E3 CRL presents a mechanistic enigma: how is the large distance gap between E2 and the substrate binding protein bridged for the ubiquitin transfer from E2 to the substrate? When assessed based on the unbound form of the substrate binding proteins, the distances between the substrate and E2 fluctuate between 36~73 Å. However, when measured in the bound-to-the-adaptor form, the distance fluctuation shrinks to 41~61 Å. This large fluctuation in the distance derives from flexible linkers between the substrate binding domain and the box domain. In all available structures of the substrate binding proteins, F-box proteins Skp2, Fbw7, β-TrCP1, Cdc4, Fbs1, TIR1; VHL-box protein pVHL; and SOCS-box proteins SOCS2, and SOCS4, the flexible linkers rotate during the simulations by up to 30–80°. In particular, movements of the linker were observed to correlate allosterically with the binding sites to the adaptor and the substrate. Unfortunately, not all of these substrate binding proteins have available structures bound to their cognate substrate peptides. We performed molecular dynamics simulations for five substrate binding proteins, Skp2, Fbw7, β-TrCP1, Cdc4 and pVHL with available bound-to-substrate structures. The simulations were for two forms: the first is the bound-to-substrate form; the second, the bound-to-both form, with both the substrate and the adaptor bound to the different domains of the substrate binding protein. We compared these simulation results with our simulations for the unbound and bound-to-adaptor forms44
. Our goal is to elucidate the molecular mechanism of the ubiquitin transfer reaction, given the large distance gap between the E2-ubiquitin and the substrate binding domain. The large conformational change in the substrate binding protein (), leads us to suspect that the E3 CRL is under conformational control.
Figure 8 A scheme showing that the rotation of the substrate binding domain (SBD) shortens the distance gap between the substrate acceptor lysine and the Ub-E2 active site. The minimum (left) and maximum distances (right) between the tip of the substrate peptide (more ...)
Inspection of the distance gap in the bound-to-both form suggests that when the substrate binding protein binds both the adaptor protein and the substrate, the distance gap between the substrate binding protein and E2 can be shortened to 39~49
, compared to 50~60
in the crystal structure. The mean distances for the unbound state are significantly larger than those for the bound form, and the mean distances for all three bound forms are similar, suggesting that linker motions can shorten the distance in the bound state, regardless of whether the substrate binding protein is bound to the adaptor, substrate or both. To what extent can the distance be shortened? Even though all bound states have similar mean distances, their significantly different minimum, maximum and range of distances, as shown in , suggest differences in the distributions of the populations of the states, thus the likelihood of CRL reaching the shortest and longest distances.
The distances between the substrate peptide and E2 could be shortened from a maximum of 47~76
to a minimum of 33~51
(), which is 7~12
shorter than the distance in the crystal structure. The substrates occupy certain space, easily filling this gap. For example, during the simulations of the bound-to-both form of β-TrCP1, the distances between the tip of the substrate β-catenin and the E2 cysteine are in the range of 33~47 Å. Because only a fraction of β-catenin was crystallized, the acceptor lysine of β-catenin, which is not included in the crystal structure and 11 residues away from the tip of the crystallized β-catenin peptide, could reach up to 40 Å away from the tip of the crystallized β-catenin, which makes the contact of the substrate acceptor lysine and the E2 active site possible. The maximum distances, which are 14~29
larger than the minimum distances, may help the polyubiquitination process. Considering the ~20
distance between the ubiquitin Lys48 and Gly76, which connect consecutive ubiquitins in the polyubiquitin chain, the 14~29
difference between the minimum and maximum distances could accommodate ligated ubiquitins. The proposed mechanism is shown in .
Unlike the substrates in the other four substrate binding proteins which have multiple acceptor lysines, β-catenin of β-TrCP1 has been reported to have only one primary acceptor lysine site. It was also reported that the β-catenin lysine-destruction motif spacing is a determinant of ubiquitination efficiency7
. In addition, our previous study19
showed that when superimposing the box domain, the substrate binding sites of Skp2, Fbw7 and Cdc4 overlap each other, whereas β-TrCP1 substrate binding sites are more than 10
away from those of other proteins, implying a different mechanism for β-TrCP1. Nonetheless, even though β-TrCP1 has large rotation angles during the simulation and the distance range between the substrate and the E2 cysteine is significantly smaller than in other proteins, linker motions of β-TrCP1 still control the conformational change. This rotation could conceivably lead to an increase in the concentration of the substrate acceptor lysine near the E2 active site. This is consistent with the lysine spacing specificity. For Skp2, Fbw7 and Cdc4, the distance ranges between the substrate and E2 cysteine are much larger, which can provide sufficient space for the multiple acceptor lysines.
Our analysis of correlated movements supports linker motions as a key player in controlling the positioning of the substrate binding domains and thus in orienting the substrate acceptor lysines. Previously19
we observed a strong correlation between the movements of the linker and the binding sites on the two domains, particularly in the adaptor-bound form. Here, comparing the correlations between the movements of the linker and the substrate binding site in the bound-to-both forms versus other forms, β-TrCP1 has the strongest correlation among the five substrate binding proteins. This argues that when β-TrCP1 binds to both substrate and adaptor, the movements of the linker and the substrate binding domain are strongly correlated, leading to an accurate positioning of the substrate acceptor lysine for ubiquitin transfer. For Skp2, Fbw7, Cdc4 and pVHL, the movements of the linker and substrate binding sites are less correlated which suggest possible increase in the concentration of any of the potential acceptor lysines near the E2 active site.
For Skp2, Fbw7, Cdc4 and pVHL, the unbound form displays the largest linker motions. Linker motions in the bound-to-adaptor and bound-to-substrate forms decrease. However, for β-TrCP1, the form with the largest motions is not the unbound but the bound-to-substrate, suggesting that substrate binding, rather than decreasing the linker motions, allosterically enhances it22
. We calculated the conformational entropy change upon binding. As expected, unlike Skp2, Fbw7 and Cdc4 which sustain conformational entropy penalties upon binding to the substrate, β-TrCP1 has a favorable conformational entropy change, suggesting that for β-TrCP1 substrate binding may be an entropy-driven process.
In the hinge regions, near the N- and C-termini of the linker, respectively, Leu-Trp and Asn-Trp are conserved in F-box proteins; Leu and Asn may constitute linker rotation positions and Trp may serve as an anchor, stabilizing the rotation in certain angles. In the unbound state, the N-terminus hinge is the major hinge; but when bound to the substrate, the C-terminus hinge residues Asn-Trp are destabilized, serving as the major hinge. Based on these observations, we speculate that mutating Leu or Asn to Pro, or binding of small molecules to Trp, may affect the function of CRLs.
Additional linkers in other E3 components may also play key roles in facilitating the ubiquitin transfer reactions by bridging the distances between the arms and at the same time accommodating the space-demanding polyubiquitin chain. The E3 CRL has four components, the substrate binding protein, the adaptor protein, cullin, and the RING-box protein. Recently, based on two crystal structure conformers, Duda et al observed that the linker in the RING-box protein Rbx118
is flexible, which suggests that this linker may also contribute to the E3 CRL conformational change during ubiquitination. We propose that CRL works like a two-arm machine. One arm is the Rbx1 whose flexibility could be further enhanced by Nedd8, and the other arm is the substrate binding protein, which is intrinsically flexible. Our simulations indicate that Nedd8 can allosterically affect the Rbx1 arm shortening the 50 Å gap by ~40 Å (data not shown). Here we show that the substrate binding protein arm can shorten the gap by ~10 Å. Thus these two arms may work cooperatively to reduce the distance gap between the substrate and the E2’s cysteine.
We expect additional flexible links in other E3 CRL components, i.e. the adaptor and cullin. In crystal structures of the adaptor proteins Skp1, Elongin C and ASK1, there are disordered loops in the binding sites suggesting flexibility in the binding site of the adaptor to the substrate binding protein and the adaptor binding site to cullin. These flexible loops could act in concert with flexible linkers in substrate binding proteins and in the RING-box proteins. Furthermore, cullin is comprised of an N-terminal domain (NTD), which binds to the adaptor, and a C-terminal domain (CTD), which binds to the RING-box protein (, ). While it has been reported that the introduction of an artificial linker to join the NTD and CTD of cul1in can disrupt ubiquitination14
, it does not exclude the possibility of flexible linkers in cullin; however, it does suggest a cooperative, allostery-driven E3 CRL. There are three repeats in NTD and these repeats are connected by linkers and the NTD and CTD are also connected by a linker. Cullin serves as a scaffold for the E3 CRL; as such, we suspect that even a small conformational change in a cullin linker may induce a large distance change between the substrate and the E2 ubiquitin binding sites.
In conclusion, our simulations of substrate binding proteins provide a mechanism for the E3 CRL ubiquitin transfer: the linker cooperatively rotates the substrate binding domain and substrate, shortening their distances to the E2 active site, or increasing their distance allowing polyubiquitination. As such, the linker could be a new therapeutic target for E3 CRL-related human diseases. Allostery is a key mechanism in protein function: it is a cooperative event, up-or down-regulating protein activities22; 45–48
. In our case here, the linker can be targeted directly or allosterically34; 49
. Point mutations or drug design targeting alteration of the linker motions may affect the overall function of CRLs.