Kathryn R. Ely

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. Copyright © 2005 John Wiley & Sons, Ltd.

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.

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 Å structure reveals critical side chain movements in a hydrophobic pocket in the active site. Notably, the side chain of tyrosine204 is rotated by ∼90° 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. Copyright © 2003 John Wiley & Sons, Ltd.

Tumor necrosis factor (TNF) signaling is controlled by receptors and intracellular signaling pathways that activate the NF‐κB 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. Copyright © 2002 John Wiley & Sons, Ltd.

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).

Tumor necrosis factor receptors (TNFR) are single transmembrane-spanning glycoproteins that bind cytokines and trigger multiple signal transduction pathways. Many of these TNFRs rely on interactions with TRAF proteins that bind to the intracellular domain of the receptors. CD40 is a member of the TNFR family that binds to several different TRAF proteins. We have determined the crystal structure of a 20-residue fragment from the cytoplasmic domain of CD40 in complex with the TRAF domain of TRAF3. The CD40 fragment binds as a hairpin loop across the surface of the TRAF domain. Residues shown by mutagenesis and deletion analysis to be critical for TRAF3 binding are involved either in direct contact with TRAF3 or in intramolecular interactions that stabilize the hairpin. Comparison of the interactions of CD40 with TRAF3 vs. TRAF2 suggests that CD40 may assume different conformations when bound to different TRAF family members. Thismolecular adaptationmay influence binding affinity and specific cellular triggers.