Designing active RNF4 monomers by introducing a tryptophan: avidity towards E2∼Ub conjugates dictates the activity of ubiquitin RING E3 ligases.

Ubiquitin RING E3 ligases (E3s) catalyze ubiquitin (Ub) transfer to their substrates by engaging E2∼Ub intermediates with the help of their RING domains. Different E3s have been found to contain a conserved tryptophan residue in their RING that plays an essential role in E2 binding and, hence, enzymatic activity. Many active E3s, however, lack this specific residue. We mined through the existing data to observe that the conservation of the tryptophan and quaternary organization of the RING domains are remarkably correlated. Monomeric RINGs possess the tryptophan while all well-characterized dimeric RINGs, except RNF8, contain other amino acid residues. Biochemical analyses on representative E3s and their mutants reveal that the tryptophan is essential for optimal enzymatic activity of monomeric RINGs whereas dimeric E3s with tryptophan display hyperactivity. Most critically, the introduction of the tryptophan restores the activity of inactive monomeric RNF4 mutants, an obligatory dimeric E3. Binding studies indicate that monomeric RINGs retained the tryptophan for their optimal functionality to compensate for weak Ub binding. On the other hand, tryptophan was omitted from dimeric RINGs during the course of evolution to prevent unwanted modifications and allow regulation of their activity through oligomerization.

Structural insights into the nanomolar affinity of RING E3 ligase ZNRF1 for Ube2N and its functional implications.

RING (Really Interesting New Gene) domains in ubiquitin RING E3 ligases exclusively engage ubiquitin (Ub)-loaded E2s to facilitate ubiquitination of their substrates. Despite such specificity, all RINGs characterized till date bind unloaded E2s with dissociation constants (Kds) in the micromolar to the sub-millimolar range. Here, we show that the RING domain of E3 ligase ZNRF1, an essential E3 ligase implicated in diverse cellular pathways, binds Ube2N with a Kd of ∼50 nM. This high-affinity interaction is exclusive for Ube2N as ZNRF1 interacts with Ube2D2 with a Kd of ∼1 µM, alike few other E3s. The crystal structure of ZNRF1 C-terminal domain in complex with Ube2N coupled with mutational analyses reveals the molecular basis of this unusual affinity. We further demonstrate that the ubiquitination efficiency of ZNRF1 : E2 pairs correlates with their affinity. Intriguingly, as a consequence of its high E2 affinity, an excess of ZNRF1 inhibits Ube2N-mediated ubiquitination at concentrations ≥500 nM instead of showing enhanced ubiquitination. This suggests a novel mode of activity regulation of E3 ligases and emphasizes the importance of E3-E2 balance for the optimum activity. Based on our results, we propose that overexpression-based functional analyses on E3 ligases such as ZNRF1 must be approached with caution as enhanced cellular levels might result in aberrant modification activity.

Role of E2-RING Interactions in Governing RNF4-Mediated Substrate Ubiquitination.

Members of the really interesting new gene (RING) E3 ubiquitin ligase family bind to both substrate and ubiquitin-charged E2 enzyme, promoting the transfer of ubiquitin from the E2 to substrate. Either a single ubiquitin or one of the several types of polyubiquitin chains can be conjugated to substrate proteins, with different types of ubiquitin modifications signaling the distinct outcomes. E2 enzymes play a central role in governing the nature of the ubiquitin modification, although the essential features of the E2 that differentiate mono- versus polyubiquitinating E2 enzymes remain unclear. RNF4 is a compact RING E3 ligase that directs the ubiquitination of polySUMO chains in concert with several different E2 enzymes. RNF4 monoubiquitinates polySUMO substrates in concert with RAD6B and polyubiquitinates substrates together with UBCH5B, a promiscuous E2 that can function with a broad range of E3 ligases. We find that the divergent ubiquitination activities of RAD6B and UBCH5B are governed by differences at the RING-binding surface of the E2. By mutating the RAD6B RING-binding surface to resemble that of UBCH5B, RAD6B can be transformed into a highly active UBCH5B-like E2 that polyubiquitinates SUMO chains in concert with RNF4. The switch in RAD6B activity correlates with increased affinity of the E2 for RNF4. These results point to an important role of the affinity between an E3 and its cognate E2 in governing the multiplicity of substrate ubiquitination.

RING E3-Catalyzed E2 Self-Ubiquitination Attenuates the Activity of Ube2E Ubiquitin-Conjugating Enzymes.

Ubiquitination of a target protein is accomplished through sequential actions of the E1, E2s, and the E3s. E2s dictate the modification topology while E3 ligases confer substrate specificity and recruit the cognate E2. Human genome codes for ~35 different E2 proteins; all of which contain the characteristic ubiquitin-conjugating UBC core domain sufficient for catalysis. Many of these E2 enzymes also have N- or C-terminal extensions; roles of which are not very well understood. We show that the N-terminal extension of Ube2E1 undergoes intramolecular auto-ubiquitination. This self-ubiquitination activity is enhanced in the presence of interacting RING E3 ligases and results in a progressive attenuation of the E2 activity toward substrate/E3 modification. We also find that the N-terminal ubiquitination sites are conserved in all the three Ube2Es and replacing them with arginine renders all three full-length Ube2Es equally active as their core UBC domains. Based on these results, we propose that E3-catalyzed self-ubiquitination acts as a key regulatory mechanism that controls the activity of Ube2E class of ubiquitin E2s.

Studies on Escherichia coli HflKC suggest the presence of an unidentified λ factor that influences the lysis-lysogeny switch.

BACKGROUND: The lysis-lysogeny decision in the temperate coliphage λ is influenced by a number of phage proteins (CII and CIII) as well as host factors, viz. Escherichia coli HflB, HflKC and HflD. Prominent among these are the transcription factor CII and HflB, an ATP-dependent protease that degrades CII. Stabilization of CII promotes lysogeny, while its destabilization induces the lytic mode of development. All other factors that influence the lytic/lysogenic decision are known to act by their effects on the stability of CII. Deletion of hflKC has no effect on the stability of CII. However, when λ infects ΔhflKC cells, turbid plaques are produced, indicating stabilization of CII under these conditions. RESULTS: We find that CII is stabilized in ΔhflKC cells even without infection by λ, if CIII is present. Nevertheless, we also obtained turbid plaques when a ΔhflKC host was infected by a cIII-defective phage (λcIII67). This observation raises a fundamental question: does lysogeny necessarily correlate with the stabilization of CII? Our experiments indicate that CII is indeed stabilized under these conditions, implying that stabilization of CII is possible in ΔhflKC cells even in the absence of CIII, leading to lysogeny. CONCLUSION: We propose that a yet unidentified CII-stabilizing factor in λ may influence the lysis-lysogeny decision in ΔhflKC cells.

Structural insights into the assembly and function of the SAGA deubiquitinating module.

SAGA is a transcriptional coactivator complex that is conserved across eukaryotes and performs multiple functions during transcriptional activation and elongation. One role is deubiquitination of histone H2B, and this activity resides in a distinct subcomplex called the deubiquitinating module (DUBm), which contains the ubiquitin-specific protease Ubp8, bound to Sgf11, Sus1, and Sgf73. The deubiquitinating activity depends on the presence of all four DUBm proteins. We report here the 1.90 angstrom resolution crystal structure of the DUBm bound to ubiquitin aldehyde, as well as the 2.45 angstrom resolution structure of the uncomplexed DUBm. The structure reveals an arrangement of protein domains that gives rise to a highly interconnected complex, which is stabilized by eight structural zinc atoms that are critical for enzymatic activity. The structure suggests a model for how interactions with the other DUBm proteins activate Ubp8 and allows us to speculate about how the DUBm binds to monoubiquitinated histone H2B in nucleosomes.

Specific hydrophobic residues in the alpha4 helix of lambdaCII are crucial for maintaining its tetrameric structure and directing the lysogenic choice.

The CII protein of the temperate bacteriophage lambda is the decision-making factor that determines the viral lytic/lysogenic choice. It is a homotetrameric transcription activator that recognizes and binds specific direct repeat sequences TTGCN(6)TTGC in the lambda genome. The quaternary structure of CII is held by a four-helix bundle. It is known that the tetrameric organization of CII is necessary for its activity, but the molecular mechanism behind this requirement is not known. By specific site-directed mutagenesis of hydrophobic residues in the alpha4 helix of CII that constitutes the four-helix bundle, we found that residues leu70, val74 and leu78 were crucial for maintaining the tetrameric structure of the protein. When any of these residues was substituted by a polar one, CII lost its activity and failed to promote lysogeny. This loss of activity was accompanied by the inability of CII to form tetramers, to bind DNA or to activate transcription.

The structure and conformation of Lys63-linked tetraubiquitin.

Ubiquitination involves the covalent attachment of the ubiquitin (Ub) C-terminus to the lysine side chain of a substrate protein by an isopeptide bond. The modification can comprise a single Ub moiety or a chain of Ub molecules joined by isopeptide bonds between the C-terminus of one Ub with one of the seven lysine residues in the next Ub. Modification of substrate proteins with Lys63-linked poly-Ub plays a key nondegradative signaling role in many biological processes, including DNA repair and nuclear factor-kappaB activation, whereas substrates modified by Lys48-linked chains are targeted to the proteasome for degradation. The distinct signaling properties of alternatively linked Ub chains presumably stem from structural differences that can be distinguished by effector proteins. We have determined the crystal structure of Lys63 tetra-Ub at a resolution of 1.96 A and performed small-angle X-ray scattering experiments and molecular dynamics simulations to probe the conformation of Lys63 tetra-Ub in solution. The chain adopts a highly extended conformation in the crystal, in contrast with the compact globular fold of Lys48 tetra-Ub. Small-angle X-ray scattering experiments show that the Lys63 tetra-Ub chain is dynamic in solution, adopting an ensemble of conformations that are more compact than the extended form in the crystal. The results of these studies provide a basis for understanding the differences in the behavior and recognition of Lys63 poly-Ub chains.

Oxalone and lactone moieties of podophyllotoxin exhibit properties of both the B and C rings of colchicine in its binding with tubulin.

Thermodynamics of podophyllotoxin binding to tubulin and its multiple points of attachment with tubulin has been studied in detail using isothermal titration calorimetry. The calorimetric enthalpy of the association of podophyllotoxin with tubulin is negative and occurs with a negative heat capacity change (DeltaC(p) = -2.47 kJ mol(-)(1) K(-)(1)). The binding is unique with a simultaneous participation of both hydrophobic and hydrogen-bonding forces with unfavorable negative entropic contribution at higher temperature, favored with an enthalpy-entropy compensation. Interestingly, the binding of 2-methoxy-5-(2',3',4'-trimethoxyphenyl)tropone (AC, a colchicine analogue without the B ring) with tubulin is enthalpy-favored. However, the podophyllotoxin-tubulin association depending upon the temperature of the reaction has a favorable entropic and enthalpic component, which resembles both B- and C-ring properties of colchicine. On the basis of the crystal structure of the podophyllotoxin-tubulin complex, distance calculations have indicated a possible interaction between threonine 179 of alpha-tubulin and the hydroxy group on the D ring of podophyllotoxin. To confirm the involvement of the oxalone moiety as well as the lactone ring of podophyllotoxin in tubulin binding, analogues of podophyllotoxin are synthesized with methoxy substitution at the 4' position of ring D along with its isomer and another analogue epimerized at ring E. From these results, involvement of oxalone as well as the lactone ring of the drug in a specific orientation inclusive of ring A is indicated for podophyllotoxin-tubulin binding. Therefore, podophyllotoxin, like colchicine, behaves as a bifunctional ligand having properties of both the B and C rings of colchicine by making more than one point of attachment with the protein tubulin.

Structure of lambda CII: implications for recognition of direct-repeat DNA by an unusual tetrameric organization.

The temperate coliphage lambda, after infecting its host bacterium Escherichia coli, can develop either along the lytic or the lysogenic pathway. Crucial to the lysis/lysogeny decision is the homotetrameric transcription-activator protein CII (4 x 11 kDa) of the phage that binds to a unique direct-repeat sequence T-T-G-C-N6-T-T-G-C at each of the three phage promoters it activates: p(E), p(I), and p(aQ). Several regions of CII have been identified for its various functions (DNA binding, oligomerization, and susceptibility to host protease), but the crystal structure of the protein long remained elusive. Here, we present the three-dimensional structure of CII at 2.6-angstroms resolution. The CII monomer is comprised of four alpha helices and a disordered C terminus. The first three helices (alpha1-alpha3) form a compact domain, whereas the fourth helix (alpha4) protrudes in different orientations in each subunit. A four-helix bundle, formed by alpha4 from each subunit, holds the tetramer. The quaternary structure can be described as a dimer of dimers, but the tetramer does not exhibit a closed symmetry. This unusual quaternary arrangement allows the placement of the helix-turn-helix motifs of two of the four CII subunits for interaction with successive major grooves of B-DNA, from one face of DNA. This structure provides a simple explanation for how a homotetrameric protein may recognize a direct-repeat DNA sequence rather than the inverted-repeat sequences of most prokaryotic activators.

An agarose-acrylamide composite native gel system suitable for separating ultra-large protein complexes.

An agarose-acrylamide composite native gel (CNG) system has been developed for separating protein complexes of ultra-large molecular sizes (over 500kDa) and for analyzing protein-protein interactions in their native states. Various native gel conditions were explored and techniques were improved to facilitate the formation and performance of the CNG system. We demonstrate here that the CNG technique is capable of resolving a complex of RNA polymerase II and an associated factor from the free components, which had not been previously achieved with other methods. Furthermore, this CNG electrophoresis can be conveniently coupled to second-dimension sodium dodecyl sulfate-polyacrylamide gel electrophoresis for identification of protein components within discrete complexes separated during the CNG run. The CNG technique is particularly suitable for capturing dynamic protein-protein interactions as exemplified here by the formation and demonstration of RNA polymerase II-Fcp1 complex.

-NH-dansyl isocolchicine exhibits a significantly improved tubulin-binding affinity and microtubule inhibition in comparison to isocolchicine by binding tubulin through its A and B rings.

Structure-activity relationship studies have established that the A and C rings of colchicine comprise the minimum structural feature necessary for high affinity drug-tubulin binding. Thus, colchicine acts as a bifunctional ligand by making two points of attachment to the protein. Furthermore, analogues belonging to the iso series of colchicine are virtually inactive in binding to tubulin and inhibiting microtubule assembly. In the present study, we found that the substitution of a hydrophobic dansyl group on the B-ring side chain (C7 position) of isocolchicine reverses the structural alterations at the C ring and the newly synthesized -NH-dansyl isocolchicine restores the lost biological activity of the compound. It inhibits microtubule assembly efficiently with an IC(50) value of 10 microM and competes with [(3)H]colchicine for binding to tubulin. Moreover, although -NH-dansyl colchicine binding to tubulin involves two steps, the -NH-dansyl isocolchicine-tubulin interaction has been found to occur via a one-step process. Also, the affinity constant of the -NH-dansyl isocolchicine-tubulin interaction is roughly only 3 times lower than that of the -NH-dansyl colchicine-tubulin interaction. These results suggest that the enhanced microtubule inhibitory ability of -NH-dansyl isocolchicine is therefore related to the affinity of the drug-tubulin interaction and not to any conformational changes upon binding tubulin. We also observed that the competition of -NH-dansyl isocolchicine with [(3)H]colchicine for binding to tubulin was dependent on the tubulin concentration. In conclusion, this paper for the first time indicates that a biologically active bifuntional colchicine analogue can be designed where the drug binds tubulin through its A and B rings, while the C ring remains inactive.

Disorder-order transition of lambda CII promoted by low concentrations of guanidine hydrochloride suggests a stable core and a flexible C-terminus.

The CII protein of bacteriophage lambda, which activates the synthesis of the lambda repressor, plays a key role in the lysis-lysogeny switch. CII has a small in vivo half-life due to its proteolytic susceptibility, and this instability is a key component for its regulatory role. The structural basis of this instability is not known. While studying guanidine hydrochloride-assisted unfolding of CII, we found that low concentrations of the chaotrope (50-500 microM) have a considerable effect on the structure of this protein. This effect is manifest in an increase in molar ellipticity, an enhancement of intrinsic tryptophan fluorescence intensity and a reduction in ANS binding. At low concentrations of guanidine hydrochloride CII is stabilized, as reflected in a significant decrease in the rate of proteolysis by trypsin and resistance to thermal aggregation, while the tetrameric nature of the protein is retained. Thus low concentrations of guanidine hydrochloride promote a more structured conformation of the CII protein. On the basis of these observations, a model has been proposed for the structure of CII wherein the protein equilibrates between a compact form and a proteolytically accessible form, in which the C-terminal region assumes different structures.