrane upon concanavalin A treatment. TIMP-2 binds to the plasma membrane through its N-terminal domain by two different modes of interaction as follows: one is sensitive to a hydroxamate (HXM) inhibitor of MMPs and the other is HXM-insensitive. TIMP-2 bound to the membrane in a HXM-insensitive manner, comprising about 40-50% of TIMP-2 on the membrane, is the inhibitor of the cell surface-activated MMP-2. It, however, does not inhibit MMP-3, MMP-9, and the 45-kDa MMP-2 lacking the C-terminal domain. The inhibition of the 65-kDa MMP-2 by TIMP-2 is initiated by the interaction of their C-terminal domains. Subsequently, the MMP-2.TIMP-2 complex is released from the membrane, and the activity of MMP-2 is blocked by TIMP-2. In the presence of collagen types I, II, III, V, or gelatin, the rate of inhibition of the 65-kDa MMP-2 by the membrane-bound TIMP-2 decreased considerably. These results suggest that the pericellular activity of MMP-2 is tightly regulated by membrane-bound TIMP-2 and surrounding extracellular matrix components.     

Identification of the tissue inhibitor of metalloproteinases-2 (TIMP-2) binding site on the hemopexin carboxyl domain of human gelatinase A by site-directed mutagenesis. The hierarchical role in binding TIMP-2 of the unique cationic clusters of hemopexin modules III and IV

Cell surface activation of progelatinase A occurs in a quaternary complex with the tissue inhibitor of metalloproteinases-2 (TIMP-2) and two membrane-type matrix metalloproteinases. We have mutated the unique cationic clusters found in hemopexin modules III and IV of the carboxyl domain (C domain) of human gelatinase A to determine their role in binding TIMP-2. Twelve single, double, and triple site-directed mutations were produced that exhibited different TIMP-2 binding properties. Notably, single alanine substitutions at Lys547 and Lys617 reduced TIMP-2 binding by an order of magnitude from that of the recombinant wild-type C domain. Mutations that completely disrupted the C domain.TIMP-2 interaction were K558A/R561A, K610T/K617A, and K566A/K568A/K617A. A triple mutation, K566A/K568A/K575A, having TIMP-2 binding indistinguishable from the wild-type C domain (Kd 3.0 x 10(-8) M), showed that simple reduction of net positive charge does not reduce TIMP-2 affinity. Because the double mutation K566A/K568A also did not alter TIMP-2 binding, these data do not confirm previously reported chimera studies that indicated the importance of the triple lysine cluster at positions 566/567/568 in TIMP-2 binding. Nonetheless, a subtle role in TIMP-2 interaction for the 566/567/568-lysine triad is indicated from the enhanced reduction in TIMP-2 binding that occurs when mutations here were combined with K617A. Thus, these analyses indicate that the TIMP-2 binding surface lies at the junction of hemopexin modules III and IV on the peripheral rim of the gelatinase A C domain. This location implies that considerable molecular movement of the TIMP-2. C domain complex would be needed for the bound TIMP-2 to inhibit in cis the gelatinase A active site.     

Localization of the functional domains of human tissue inhibitor of metalloproteinases-3 and the effects of a Sorsby's fundus dystrophy mutation

A transient COS-7 cell expression system was used to investigate the functional domain arrangement of tissue inhibitor of metalloproteinases-3 (TIMP-3), specifically to assess the contribution of the amino- and carboxyl-terminal domains of the molecule to its matrix metalloproteinase (MMP) inhibitory and extracellular matrix (ECM) binding properties. Wild type TIMP-3 was entirely localized to the ECM in both its glycosylated (27 kDa) and unglycosylated (24 kDa) forms. A COOH-terminally truncated TIMP-3 molecule was found to be a non-ECM bound MMP inhibitor, whereas a chimeric TIMP molecule, consisting of the NH2-terminal domain of TIMP-2 fused to the COOH-terminal domain of TIMP-3, displayed ECM binding, albeit with a lower affinity than the wild type TIMP-3 molecule. Thus the functional domain arrangement of TIMP-3 is analogous to that seen in TIMP-1 and -2, namely that the NH2-terminal domain is responsible for MMP inhibition whereas the COOH-terminal domain is most important in mediating the specific functions of the molecule. A mutant TIMP-3 in which serine 181 was changed to a cysteine, found in Sorsby's fundus dystrophy, a hereditary macular degenerative disease, was also expressed in COS-7 cells. This gave rise to an additional 48-kDa species (possibly a TIMP-3 dimer) that retained its ability to inhibit MMPs and localize to the ECM. These data favor the hypothesis that the TIMP-3 mutations seen in Sorsby's fundus dystrophy contribute to disease progression by accumulation of mutant protein rather than by the loss of functional TIMP-3.     

Crystal structure of the complex formed by the membrane type 1-matrix metalloproteinase with the tissue inhibitor of metalloproteinases-2, the soluble progelatinase A receptor

The proteolytic activity of matrix metalloproteinases (MMPs) towards extracellular matrix components is held in check by the tissue inhibitors of metalloproteinases (TIMPs). The binary complex of TIMP-2 and membrane-type-1 MMP (MT1-MMP) forms a cell surface located 'receptor' involved in pro-MMP-2 activation. We have solved the 2.75 A crystal structure of the complex between the catalytic domain of human MT1-MMP (cdMT1-MMP) and bovine TIMP-2. In comparison with our previously determined MMP-3-TIMP-1 complex, both proteins are considerably tilted to one another and show new features. CdMT1-MMP, apart from exhibiting the classical MMP fold, displays two large insertions remote from the active-site cleft that might be important for interaction with macromolecular substrates. The TIMP-2 polypeptide chain, as in TIMP-1, folds into a continuous wedge; the A-B edge loop is much more elongated and tilted, however, wrapping around the S-loop and the beta-sheet rim of the MT1-MMP. In addition, both C-terminal edge loops make more interactions with the target enzyme. The C-terminal acidic tail of TIMP-2 is disordered but might adopt a defined structure upon binding to pro-MMP-2; the Ser2 side-chain of TIMP-2 extends into the voluminous S1' specificity pocket of cdMT1-MMP, with its Ogamma pointing towards the carboxylate of the catalytic Glu240. The lower affinity of TIMP-1 for MT1-MMP compared with TIMP-2 might be explained by a reduced number of favourable interactions.     

Three-dimensional structure of human tissue inhibitor of metalloproteinases-2 at 2.1 A resolution

The three-dimensional structure of human tissue inhibitor of metalloproteinases-2 (TIMP-2) was determined by X-ray crystallography to 2.1 A resolution. The structure of the inhibitor consists of two domains. The N-terminal domain (residues 1-110) is folded into a beta-barrel, similar to the oligonucleotide/oligosaccharide binding fold otherwise found in certain DNA-binding proteins. The C-terminal domain (residues 111-194) contains a parallel stranded beta-hairpin plus a beta-loop-beta motif. Comparison of the structure of uncomplexed human TIMP-2 with that of bovine TIMP-2 bound to the catalytic domain of human MMP-14 suggests an internal rotation between the two domains of approximately 13 degrees upon binding to the protease. Furthermore, local conformational differences in the two structures that might be induced by formation of the protease-inhibitor complex have been found. The most prominent of these involves residues 27-40 of the A-B beta-hairpin loop. Structure-based alignment of amino acid sequences of representatives of the TIMP family maps the sequence differences mainly to loop regions, and some of these differences are proposed to be responsible for the particular properties of the various TIMP species.     

The propeptide domain of membrane type 1 matrix metalloproteinase is required for binding of tissue inhibitor of metalloproteinases and for activation of pro-gelatinase A

Activation of secreted latent matrix metalloproteinases (MMPs) is accompanied by cleavage of the N-terminal propeptide, thereby liberating the active zinc from binding to the conserved cysteine in the pro-domain. It has been assumed that an analogous mechanism is responsible for the activation of membrane type 1 MMP (MT1-MMP). Using recombinant wild-type MT1-MMP cDNA and mutant cDNAs transfected into COS-1 cells lacking endogenous MT1-MMP, we have examined the function of the propeptide domain of MT1-MMP. MT1-MMP was characterized by immunoblotting, surface biotinylation, gelatin substrate zymography, and 125I-tissue inhibitor of metalloproteinases 2 (TIMP-2) binding. In contrast to wild-type MT1-MMP-transfected COS-1 cells, transfected COS-1 cells containing a deletion of the N-terminal propeptide domain of MT1-MMP or a chimeric construction (substitution of the pro-domain of MT1-MMP with that of collagenase 3) were functionally inactive in terms of binding of 125I-labeled TIMP-2 to the cell surface and initiating the activation of pro-gelatinase A. These results support the concept that in its native plasma membrane-inserted form, the pro-domain of MT1-MMP plays an essential role in TIMP-2 binding and subsequent activation of pro-gelatinase A.     

The b and delta subunits of the Escherichia coli ATP synthase interact via residues in their C-terminal regions

An affinity resin for the F1 sector of the Escherichia coli ATP synthase was prepared by coupling the b subunit to a solid support through a unique cysteine residue in the N-terminal leader. b24-156, a form of b lacking the N-terminal transmembrane domain, was able to compete with the affinity resin for binding of F1. Truncated forms of b24-156, in which one or four residues from the C terminus were removed, competed poorly for F1 binding, suggesting that these residues play an important role in b-F1 interactions. Sedimentation velocity analytical ultracentrifugation revealed that removal of these C-terminal residues from b24-156 resulted in a disruption of its association with the purified delta subunit of the enzyme. To determine whether these residues interact directly with delta, cysteine residues were introduced at various C-terminal positions of b and modified with the heterobifunctional cross-linker benzophenone-4-maleimide. Cross-links between b and delta were obtained when the reagent was incorporated at positions 155 and 158 (two residues beyond the normal C terminus) in both the reconstituted b24-156-F1 complex and the membrane-bound F1F0 complex. CNBr digestion followed by peptide sequencing showed the site of cross-linking within the 177-residue delta subunit to be C-terminal to residue 148, possibly at Met-158. These results indicate that the b and delta subunits interact via their C-terminal regions and that this interaction is instrumental in the binding of the F1 sector to the b subunit of F0.     

The Co-crystal structure of staphylococcal enterotoxin type A with Zn2+ at 2.7 A resolution. Implications for major histocompatibility complex class II binding

Superantigens form complexes with major histocompatibility complex (MHC) class II molecules and T-cell receptors resulting in extremely strong immunostimulatory properties. Staphylococcus aureus enterotoxin A (SEA) belongs to a subgroup of the staphylococcal superantigens that utilizes Zn2+ in the high affinity interaction with MHC class II molecules. A high affinity metal binding site was described previously in SEA co-crystallized with Cd2+ in which the metal ion was octahedrally co-ordinated, involving the N-terminal serine. We have now co-crystallized SEA with its native co-factor Zn2+ and determined its crystal structure at 2.7 A resolution. As expected for a Zn2+ ion, the co-ordination was found to be tetrahedral. Three of the ligands are located on the SEA surface on a C-terminal domain beta-sheet, while the fourth varies with the conditions. Further analysis of the zinc binding event was performed using titration microcalorimetry, which showed that SEA binds Zn2+ with an affinity of KD = 0.3 microM in an entropy driven process. The differential Zn2+ co-ordination observed here has implications for the mechanism of the SEA-MHC class II interaction.     

The C-terminal subdomain makes an important contribution to the DNA binding activity of the Pax-3 paired domain

The recognition of DNA targets by Pax-3 is achieved through the coordinate use of two distinct helix-turn-helix-based DNA-binding modules: a paired domain, composed of two structurally independent subdomains joined by a short linker, and a paired-type homeodomain. In mouse, the activity of the Pax-3 paired domain is modulated by an alternative splicing event in the paired domain linker region that generates isoforms (Q+ and Q-) with distinct C-terminal subdomain-mediated DNA-binding properties. In this study, we have used derivatives of a classical high affinity paired domain binding site (CD19-2/A) to derive an improved consensus recognition sequence for the Pax-3 C-terminal subdomain. This new consensus differs at six out of eight positions from the C-terminal subdomain recognition motif present in the parent CD19-2/A sequence, and includes a 5'-TT-3' dinucleotide at base pairs 15 and 16 that promotes high affinity binding by both Pax-3 isoforms. However, with a less favorable guanine at position 15, only the Q- isoform retains high affinity binding to this sequence, suggesting that this alternative splicing event might serve to stabilize binding to suboptimal recognition sequences. Finally, mutagenic analysis of the linker demonstrates that both the sequence and the spacing in this region contribute to the enhanced DNA-binding properties of the Pax-3/Q- isoform. Altogether, our studies establish a clear role for the Pax-3 C-terminal subdomain in DNA recognition and, thus, provide insights into an important mechanism by which Pax proteins achieve distinct target specificities.     

Expression of a novel form of p21Cip1/Waf1 in UV-irradiated and transformed cells

The tumor suppressor p53 and its target the CDK inhibitor p21 (Cip1/Waf1) are key components of the cellular response to DNA damage. Insight into how p21 is regulated in normal cells, and how it may be deregulated in tumor cells is important for the understanding of tumorigenesis. p21 was induced in normal human diploid fibroblasts after UV irradiation-induced DNA damage, but, at a high dose of UV irradiation, a faster mobility form of p21 on SDS-PAGE (designated p21delta) was expressed. Surprisingly, in a variety of growing transformed cell lines, the level of p21 was low but p21delta was prominent. We found that p21delta appeared to be derived through a loss of around 10 amino acids from the C-terminus of p21, which theoretically would remove the PCNA binding domain, a second cyclin binding domain and the nuclear localization signal sequence. Several characteristics distinguish p21 from p21delta. Both the full length p21 and p21delta could be stabilized by a proteasome inhibitor, but only the full length p21 was associated with Cdk2 and PCNA. Consistent with this, gel filtration chromatography revealed that all the full length p21 in the cell was complexed to other proteins, whereas a significant portion of p21delta was in monomeric form. Moreover, p21 was mainly localized to the nucleus, but p21delta was mainly localized to the cytoplasm. We propose that the decrease in p21 and increase in p21delta could contribute to the deregulation of the cell cycle, and could be a mechanism involved in cellular transformation.     

Cloning and deletion mutagenesis of the alpha2 delta calcium channel subunit from porcine cerebral cortex. Expression of a soluble form of the protein that retains [3H]gabapentin binding activity

The anti-epileptic, anti-hyperalgesic, and anxiolytic agent gabapentin (1-(aminomethyl)-cyclohexane acetic acid or Neurontin) has previously been shown to bind with high affinity to the alpha2delta subunit of voltage-dependent calcium channels (Gee, N. S. , Brown, J. P., Dissanayake, V. U. K., Offord, J., Thurlow, R., and Woodruff, G.N. (1996) J. Biol. Chem. 271, 5768-5776). We report here the cloning, sequencing, and deletion mutagenesis of the alpha2delta subunit from porcine brain. The deduced protein sequence has a 95.9 and 98.2% identity to the rat and human neuronal alpha2 delta sequences, respectively. [3H]Gabapentin binds with a KD of 37.5 +/- 10.4 nM to membranes prepared from COS-7 cells transfected with wild-type porcine alpha2 delta cDNA. Six deletion mutants (B-G) that lack the delta polypeptide, together with varying amounts of the alpha2 component, failed to bind [3H]gabapentin. C-terminal deletion mutagenesis of the delta polypeptide identified a segment (residues 960-994) required for correct assembly of the [3H]gabapentin binding pocket. Mutant L, which lacks the putative membrane anchor in the delta sequence, was found in both membrane-associated and soluble secreted forms. The soluble form was not proteolytically cleaved into separate alpha2 and delta chains but still retained a high affinity (KD = 30.7 +/- 8.1 nM) for [3H]gabapentin. The production of a soluble alpha2delta mutant supports the single transmembrane model of the alpha2 delta subunit and is an important step toward the large-scale recombinant expression of the protein.     

Rational design for the development of epidermal growth factor receptor antagonists

Epidermal growth factor (EGF) and transforming growth factor-alpha (TGF alpha) bind with similar high affinity to the human EGF receptor. Using a domain-exchange strategy we have shown that the C-terminal linear region of these molecules is involved in high affinity receptor binding. By further single amino acid substitution in this linear C-terminal region, a putative interaction site of these ligands with their receptor has been identified. This identification of a receptor binding domain in EGF/TGF alpha provides an important initial step in the development of EGF receptor antagonists with significant clinical potential.     

High resolution structure of the N-terminal domain of tissue inhibitor of metalloproteinases-2 and characterization of its interaction site with matrix metalloproteinase-3

The high resolution structure of the N-terminal domain of tissue inhibitor of metalloproteinases-2 (N-TIMP-2) in solution has been determined using multidimensional heteronuclear NMR spectroscopy, with the structural calculations based on an extensive set of constraints, including 3132 nuclear Overhauser effect-based distance constraints, 56 hydrogen bond constraints, and 220 torsion angle constraints (an average of 26.9 constraints/residue). The core of the protein consists of a five-stranded beta-barrel that is homologous to the beta-barrel found in the oligosaccharide/oligonucleotide binding protein fold. The binding site for the catalytic domain of matrix metalloproteinases-3 (N-MMP-3) on N-TIMP-2 has been mapped by determining the changes in chemical shifts on complex formation for signals from the protein backbone (15N, 13C, and 1H). This approach identified a discrete N-MMP-3 binding site on N-TIMP-2 composed of the N terminus of the protein and the loops between beta-strands AB, CD, and EF. The beta-hairpin formed from strands A and B in N-TIMP-2 is significantly longer than the equivalent structure in TIMP-1, allowing it to make more extensive binding interactions with the MMP catalytic domain. A detailed comparison of the N-TIMP-2 structure with that of TIMP-1 bound to N-MMP-3 (Gomis-Ruth, F.-X., Maskos, K., Betz, M., Bergner, A., Huber, R., Suzuki, K., Yoshida, N., Nagase, H. , Brew, K., Bourne, G. P., Bartunik, H. & Bode, W. (1997) Nature 389, 77-80) revealed that the core beta-barrels are very similar in topology but that the loop connecting beta-strands CD (P67-C72) would need to undergo a large conformational change for TIMP-2 to bind in a similar manner to TIMP-1.     

The solution structure of a fungal AREA protein-DNA complex: an alternative binding mode for the basic carboxyl tail of GATA factors

The solution structure of a complex between the DNA binding domain of a fungal GATA factor and a 13 base-pair oligonucleotide containing its physiologically relevant CGATAG target sequence has been determined by multidimensional nuclear magnetic resonance spectroscopy. The AREA DNA binding domain, from Aspergillus nidulans, possesses a single Cys2-Cys2 zinc finger module and a basic C-terminal tail, which recognize the CGATAG element via an extensive network of hydrophobic interactions with the bases in the major groove and numerous non-specific contacts along the sugar-phosphate backbone. The zinc finger core of the AREA DNA binding domain has the same global fold as that of the C-terminal DNA binding domain of chicken GATA-1. In contrast to the complex with the DNA binding domain of GATA-1 in which the basic C-terminal tail wraps around the DNA and lies in the minor groove, the structure of complex with the AREA DNA binding domain reveals that the C-terminal tail of the fungal domain runs parallel with the sugar phosphate backbone along the edge of the minor groove. This difference is principally attributed to amino acid substitutions at two positions of the AREA DNA binding domain (Val55, Asn62) relative to that of GATA-1 (Gly55, Lys62). The impact of the different C-terminal tail binding modes on the affinity and specificity of GATA factors is discussed.     

Grb2 forms an inducible protein complex with CD28 through a Src homology 3 domain-proline interaction

CD28 provides a costimulatory signal that results in optimal activation of T cells. The signal transduction pathways necessary for CD28-mediated costimulation are presently unknown. Engagement of CD28 leads to its tyrosine phosphorylation and subsequent binding to Src homology 2 (SH2)-containing proteins including the p85 subunit of phosphatidylinositol 3'-kinase (PI3K); however, the contribution of PI3K to CD28-dependent costimulation remains controversial. Here we show that CD28 is capable of binding the Src homology 3 (SH3) domains of several proteins, including Grb2. The interaction between Grb2 and CD28 is mediated by the binding of Grb2-SH3 domains to the C-terminal diproline motif present in the cytoplasmic domain of CD28. While the affinity of the C-terminal SH3 domain of Grb2 for CD28 is greater than that of the N-terminal SH3 domain, optimal binding requires both SH3 domains. Ligation of CD28, but not tyrosine-phosphorylation, is required for the SH3-mediated binding of Grb2 to CD28. We propose a model whereby the association of Grb2 with CD28 occurs via an inducible SH3-mediated interaction and leads to the recruitment of tyrosine-phosphorylated proteins such as p52(shc) bound to the SH2 domain of Grb2. The inducible interaction of Grb2 to the C-terminal region of CD28 may form the basis for PI3K-independent signaling through CD28.     

Mutation analysis of CD95 (APO-1/Fas) in childhood B-lineage acute lymphoblastic leukaemia

The delta-type phospholipase C (PLC) is thought to be evolutionally the most basal form in the mammalian PLC family. One of the delta-type isoforms, PLC-delta 1, binds to both phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) and inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) with a high affinity via its pleckstrin homology (PH) domain. We report here a missense mutation in the region encoding the C-terminal PH domain of the human PLC-delta 1. This is also the first report of a mutation in the human PLC genes. A single base substitution (G to A) causes the amino acid replacement, Arg105 to His. Site-directed mutagenesis of the glutathione-S-transferase (GST)/PLC-delta 1 fusion protein changing Arg105 to His resulted in a fourfold decrease in the affinity of specific Ins(1,4,5)P3 binding and a reduction in PtdIns(4,5)P2 hydrolysing activity to about 40% of that of the wild-type enzyme. This remarkable loss of function can be interpreted in terms of a conformational change in the PH domain.