Despite the widespread importance of RING/U-box E3 ubiquitin ligases in ubiquitin (Ub) signaling, the mechanism by which this class of enzymes facilitates Ub transfer remains enigmatic. Here we present a structural model for a RING/U-box E3:E2~Ub complex poised for Ub transfer. The model and additional analyses reveal that E3 binding biases dynamic E2~Ub ensembles toward closed conformations with enhanced reactivity for substrate lysines. We identify a key hydrogen bond between a highly conserved E3 sidechain and an E2 backbone carbonyl, observed in all structures of active RING/U-Box E3/E2 pairs, as the linchpin for allosteric activation of E2~Ub. The conformational biasing mechanism is generalizable across diverse E2s and RING/U-box E3s, but is not shared by HECT-type E3s. The results provide a structural model for a RING/U-box E3:E2~Ub ligase complex and identify the long sought-after source of allostery for RING/U-Box activation of E2~Ub conjugates.

The structural basis for binding of the acidic transcription activator Gcn4 and one activator-binding domain of the Mediator subunit Gal11/Med15 was examined by NMR. Gal11 activator-binding domain 1 has a four-helix fold with a small shallow hydrophobic cleft at its center. In the bound complex, eight residues of Gcn4 adopt a helical conformation allowing three Gcn4 aromatic/aliphatic residues to insert into the Gal11 cleft. The protein-protein interface is dynamic and surprisingly simple, involving only hydrophobic interactions. This allows Gcn4 to bind Gal11 in multiple conformations and orientations, an example of a “fuzzy complex” where the Gcn4-Gal11 interface cannot be described by a single conformation. Gcn4 uses a similar mechanism to bind two other unrelated activator-binding domains. Functional studies in yeast show the importance of residues at the protein interface, define the minimal requirements for a functional activator, and suggest a mechanism by which activators bind to multiple unrelated targets.

The human epidermal growth factor receptor (HER) tyrosine kinases homo- and hetero-dimerize to activate downstream signaling pathways. HER3 is a catalytically impaired member of the HER family that contributes to the development of several human malignancies and is mutated in a subset of cancers. HER3 signaling depends on heterodimerization with a catalytically active partner, in particular EGFR (the founding family member, also known as HER1) or HER2. The activity of homodimeric complexes of catalytically active HER family members depends on allosteric activation between the two kinase domains. To determine the structural basis for HER3 signaling through heterodimerization with a catalytically active HER receptor, we solved the crystal structure of the heterodimeric complex formed by the isolated kinase domains of EGFR and HER3. The structure visualized HER3 as an allosteric activator of EGFR and revealed a conserved role of the allosteric mechanism in activation of HER family members through heterodimerization. To understand the effects of cancer-associated HER3 mutations at the molecular level, we solved the structures of two HER3 kinase mutants, each in a heterodimeric complex with the kinase domain of EGFR. These structures, combined with biochemical analysis and molecular dynamics simulations, indicated that the cancer-associated HER3 mutations enhanced the allosteric potential of HER3 by redesigning local interactions at the dimerization interface.

The human epidermal growth factor receptor 3 (HER3) is a receptor tyrosine kinase that lacks catalytic activity, but is essential for cellular homeostasis due to its ability to allosterically activate EGFR/HER2. Though catalytically inactive, HER3 binds ATP tightly, hinting at a possible role of the nucleotide-binding pocket in modulating HER3 function. We report a structure of the HER3 pseudokinase bound to the ATP-competitive inhibitor bosutinib. Previously solved structures show that bosutinib can potently interact with multiple kinase domain conformations. In complex with HER3, bosutinib binds to yet another conformation, which is nearly identical to that observed in the HER3/ATP complex. Interestingly, occupation of the ATP-binding site by bosutinib improves the ability of HER3 to act as an allosteric activator of EGFR in vitro by increasing the affinity of the HER3/EGFR heterodimer in a membrane-dependent manner.

New approaches are currently being developed to expose biochemistry and molecular biology undergraduates to a more interactive learning environment. Here, we propose a unique project-based laboratory module, which incorporates exposure to biophysical chemistry approaches to address problems in protein chemistry. Each of the experiments described herein contributes to the stepwise process of isolating, identifying, and analyzing a protein involved in a central biological process, prokaryotic translation. Students are provided with expression plasmids that harbor an unknown translation factor, and it is their charge to complete a series of experiments that will allow them to develop hypotheses for discovering the identity of their unknown (from a list of potential candidates). Subsequent to the identification of their unknown translation factor, a series of protein unfolding exercises are performed employing circular dichroism and fluorescence spectroscopies, allowing students to directly calculate thermodynamic parameters centered around determining the equilibrium constant for unfolding as a function of denaturant (temperature or chemical). The conclusion of this multi-part laboratory exercise consists of both oral and written presentations, emphasizing synthesis of the roles of each translation factor during the stepwise process of translation.