UBE4B promotes Hdm2-mediated degradation of the tumor suppressor p53.

The TP53 gene (encoding the p53 tumor suppressor) is rarely mutated, although frequently inactivated, in medulloblastoma and ependymoma. Recent work in mouse models showed that the loss of p53 accelerated the development of medulloblastoma. The mechanism underlying p53 inactivation in human brain tumors is not completely understood. We show that ubiquitination factor E4B (UBE4B), an E3 and E4 ubiquitin ligase, physically interacts with p53 and Hdm2 (also known as Mdm2 in mice). UBE4B promotes p53 polyubiquitination and degradation and inhibits p53-dependent transactivation and apoptosis. Notably, silencing UBE4B expression impairs xenotransplanted tumor growth in a p53-dependent manner and overexpression of UBE4B correlates with decreased expression of p53 in these tumors. We also show that UBE4B overexpression is often associated with amplification of its gene in human brain tumors. Our data indicate that amplification and overexpression of UBE4B represent previously undescribed molecular mechanisms of inactivation of p53 in brain tumors.

Catalytic proficiency of ubiquitin conjugation enzymes: balancing pK(a) suppression, entropy, and electrostatics.

Biological organisms orchestrate coordinated responses to external stimuli through temporal fluctuations in protein-protein interaction networks using molecular mechanisms such as the synthesis and recognition of polyubiquitin (polyUb) chains on signaling adaptor proteins. One of the pivotal chemical steps in ubiquitination involves reaction of a lysine amino group with a thioester group on an activated E2, or ubiquitin conjugation enzyme, to form an amide bond between Ub and a target protein. In this study, we demonstrate a nominal 14-fold range for the rate of the chemical step, k(cat), catalyzed by different E2 enzymes using non-steady-state, single-turnover assays. However, the observed range for k(cat) is as large as ∼100-fold for steady-state, single-turnover assays. Biochemical assays were used in combination with measurement of the underlying protein-protein interaction kinetics using NMR line-shape and ZZ-exchange analyses to determine the rate of polyUb chain synthesis catalyzed by the heterodimeric E2 enzyme Ubc13-Mms2. Modest variations in substrate affinity and k(cat) can achieve functional diversity in E2 mechanism, thereby influencing the biological outcomes of polyubiquitination. E2 enzymes achieve reaction rate enhancements through electrostatic effects such as suppression of substrate lysine pK(a) and stabilization of transition states by the preorganized, polar enzyme active site as well as the entropic effects of binding. Importantly, modestly proficient enzymes such as E2s maintain the ability to tune reaction rates; this may confer a biological advantage for achieving specificity in the diverse cellular roles for which these enzymes are involved.

Dynamics of the RING domain from human TRAF6 by 15N NMR spectroscopy: implications for biological function.

Activation of transcription factor NF-kappaB requires Lys63-linked polyubiquitination of the E3 ubiquitin ligase TRAF6 via protein-protein interactions mediated by a RING domain. In this study, intra- and intermolecular chemical exchange processes of the TRAF6 RING domain were analyzed by (15)N NMR spectroscopy. Micro- to millisecond time scale motions were assessed through R 1, R 2, NOE, and cross-correlated relaxation measurements, and the kinetics of these motions were quantified with relaxation dispersion. The relaxation experiments indicate that the protein core is rigid, consistent with the functional requirement that RING domains form a binding scaffold for E2 ubiquitin conjugation enzymes. Chemical exchange is observed at the C-terminal end of the main alpha-helix of the RING domain. The C-terminal end of the main alpha-helix from the RING domain is involved in E2-E3 interactions, and modulation of slow motions for this region of the helix may be a general mechanism by which these interactions achieve ubiquitin transfer. Chemical shift mapping indicates that the TRAF6 RING domain does not self-associate in solution. Numerous RING domains are homo- or heterodimeric, and this is thought to be a functional necessity for recruitment of substrates for ubiquitination, or recruitment of multiple E2 enzymes for efficient substrate ubiquitination. However, lack of self-association for the RING domain from TRAF6, and the observation that the intact protein is a trimer, suggests that close association of RING domains within a homodimeric scaffold may not be a fundamental requirement for biological function.

Structure, interactions, and dynamics of the RING domain from human TRAF6.

A key step in the signaling cascade responsible for activation of the transcription factor NF-kappaB involves Lys63-linked polyubiquitination of TRAF6. Covalent attachment of ubiquitin (Ub) to TRAF6, and subsequent poly(Ub) chain synthesis, is catalyzed by the hUev1a-hUbc13 heterodimer. hUbc13 is a catalytically competent E2 enzyme, and hUev1a is an E2-like protein that binds substrate Ub. The hUev1a-hUbc13 heterodimer is targeted to TRAF6 through interactions between hUbc13 and the N-terminal RING domain from TRAF6. Nuclear magnetic resonance (NMR) spectroscopy was used to determine the solution state structure of the RING domain from human TRAF6, and the interaction between hUbc13 and TRAF6 was characterized using NMR chemical shift mapping. The main-chain dynamics of the RING domain from TRAF6 were studied using (15)N NMR relaxation. Analysis of the main-chain dynamics data indicates that residues within the alpha-helix and beta-sheet of the RING domain are as rigid as regions of canonical secondary structure in larger proteins, consistent with the biological role of RING-domain E3 proteins, which requires that the E3 contain a recognition site for recruitment of E2 ubiquitin conjugation enzymes.

Structure and interactions of the ubiquitin-conjugating enzyme variant human Uev1a: implications for enzymatic synthesis of polyubiquitin chains.

Lys(63)-linked polyubiquitination of TRAF2 or TRAF6 is an essential step within the signal transduction cascade responsible for activation of p38, c-Jun N-terminal kinase, and the transcription factor NF-kappaB. Attachment of ubiquitin (Ub) to a TRAF, and conjugation of Ub molecules to form a polyUb chain, is catalyzed by a heterodimer composed of a catalytically active E2 (hUbc13), involved in covalent bond transfer, and hUev1a, an E2-like protein involved in substrate Ub binding. Given the key biochemical processes in which hUev1a is involved, it is important to determine the molecular basis of the catalytic mechanism for Lys(63)-linked protein ubiquitination. Nuclear magnetic resonance (NMR) spectroscopy was used to determine the structure of hUev1a and its interactions with Ub and hUbc13. A structural model for the Ub-hUev1a-hUbc13-Ub tetramer was developed to gain chemical insight into the synthesis of Lys(63)-linked Ub chains. We propose that a network of hydrogen bonds involving hUbc13-Asp(81) and Ub-Glu(64) positions Ub-Lys(63) proximal to the active site. Interestingly, restrained molecular dynamics simulations in implicit solvent indicate that deprotonation of Ub-Lys(63) does not involve a general Asp or Glu base and may occur when the amino group approaches the thioester carbonyl carbon near the Bürgi-Dunitz trajectory.

Structural basis for non-covalent interaction between ubiquitin and the ubiquitin conjugating enzyme variant human MMS2.

Modification of proteins by post-translational covalent attachment of a single, or chain, of ubiquitin molecules serves as a signaling mechanism for a number of regulatory functions in eukaryotic cells. For example, proteins tagged with lysine-63 linked polyubiquitin chains are involved in error-free DNA repair. The catalysis of lysine-63 linked polyubiquitin chains involves the sequential activity of three enzymes (E1, E2, and E3) that ultimately transfer a ubiquitin thiolester intermediate to a protein target. The E2 responsible for catalysis of lysine-63 linked polyubiquitination is a protein heterodimer consisting of a canonical E2 known as Ubc13, and an E2-like protein, or ubiquitin conjugating enzyme variant (UEV), known as Mms2. We have determined the solution structure of the complex formed by human Mms2 and ubiquitin using high resolution, solution state nuclear magnetic resonance (NMR) spectroscopy. The structure of the Mms2-Ub complex provides important insights into the molecular basis underlying the catalysis of lysine-63 linked polyubiquitin chains.

Main chain and side chain dynamics of the ubiquitin conjugating enzyme variant human Mms2 in the free and ubiquitin-bound States.

Protein ubiquitination involves a cascade of enzymatic steps where ubiquitin (Ub) is sequentially transferred as a thiolester intermediate from an E1 enzyme to an E2 enzyme and finally to the protein target with the help of a Ub-protein ligase. Protein ubiquitination brought about by the Ubc13-Mms2 (E2-E2) complex has a unique role in the cell, unrelated to protein degradation. The Mms2-Ubc13 heterodimer links Ub molecules to one another through an isopeptide bond between its own C-terminus and Lys-63 on another Ub. The role of Mms2 is to orient a target-bound Ub molecule such that its Lys-63 is proximal to the C-terminus of the Ub molecule that is covalently linked to the active site of Ubc13. To gain insight into the influence of protein dynamics on the affinity of Ub for Mms2, we have determined pico- to nanosecond time scale fluctuations of the main chain and methyl side chains of human Mms2 in the free and Ub-bound states using solution state (15)N and (2)H nuclear magnetic resonance relaxation measurements. Analysis of the relaxation data allows for a semiquantitative estimation of the conformational entropy change (TDeltaS(NMR)) for the main chain and side chain methyl groups of Mms2 upon binding Ub. The value of TDeltaS(NMR) for the main chain and side chain methyl groups of Mms2 is -8 +/- 2 and -2 +/- 2 kcal mol(-)(1), respectively. The experimental DeltaG(binding) for the Mms2.Ub complex is -6 kcal mol(-)(1). Estimation of DeltaG(binding) using an empirical structure-based approach that does not account for changes in main chain entropy yields a value of -17 +/- 2 kcal mol(-)(1). However, inclusion of TDeltaS(NMR) for the main chain of Mms2 increases the estimated DeltaG(binding) to -9 +/- 3 kcal mol(-)(1). Assuming that changes in Ub main chain dynamics contribute to TDeltaS(NMR) to the same extent as Mms2, the estimated DeltaG(binding) is further reduced to -1 +/- 4 kcal mol(-)(1), a value close to the experimental DeltaG(binding).