Structural studies of the SET domain from RIZ1 tumor suppressor.

RIZ1 is a transcriptional regulator and tumor suppressor that catalyzes methylation of lysine 9 of histone H3. It contains a distinct SET domain, sometimes referred to as PR (PRDI-BF1 and RIZ1 homology) domain, that is responsible for its catalytic activity. We determined the solution structure of the PR domain from RIZ1 and characterized its interaction with S-adenosyl-l-homocysteine (SAH) and a peptide from histone H3. Despite low sequence identity with canonical SET domains, the PR domain displays a typical SET fold including a pseudo-knot at the C-terminus. The N-flanking sequence of RIZ1 PR domain adopts a novel conformation and interacts closely with the SET fold. The C-flanking sequence contains an alpha-helix that points away from the protein face that harbors active site in other SET domains. The SET fold of RIZ1 does not have detectable affinity for SAH but it interacts with a synthetic peptide comprising residues 1-20 of histone H3.

Protein-protein interactions in TRAF3.

TNF-receptor-associated factors (TRAFs) are intracellular proteins that bind to the cytoplasmic portion of TNF receptors and mediate downstream signaling. The six known TRAF proteins play overlapping yet distinct roles in controlling immune responses as well as cellular processes such as activation of NF-kappaB and JNK signaling pathways. For example, CD40 binds to TRAF2, TRAF3 and TRAF6 to control B cell differentiation, proliferation and growth. In contrast, binding of lymphotoxin-beta receptor (LTbetaR) to TRAF2 and TRAF5 propagates signals leading to activation of NF-kappaB, while binding to TRAF3 induces negative regulation of this pathway and leads to apoptosis in tumor cells. Binding recognition is mediated by specific contacts of a consensus recognition sequence in the partner with residues in a hydrophobic crevice on the TRAF molecule. Since each of these protein-protein interactions occurs within this same binding crevice, it appears that TRAF-mediated cellular mechanisms may be regulated, in part, by the level of expression or recruitment of the adaptor proteins or receptors that are competing for the crevice. The specific contacts of CD40, LTbetaR and BAFF-R have been defined in crystal structures of the complex with TRAF3. In addition, the downstream regulator TANK and the viral oncogenic protein LMP1 from the Epstein Barr virus also bind to the same TRAF crevice and these contacts have also been described crystallographically. Comparison of these five crystal structures has revealed that the recognition motifs in each of these proteins are accommodated in one TRAF3 binding crevice and that the binding interface is structurally and functionally adaptive. In this chapter, the molecular details of the interactions will be described and correlated with the functional implications for multiple TRAF3 roles in cellular regulation.

Molecular determinants for interaction of SHEP1 with Cas localize to a highly solvent-protected region in the complex.

Protein-protein interactions between SHEP and Cas proteins influence cellular signaling through tyrosine kinases, as well as integrin-mediated signaling, and may be linked to antiestrogen resistance. Data from past studies suggests that association between SHEP and Cas proteins is critical for these cellular effects. In this study, the interacting domains of each protein were co-expressed in bacteria and a soluble stable complex was purified. Deuterium exchange mass spectrometry was used to define regions that are buried when SHEP1 is in complex with Cas. The results reveal four segments in SHEP1 that are highly protected, including a region (residues 619-640) that contains a key residue, tyrosine 635, required for association with Cas. This region is predominately hydrophilic, yet remains protected from solvent in the complex.

Molecular basis for regulation of Src by the docking protein p130Cas.

The docking protein p130Cas (Cas) becomes tyrosine-phosphorylated in its central substrate domain in response to extracellular stimuli such as integrin-mediated cell adhesion, and transmits signals through interactions with various intracellular signaling molecules such as the adaptor protein Crk. Src-family kinases (SFKs) bind a specific site in the carboxyl-terminal region of Cas and subsequently SFKs phosphorylate progressively the substrate domain in Cas. In this study crystallography, mutagenesis and binding assays were used to understand the molecular basis for Cas interactions with SFKs. Tyrosine phosphorylation regulates binding of Cas to SFKs, and the primary site for this phosphorylation, Y762, has been proposed. A phosphorylated peptide corresponding to Cas residues 759MEDpYDYVHL767 containing the key phosphotyrosine was crystallized in complex with the SH3-SH2 domain of the SFK Lck. The results provide the first structural data for this protein-protein interaction. The motif in Cas 762pYDYV binds to the SH2 domain in a mode that mimics high-affinity ligands, involving dual contacts of Y762 and V765 with conserved residues in SFK SH2 domains. In addition, Y764 is in position to make an electrostatic contact after phosphorylation with a conserved SFK arginine that mediates interactions with other high-affinity SH2 binders. These new molecular data suggest that Cas may regulate activity of Src as a competing ligand to displace intramolecular interactions that occur in SFKs (between the C-terminal tail and the SH2 domain) and restrain and down-regulate the kinase in an inactive form.

Structural analysis of Siah1-Siah-interacting protein interactions and insights into the assembly of an E3 ligase multiprotein complex.

Siah1 is the central component of a multiprotein E3 ubiquitin ligase complex that targets beta-catenin for destruction in response to p53 activation. The E3 complex comprises, in addition to Siah1, Siah-interacting protein (SIP), the adaptor protein Skp1, and the F-box protein Ebi. Here we show that SIP engages Siah1 by means of two elements, both of which are required for mediating beta-catenin destruction in cells. An N-terminal dimerization domain of SIP sits across the saddle-shaped upper surface of Siah1, with two extended legs packing against the sides of Siah1 by means of a consensus PXAXVXP motif that is common to a family of Siah-binding proteins. The C-terminal domain of SIP, which binds to Skp1, protrudes from the lower surface of Siah1, and we propose that this surface provides the scaffold for bringing substrate and the E2 enzyme into apposition in the functional complex.

LMP1 protein from the Epstein-Barr virus is a structural CD40 decoy in B lymphocytes for binding to TRAF3.

Epstein-Barr virus is a human herpesvirus that causes infectious mononucleosis and lymphoproliferative malignancies. LMP1 (latent membrane protein-1), which is encoded by this virus and which is essential for transformation of B lymphocytes, acts as a constitutively active mimic of the tumor necrosis factor receptor (TNFR) CD40. LMP1 is an integral membrane protein containing six transmembrane segments and a cytoplasmic domain at the C terminus that binds to intracellular TNFR-associated factors (TRAFs). TRAFs are intracellular co-inducers of downstream signaling from CD40 and other TNFRs, and TRAF3 is required for activation of B lymphocytes by LMP1. Cytoplasmic C-terminal activation region 1 of LMP1 bears a motif (PQQAT) that conforms to the TRAF recognition motif PVQET in CD40. In this study, we report the crystal structure of this portion of LMP1 C-terminal activation region-1 (204PQQATDD210) bound in complex with TRAF3. The PQQAT motif is bound in the same binding crevice on TRAF3 where CD40 is bound, providing a molecular mechanism for LMP1 to act as a CD40 decoy for TRAF3. The LMP1 motif is presented in the TRAF3 crevice as a close structural mimic of the PVQET motif in CD40, and the intermolecular contacts are similar. However, the viral protein makes a unique contact: a hydrogen bond network formed between Asp210 in LMP1 and Tyr395 and Arg393 in TRAF3. This intermolecular contact is not made in the CD40-TRAF3 complex. The additional hydrogen bonds may stabilize the complex and strengthen the binding to permit LMP1 to compete with CD40 for binding to the TRAF3 crevice, influencing downstream signaling to B lymphocytes and contributing to dysregulated signaling by LMP1.

Characterization of the PR domain of RIZ1 histone methyltransferase.

RIZ1 (PRDM2) and PRDI-BF1 (PRDM1) are involved in B cell differentiation and the development of B cell lymphomas. These proteins are expressed in two forms that differ by the presence or absence of a PR domain. The protein product that retains the PR domain is anti-tumorigenic while the product that lacks the PR domain is oncogenic and over-expressed in tumor cells. The conserved PR domain is homologous to the SET domain from a family of histone methyltransferases. RIZ1 is also a histone methyltransferase and methylates lysine 9 in histone H3. This activity has been mapped to the PR domain. In the present study, deuterium exchange mass spectrometry was used to define the structural boundaries of the RIZ1 PR domain and to map sites of missense mutations that occur in human cancers and reduce methyltransferase activity. Flexible segments were selectively deleted to produce protein products that crystallize for structural studies. Segments at the carboxyl terminus of the PR domain that are involved in methylation of H3 were shown to be flexible, similar to SET domains, suggesting that the PR and SET methyltransferases may belong to an emerging class of proteins that contain mobile functional regions.

The serine-rich domain from Crk-associated substrate (p130cas) is a four-helix bundle.

p130(cas) (Crk-associated substrate) is a docking protein that is involved in assembly of focal adhesions and concomitant cellular signaling. It plays a role in physiological regulation of cell adhesion, migration, survival, and proliferation, as well as in oncogenic transformation. The molecule consists of multiple protein-protein interaction motifs, including a serine-rich region that is positioned between Crk and Src-binding sites. This study reports the first structure of a functional domain of Cas. The solution structure of the serine-rich region has been determined by NMR spectroscopy, demonstrating that this is a stable domain that folds as a four-helix bundle, a protein-interaction motif. The serine-rich region bears strong structural similarity to four-helix bundles found in other adhesion components like focal adhesion kinase, alpha-catenin, or vinculin. Potential sites for phosphorylation and interaction with the 14-3-3 family of cellular regulators are identified in the domain and characterized by site-directed mutagenesis and binding assays. Mapping the degree of amino acid conservation onto the molecular surface reveals a patch of invariant residues near the C terminus of the bundle, which may represent a previously unidentified site for protein interaction.

Crystallization of the SH2-binding site of p130Cas in complex with Lck, a Src-family kinase.

Cas-family proteins serve as docking proteins in integrin-mediated signal transduction. The founding member of this family, p130Cas, becomes tyrosine-phosphorylated in response to extracellular stimuli such as integrin-mediated cell adhesion and ligand engagement of receptor tyrosine kinases. Cas proteins are large multidomain molecules that transmit signals as intermediaries through interactions with signaling molecules such as FAK and other tyrosine kinases, as well as tyrosine phosphatases. After Cas is tyrosine-phosphorylated, it acts as a docking protein for binding SH2 domains of Src-family kinases. In order to examine the structural basis for a key step in propagation of signals by Cas, one of the major SH2-binding sites of Cas has been crystallized in complex with the SH3-SH2 regulatory domains of the Src-family kinase Lck. Crystallization conditions were identified by high-throughput screening and optimized with multiple rounds of seeding. The crystals formed at 295 K in space group P2(1)2(1)2(1), with unit-cell parameters a = 77.4, b = 107.3, c = 166.4 A, and diffract to 2.7 A resolution.

Key molecular contacts promote recognition of the BAFF receptor by TNF receptor-associated factor 3: implications for intracellular signaling regulation.

B cell-activating factor belonging to the TNF family receptor (BAFF-R), a member of the TNFR superfamily, plays a role in autoimmunity after ligation with BAFF ligand (also called TALL-1, BLyS, THANK, or zTNF4). BAFF/BAFF-R interactions are critical for B cell regulation, and signaling from this ligand-receptor complex results in NF-kappaB activation. Most TNFRs transmit signals intracellularly by recruitment of adaptor proteins called TNFR-associated factors (TRAFs). However, BAFF-R binds only one TRAF adaptor, TRAF3, and this interaction negatively regulates activation of NF-kappaB. In this study, we report the crystal structure of a 24-residue fragment of the cytoplasmic portion of BAFF-R bound in complex with TRAF3. The recognition motif (162)PVPAT(166) in BAFF-R is accommodated in the same binding crevice on TRAF3 that binds two related TNFRs, CD40 and LTbetaR, but is presented in a completely different structural framework. This region of BAFF-R assumes an open conformation with two extended strands opposed at right angles that each make contacts with TRAF3. The recognition motif is located in the N-terminal arm and intermolecular contacts mediate TRAF recognition. In the C-terminal arm, key stabilizing contacts are made, including critical hydrogen bonds with Gln(379) in TRAF3 that define the molecular basis for selective binding of BAFF-R solely to this member of the TRAF family. A dynamic conformational adjustment of Tyr(377) in TRAF3 occurs forming a new intermolecular contact with BAFF-R that stabilizes the complex. The structure of the complex provides a molecular explanation for binding affinities and selective protein interactions in TNFR-TRAF interactions.

Organization of functional domains in the docking protein p130Cas.

The docking protein p130Cas becomes phosphorylated upon cell adhesion to extracellular matrix proteins, and is thought to play an essential role in cell transformation. Cas transmits signals through interactions with the Src-homology 3 (SH3) and Src-homology 2 domains of FAK or v-Crk signaling molecules, or with 14-3-3 protein, as well as phosphatases PTP1B and PTP-PEST. The large (130kDa), multi-domain Cas molecule contains an SH3 domain, a Src-binding domain, a serine-rich protein interaction region, and a C-terminal region that participates in protein interactions implicated in antiestrogen resistance in breast cancer. In this study, as part of a long-term goal to examine the protein interactions of Cas by X-ray crystallography and nuclear magnetic resonance spectroscopy, molecular constructs were designed to express two adjacent domains, the serine-rich domain and the Src-binding domain, that each participate in intermolecular contacts dependent on protein phosphorylation. The protein products are soluble, homogeneous, monodisperse, and highly suitable for structural studies to define the role of Cas in integrin-mediated cell signaling.

Structurally distinct recognition motifs in lymphotoxin-beta receptor and CD40 for tumor necrosis factor receptor-associated factor (TRAF)-mediated signaling.

Lymphotoxin-beta receptor (LTbetaR) and CD40 are members of the tumor necrosis factor family of signaling receptors that regulate cell survival or death through activation of NF-kappaB. These receptors transmit signals through downstream adaptor proteins called tumor necrosis factor receptor-associated factors (TRAFs). In this study, the crystal structure of a region of the cytoplasmic domain of LTbetaR bound to TRAF3 has revealed an unexpected new recognition motif, 388IPEEGD393, for TRAF3 binding. Although this motif is distinct in sequence and structure from the PVQET motif in CD40 and PIQCT in the regulator TRAF-associated NF-kappaB activator (TANK), recognition is mediated in the same binding crevice on the surface of TRAF3. The results reveal structurally adaptive "hot spots" in the TRAF3-binding crevice that promote molecular interactions driving specific signaling after contact with LTbetaR, CD40, or the downstream regulator TANK.

Anastellin, an FN3 fragment with fibronectin polymerization activity, resembles amyloid fibril precursors.

Anastellin is a carboxy-terminal fragment of the first FN3 domain from human fibronectin. It is capable of polymerizing fibronectin in vitro, and it displays anti-tumor, anti-metastatic and anti-angiogenic properties in vivo. We have determined the structure of anastellin using nuclear magnetic resonance spectroscopy and identified residues critical for its activity. Anastellin exhibits dynamic fluctuations and conformational exchange in solution. Its overall topology is very similar to the corresponding region of full-length FN3 domains. However, its hydrophobic core becomes solvent-accessible and some of its beta-strands lose their protection against hydrogen bonding to beta-strands from other molecules. These features seem to be relevant for the fibronectin polymerization activity of anastellin and resemble the characteristics of amyloid fibril precursors. We suggest that this analogy is not random and may reflect similarities between fibronectin and amyloid fibril formation.

Conformational restrictions in the active site of unliganded human caspase-3.

Caspases are cysteine proteases that play a critical role in the initiation and regulation of apoptosis. These enzymes act in a cascade to promote cell death through proteolytic cleavage of intracellular proteins. Since activation of apoptosis is implicated in human diseases such as cancer and neurodegenerative disorders, caspases are targets for drugs designed to modulate their action. Active caspases are heterodimeric enzymes with two symmetrically arranged active sites at opposite ends of the molecule. A number of crystal structures of caspases with peptides or proteins bound at the active sites have defined the mechanism of action of these enzymes, but molecular information about the active sites before substrate engagement has been lacking. As part of a study of peptidyl inhibitors of caspase-3, we crystallized a complex where the inhibitor did not bind in the active site. Here we present the crystal structure of the unoccupied substrate-binding site of caspase-3. No large conformational differences were apparent when this site was compared with that in enzyme-inhibitor complexes. Instead, the 1.9 A structure reveals critical side chain movements in a hydrophobic pocket in the active site. Notably, the side chain of tyrosine204 is rotated by approximately 90 degrees so that the phenol group occupies the S2 subsite in the active site. Thus, binding of substrate or inhibitors is impeded unless rotation of this side chain opens the area. The positions of these side chains may have important implications for the directed design of inhibitors of caspase-3 or caspase-7.

Structural analysis of Siah1 and its interactions with Siah-interacting protein (SIP).

Seven in absentia homologue (Siah) family proteins bind ubiquitin-conjugating enzymes and target proteins for proteasome-mediated degradation. Recently we identified a novel Siah-interacting protein (SIP) that is a Sgt1-related molecule that provides a physical link between Siah family proteins and the Skp1-Cullin-F-box ubiquitin ligase component Skp1. In the present study, a structure-based approach was used to identify interacting residues in Siah that are required for association with SIP. In Siah1 a large concave surface is formed across the dimer interface. Analysis of the electrostatic surface potential of the Siah1 dimer reveals that the beta-sheet concavity is predominately electronegative, suggesting that the protein-protein interactions between Siah1 and SIP are mediated by ionic contacts. The structural prediction was confirmed by site-directed mutagenesis of these electronegative residues, resulting in loss of binding of Siah1 to SIP in vitro and in cells. The results also provide a structural basis for understanding the mechanism by which Siah family proteins interact with partner proteins such as SIP.

Structurally adaptive hot spots at a protein interaction interface on TRAF3.

Tumor necrosis factor (TNF) signaling is controlled by receptors and intracellular signaling pathways that activate the NF-kappaB transcription factor. The resulting signals elicit immune responses and have important implications for disorders such as autoimmunity or allergic reactions. TNF-receptor-associated factors (TRAFs) bind to the cytoplasmic portion of TNFRs as well as downstream regulators and thus are co-inducers of the signal transduction. TRAF3 binds to diverse receptors and regulators by accomodating a conserved motif that is embedded in completely different structural frameworks. Thus, the protein-protein contact region on TRAF3 represents a binding interface that is structurally and functionally adaptive. In this report, three 'hot spots' at the TRAF3 protein-interaction interface are defined that provide the principal contact regions for different binding partners. The side-chains of residues at these 'hot spots' are flexible and undergo movements on binding the different partners. These side chain rearrangements provide a structural adaptability that promotes interaction with a variety of distinct proteins. It is proposed that similar adaptive 'hot spots' are also present on the binding surfaces of TRAF1, TRAF2 and TRAF5.

Crystallization and preliminary X-ray analysis of the TRAF domain of TRAF3.

Tumor necrosis factor receptors (TNFR) signal events in immune responses, Ig class switching, activation of NF-kappaB or regulation of apoptosis. TNFR-associated factors (TRAFs) are adaptor proteins that connect TNFRs to downstream signaling pathways, including the NF-kappaB and c-JUN N-terminal kinase (JNK) pathways. Members of the TRAF family exist as trimers and share a conserved TRAF domain that mediates binding to the cytoplasmic domains of TNFRs. The TRAF domain from TRAF3 has been crystallized. In addition, an N-terminally truncated form of the domain has been crystallized in space group P321 with a shortened c axis and markedly improved diffraction (2.5 A resolution).

BAG4/SODD protein contains a short BAG domain.

BAG (Bcl-2-associated athanogene) proteins are molecular chaperone regulators that affect diverse cellular pathways. All members share a conserved motif, called the BAG domain (BD), which binds to Hsp70/Hsc70 family proteins and modulates their activity. We have determined the solution structure of BD from BAG4/SODD (silencer of death domains) by multidimensional nuclear magnetic resonance methods and compared it to the corresponding domain in BAG1 (Briknarová, K., Takayama, S., Brive, L., Havert, M. L., Knee, D. A., Velasco, J., Homma, S., Cabezas, E., Stuart, J., Hoyt, D. W., Satterthwait, A. C., Llinás, M., Reed, J. C., and Ely, K. R. (2001) Nat. Struct. Biol. 8, 349-352). The difference between BDs from these two BAG proteins is striking, and the structural comparison defines two subfamilies of mammalian BD-containing proteins. One subfamily includes the closely related BAG3, BAG4, and BAG5 proteins, and the other is represented by BAG1, which contains a structurally and evolutionarily distinct BD. BDs from both BAG1 and BAG4 are three-helix bundles; however, in BAG4, each helix in this bundle is three to four turns shorter than its counterpart in BAG1, which reduces the length of the domain by one-third. BAG4 BD thus represents a prototype of the minimal functional fragment that is capable of binding to Hsc70 and modulating its chaperone activity.

Downstream regulator TANK binds to the CD40 recognition site on TRAF3.

TRAFs (tumor necrosis factor receptor [TNFR]-associated factors) bind to the cytoplasmic portion of liganded TNFRs and stimulate activation of NF-kappaB or JNK pathways. A modulator of TRAF signaling, TANK, serves as either an enhancer or an inhibitor of TRAF-mediated signaling pathways. The crystal structure of a region of TANK bound to TRAF3 has been determined and compared to a similar CD40/TRAF3 complex. TANK and CD40 bind to the same crevice on TRAF3. The recognition motif PxQxT is presented in a boomerang-like structure in TANK that is markedly different from the hairpin loop that forms in CD40 upon binding to TRAF3. Critical TANK contact residues were confirmed by mutagenesis to be required for binding to TRAF3 or TRAF2. Binding affinity, measured by isothermal titration calorimetry and competition assays, demonstrated that TANK competes with CD40 for the TRAF binding site.

A glucocorticoid-responsive mutant androgen receptor exhibits unique ligand specificity: therapeutic implications for androgen-independent prostate cancer.

The cortisol/cortisone-responsive AR (AR(ccr)) has two mutations (L701H and T877A) that were found in the MDA PCa human prostate cancer cell lines established from a castrated patient whose metastatic tumor exhibited androgen-independent growth. Cortisol and cortisone bind to the AR(ccr) with high affinity. In the present study, we characterized the structural determinants for ligand binding to the AR(ccr). Our data revealed that many of the C17, C19, and C21 circulating steroids, at concentrations that are found in vivo, functioned as effective activators of the AR(ccr) but had little or no activity via the wild-type AR or GRalpha. Among the synthetic glucocorticoids tested, dexamethasone activated both GRalpha and AR(ccr), whereas triamcinolone was selective for GRalpha. In MDA PCa 2b cells, growth and prostate-specific antigen production were stimulated by potent AR(ccr) agonists such as cortisol or 9alpha-fluorocortisol but not by triamcinolone (which did not bind to or activate the AR(ccr)). Of the potential antagonists tested, bicalutamide (casodex) and GR antagonist RU38486 showed inhibitory activity. We postulate that corticosteroids provide a growth advantage to prostate cancer cells harboring the promiscuous AR(ccr) in androgen-ablated patients and contribute to their transition to androgen-independence. We predict that triamcinolone, a commonly prescribed glucocorticoid, would be a successful therapeutic agent for men with this form of cancer, perhaps in conjunction with the antagonist casodex. We hypothesize that triamcinolone administration would inhibit the hypothalamic-pituitary-adrenal axis, thus suppressing endogenous corticosteroids, which stimulate tumor growth. Triamcinolone, by itself, would not activate the AR(ccr) or promote tumor growth but would provide glucocorticoid activity essential for survival.