Kathryn R. Ely

Crystal structure of the tenth type III cell adhesion module of human fibronectin.

The crystal structure of the cell adhesion module of fibronectin (FNIII10) has been determined at 1.8 A resolution. A recombinant fragment corresponding to the tenth type III module of human fibronectin was crystallized in space group P2(1) with a = 30.7, b = 35.1 and c = 37.7 A and beta = 107 degrees. The structure was determined by molecular replacement and refined by least squares methods. The crystallographic R-factor for the final model of the 91 amino acid module plus 56 solvent atoms is 0.18 for 10 to 1.8 A data. The module consists of two layers of beta-sheet, one with three antiparallel strands and the other with four antiparallel strands. The beta-sheets enclose a hydrophobic core of 24 amino acid side-chains. The module contains the RGD cell recognition sequence in a flexible loop connecting two beta-strands. The tertiary structure of the FNIII10 module has been used to develop a structure-based sequence alignment of 17 type III modules in fibronectin based on the striking conservation of homologous hydrophobic residues. A similar pattern of homologous alternating hydrophobic residues is also evident in a comparison of type III modules in proteins unrelated to fibronectin such as cytokine receptors and muscle proteins.

Differential Requirements for Tumor Necrosis Factor Receptor-associated Factor Family Proteins in CD40-mediated Induction of NF-κB and Jun N-terminal Kinase Activation

CD40 is a member of the tumor necrosis factor receptor family that mediates a number of important signaling events in B-lymphocytes and some other types of cells through interaction of its cytoplasmic (ct) domain with tumor necrosis factor receptor-associated factor (TRAF) proteins. Alanine substitution and truncation mutants of the human CD40ct domain were generated, revealing residues critical for binding TRAF2, TRAF3, or both of these proteins. In contrast to TRAF2 and TRAF3, direct binding of TRAF1, TRAF4, TRAF5, or TRAF6 to CD40 was not detected. However, TRAF5 could be recruited to wild-type CD40 in a TRAF3-dependent manner but not to a CD40 mutant (Q263A) that selectively fails to bind TRAF3. CD40 mutants with impaired binding to TRAF2, TRAF3, or both of these proteins completely retained the ability to activate NF-κB and Jun N-terminal kinase (JNK), implying that CD40 can stimulate TRAF2- and TRAF3-independent pathways for NF-κB and JNK activation. A carboxyl-truncation mutant of CD40 lacking the last 32 amino acids required for TRAF2 and TRAF3 binding, CD40(Δ32), mediated NF-κB induction through a mechanism that was suppressible by co-expression of TRAF6(ΔN), a dominant-negative version of TRAF6, but not by TRAF2(ΔN), implying that while TRAF6 does not directly bind CD40, it can participate in CD40 signaling. In contrast, TRAF6(ΔN) did not impair JNK activation by CD40(Δ32). Taken together, these findings reveal redundancy in the involvement of TRAF family proteins in CD40-mediated NF-κB induction and suggest that the membrane-proximal region of CD40 may stimulate the JNK pathway through a TRAF-independent mechanism.

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 β-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 β-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.

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-κB 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-κB. 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 162PVPAT166 in BAFF-R is accommodated in the same binding crevice on TRAF3 that binds two related TNFRs, CD40 and LTβR, 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 Gln379 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 Tyr377 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.

Crystal structure of the MS2 coat protein dimer: implications for RNA binding and virus assembly.

BACKGROUND The coat protein in RNA bacteriophages binds and encapsidates viral RNA, and also acts as translational repressor of viral replicase by binding to an RNA hairpin in the RNA genome. Because of its dual function, the MS2 coat protein is an interesting candidate for structural studies of protein-RNA interactions and protein-protein interactions. In this study, unassembled MS2 coat protein dimers were selected to analyze repressor activity and virus assembly. RESULTS The crystal structure of a mutant MS2 coat protein that is defective in viral assembly yet retains repressor activity has been determined at 2.0 A resolution. The unassembled dimer is stabilized by interdigitation of alpha-helices, and the formation of a 10-stranded antiparallel beta-sheet across the interface between monomers. The substitution of arginine for tryptophan at residue 82 results in the formation of two new inter-subunit hydrogen bonds that further stabilize the dimer. Residues that influence RNA recognition, identified by molecular genetics, were located across the beta-sheet. Two of these residues (Tyr85 and Asn87) are displaced in the unliganded dimer and are located in the same beta-strand as the Trp-->Arg mutation. CONCLUSIONS When compared with the structure of the coat protein in the assembled virus, differences in orientation of residues 85 and 87 suggest conformational adjustment on binding RNA in the first step of viral assembly. The substitution at residue 82 may affect virus assembly by imposing conformational restriction on the loop that makes critical inter-subunit contacts in the capsid.

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.

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.

Structural Analysis of Siah1 and Its Interactions with Siah-interacting Protein (SIP)

Seven inabsentia 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 β-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 SIPin 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.

Degrading liaisons: Siah structure revealed

The structure of the substrate-binding domain of Siah provides insight into how this protein interacts with a diverse set of substrate proteins.

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.