Ubiquitination of proteins provides a powerful and versatile post-translational signal in the eukaryotic cell. The formation of a thioester bond between ubiquitin (Ub) and the active site of a ubiquitin-conjugating enzyme (E2) is critical for Ub transfer to substrates. Assembly of a functional ubiquitin ligase (E3) complex poised for Ub transfer involves recognition and binding of an E2~Ub conjugate. Therefore, full characterization of the structure and dynamics of E2~Ub conjugates is required for further mechanistic understanding of Ub transfer reactions. Here we present characterization of the dynamic behavior of E2~Ub conjugates of two human enzymes, UbcH5c~Ub and Ubc13~Ub, in solution as determined by NMR and SAXS. Within each conjugate, Ub retains great flexibility with respect to the E2, indicative of highly dynamic species that adopt manifold orientations. The population distribution of Ub conformations is dictated by the identity of the E2: UbcH5c~Ub populates an array of extended conformations and the population of Ubc13~Ub conjugates favors a closed conformation in which the hydrophobic surface of Ub faces Helix 2 of Ubc13. We propose that the varied conformations adopted by Ub represent available binding modes of the E2~Ub species and thus provide insight into the diverse E2~Ub protein interactome, particularly regarding interaction with Ub ligases. Keywords ubiquitin; ubiquitin conjugating enzyme; ubiquitination; UbcH5; Ubc13; NMR; spin label; SAXS Covalent attachment of the 8.6 kDa Ubiquitin (Ub) to target proteins is an essential step in eukaryotic signaling pathways. The type of Ub modification can vary, inducing distinct signals. For example, mono-ubiquitination may elicit a signal for protein transport while poly-ubiquitination (attachment of a chain of Ubs to the target) may mark a protein for proteasome-mediated degradation. Covalent attachment of Ub to a substrate proceeds through a multi-enzyme process consisting of a Ub-activating enzyme (E1), a Ubconjugating enzyme (E2), and a Ub ligase (E3) (1). The human genome contains two Ub E1s, ~35 E2s, and many hundreds of E3s (2). Despite the large number of E2s, they share significant similarity at both the sequence and structure levels. The E2 plays a central role in the ubiquitination cascade, shuttling Ub from an E1 to an E3/substrate complex. The E2 (6), and 3JW0 (7)). In each case, the backbone of the E2 and Ub moieties are not significantly altered from their free structures. However, each structure presents a unique relative orientation of the E2 and Ub units.Despite the wealth of atomic-level structural information on E2s, on E2/E3 complexes, and a growing number of E2~Ub conjugates, the ways in which E3s catalyze Ub transfer from an E2~Ub remain poorly understood. It is clear that one function of the E3 is to bind a substrate simultaneously with binding an E2~Ub, thereby bringing the two components into proximity. However, even in the absence of a protein substrate, E3s have been shown to enhance the rate at which Ub is released ...

Structural features of neurons create challenges for effective production and distribution of essential metabolic energy. We investigated how metabolic energy is distributed between cellular compartments in photoreceptors. In avascular retinas, aerobic production of energy occurs only in mitochondria that are located centrally within the photoreceptor. Our findings indicate that metabolic energy flows from these central mitochondria as phosphocreatine toward the photoreceptor's synaptic terminal in darkness. In light, it flows in the opposite direction as ATP toward the outer segment. Consistent with this model, inhibition of creatine kinase in avascular retinas blocks synaptic transmission without influencing outer segment activity. Our findings also reveal how vascularization of neuronal tissue can influence the strategies neurons use for energy management. In vascularized retinas, mitochondria in the synaptic terminals of photoreceptors make neurotransmission less dependent on creatine kinase. Thus, vasculature of the tissue and the intracellular distribution of mitochondria can play key roles in setting the strategy for energy distribution in neurons.energy metabolism | phototransduction A significant energy distribution problem can arise from the relative locations of mitochondria, ion pumps, and synapses in neurons. In photoreceptors, ion pumps occupy the intervening space between the centrally located mitochondria and the synaptic terminal. Ion pumping in dark-adapted photoreceptors consumes ∼20× more energy than neurotransmission (1). Therefore, the pumps could intercept all the metabolic energy made by the mitochondria before it can reach the synaptic terminal. In the vascularized retinas of mice, rats, and humans (2-4) this problem is solved by the presence of additional mitochondria in the terminal. However, in the avascular retinas of zebrafish, salamanders, rabbits, and guinea pigs there are no mitochondria in the terminals (2, 4, 5), which creates a need to partition some of the energy made by the central mitochondria into a protected form that can bypass the ion pumps to support the essential energy demands of the synaptic terminal.Energy consumption within retinal photoreceptors is compartmentalized and light-dependent. During illumination, phototransduction and light adaptation consume energy in the outer segment (OS). In darkness, energy is consumed by ion pumps in the inner segment and by glutamate release at the synaptic terminal (1). Energy demands and O 2 consumption are far greater in darkness than in light (1, 6-8).Metabolic energy is distributed in most cells as either ATP or phosphocreatine (PCr). There are 2 isoforms of creatine kinase (CK) in neurons, ubiquitous mitochondrial creatine kinase (uMtCK), and brain-type cytoplasmic creatine kinase (CK-B). uMtCK creates PCr from ATP at mitochondria (9), and CK-B can recreate ATP from PCr at sites of energy demand. In this way uMtCK and CK-B can collaborate to transfer metabolic energy between neuronal compartments (10, 11). This paper descr...