E3 Interactions

After E2/ubiquitin thiol ester formation, the ubiquitin must be transferred to the substrate, which is sometimes another ubiquitin. An E3 is usually required for this reaction in vitro, and is always required in vivo. There are three known types of E3s the RING domain, HECT domain, and U-box (UED2 homology) families. RING and U-box E3s act as bridging factors for E2s and substrates, but HEGT E3s use a different mechanism, adding an extra step to the pathway (Section 5.6.3.3). 

The small RING domain coordinates two zinc ions in a cross-brace arrangement [104]. The domain is defined by the presence of eight zinc-binding groups (cysteines and histidines) with a conserved spacing, such that the distance between 

The closest approach of a RING-domain residue to the active-site cysteine of UbcH7 is about 15 A, arguing against a role for RING E3s in chemical catalysis [106]. Instead, RING E3s have been proposed to facilitate ubiquitination by inducing physical proximity of the E2/ubiquitin thiol ester and the substrate [23, 30, 106, 109]. Catalysis would result from the increased local concentrations of the two reactants (discussed further below). 

RING/E2 interactions have also been studied with BRCAl. This E3 is unique in that it must heterodimerize with a second RING-domain protein, BARDl, in order to display maximal E3 activity [110]. Even though the heterodimer interface leaves 

A model of the full SCF/E2 complex [112] shows that the end of Skp2 which binds the substrate is pointed toward the Rbxl-bound E2, with a 50-A gap between the two. Models based on two other SCF structures show similar distances between the F-box protein and the E2 [109, 115]. Whether this gap can be bridged by the bound substrate is currently unclear. It has been suggested that the E2 may bind to Rbxl somewhat differently than UbcH7 is observed to bind in the c-Cbl RING/ UbcH7 complex, but it is not obvious that this can lead to a 20 A movement of the E2 toward the bound substrate as suggested [109]. 

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One point that arises from study of these E2-E3 structures, is that there is a degree of specificity in their physical interactions. The biochemical data extends the idea of physical specificity in E2-E3 interactions to functional specificity. An example of this is the Brcal-Bardl heterodimer, which interacts physically with UbcH7 and UbcHSC, but is only active with UbcHSC. There appear to be two consequences of E2 specificity variability in the strength of a particular E3 response and variability in the type of ubiquitin modification. 

The surface of UbcH7 that contacts c-Cbl does not overlap with the E2 surface that contacts ubiquitin (Figures. 5.4 and 5.5 see also Ref [108]), confirming that the E2/ubiquitin thiol ester can associate with a RING E3. The E2 surface that contacts c-Cbl does, however, overlap the E2 surface implicated in E1/E2 interactions (Section 5.6.1). Thus, the El may have to depart from the E2/ubiquitin complex before E2/E3 interactions can take place. 

Xie Y, Varshavsky A (1999) The E2-E3 interaction in the N-end rule pathway the RING-H2 finger of E3 is required for the synthesis of multiubiquitin chain. EMBO J 18 6832-6844... 

Similarly, the regulation of PDK activity is modified in adult muscle PDC. For example, PDK activity is inhibited by pyruvate and propionate (metabolites elevated during anaerobic metabolism) and is less sensitive to stimulation by elevated NADH/NAD+ and acetyl CoA/CoA ratios (Fig. 14.2) (Thissen et al, 1986 Chen et al, 1998). The effects of NADH and acetyl CoA on PDK activity are mediated by the degree of E3-catalysed oxidation and E2-catalysed acetylation of the inner lipoyl domain of E2 (Roche and Cate, 1977 Rahmatullah and Roche, 1985, 1987 Ravindran et al, 1996 Yang et al, 1998), so that the regulation of this phenomenon is complex and involves multiple interacting components. 

We utilized this technique to analyze Scrapper gene-dehcient (SCR-KO) mice.21 SCRAPPER, a protein that we have recently reported, is localized at synapses in neurons. It is a ubiquitin E3 ligase that is involved in the decomposition of RIM (Rab3-interacting molecule) 1, an important regulator of synaptic plasticity, and thus regulates synaptic transmissions.22... 

H. Ge, UPA, a universal protein array system for quantitative detection of protein-protein, protein-DNA, protein-RNA and protein-ligand interactions. Nucl. Acids Res. 28, e3 (2000). 

Adsorption. This step depends on the possible interaction between molecules and the catalyst surface. When the reactants reach the active sites, they chemisorb on adjacent active sites. The chemisorption may be dissociative and the adjacent active sites may be of the same or different origin. The chemisorbed species react and the kinetics generally follow an exponential dependence on temperature, exp( EfRT), where E3 is the activation energy of chemisorption. 

Further characterization of the dendrimer-coupled protein complexes was studied by hydrophobic interaction chromatography carried out by purification over an octyl-Sepharose column. The products obtained by reaction of SIAB with ALP and its complex with third-generation dendrimer, ALP-E3, were... 

Fig. 4.1. Fundamentals of the ubiquitin system. Adapted from Ref [5]. Figure 4.1 shows the fundamentals of the ubiquitin system. (1) Ubiquitin is synthesized in linear chains or as the N-terminal fusion with small ribosomal subunits that are cleaved by de-ubiquitylating enzymes to form the active protein. Ubiquitin is then activated in an ATP-dependent manner by El where a thiolester linkage is formed. It is then transthiolated to the active-site cysteine of an E2. E2s interact with E3s and with substrates and mediate either the indirect (in the case of HECT E3s) or direct transfer of ubiquitin to substrate. A number of factors can affect this process. We know that interactions with Hsp70 can facilitate ubiquitylation in specific instances and competition for lysines on substrates with the processes of acetylation and sumoylation may be inhibitory in certain instances. (2) For efficient proteasomal targeting to occur chains of ubiquitin linked internally through K48 must be formed. This appears to involve multiple...

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