Activation of UbcH5c∼Ub Is the Result of a Shift in Interdomain Motions of the Conjugate Bound to U‑Box E3 Ligase E4B

Post-translational modification of proteins with ubiquitin is mediated by dynamic multienzyme machinery (E1, E2, and E3). E3 ubiquitin ligases play a key role acting as both scaffolds to bring reactants together and activators to catalyze ubiquitin (Ub) transfer from E2∼Ub conjugates to substrates. Our recent studies provided insights into the mechanism of the activation event; binding of an E3 to an E2∼Ub conjugate was found to affect the motions of E2∼Ub and allosterically stimulate Ub transfer. This proposed mechanism implies that the dynamics of the conjugate, which has been shown to occupy a wide range of E2∼Ub orientations, will be altered significantly upon binding of E3. To directly assess the effect of E3 binding on E2∼Ub dynamics, we undertook an in-depth comparative analysis of ¹⁵N nuclear magnetic resonance relaxation of UbcH5c∼Ub in the absence and presence of the E3 ligase, E4B. Challenges encountered in deciphering interdomain motions for this ternary complex are discussed along with the limitations of the current approaches. Notably, although a reduction in interdomain dynamics of UbcH5c∼Ub is observed upon binding to E4B, Ub retains an extensive degree of flexibility. These results provide strong support for our dynamic model of a significant orientational bias of Ub toward a more closed conformation in the E3/E2∼Ub complex

Measuring And Modeling Highly Accurate \u3csup\u3e15\u3c/sup\u3eN Chemical Shift Tensors In A Peptide.

NMR studies measuring chemical shift tensors are increasingly being employed to assign structure in difficult-to-crystallize solids. For small organic molecules, such studies usually focus on 13C sites, but proteins and peptides are more commonly described using 15N amide sites. An important and often neglected consideration when measuring shift tensors is the evaluation of their accuracy against benchmark standards, where available. Here we measure 15N tensors in the dipeptide glycylglycine at natural abundance using the slow-spinning FIREMAT method with SPINAL-64 decoupling. The accuracy of these 15N tensors is evaluated by comparing to benchmark single crystal NMR 15N measurements and found to be statistically indistinguishable. These FIREMAT experimental results are further used to evaluate the accuracy of theoretical predictions of tensors from four different density functional theory (DFT) methods that include lattice effects. The best theoretical approach provides a root mean square (rms) difference of ±3.9 ppm and is obtained from a fragment-based method and the PBE0 density functional

Redefining the DNA-Binding Domain of Human XPA

Xeroderma pigmentosum complementation group A (XPA) protein plays a critical role in the repair of DNA damage via the nucleotide excision repair (NER) pathway. XPA serves as a scaffold for NER, interacting with several other NER proteins as well as the DNA substrate. The critical importance of XPA is underscored by its association with the most severe clinical phenotypes of the genetic disorder Xeroderma pigmentosum. Many of these disease-associated mutations map to the XPA_98–219 DNA-binding domain (DBD) first reported ∼20 years ago. Although multiple solution NMR structures of XPA_98–219 have been determined, the molecular basis for the interaction of this domain with DNA is only poorly characterized. In this report, we demonstrate using a fluorescence anisotropy DNA-binding assay that the previously reported XPA DBD binds DNA with substantially weaker affinity than the full-length protein. In-depth analysis of the XPA sequence suggested that the original DBD construct lacks critical basic charge and helical elements at its C-terminus. Generation and analysis of a series of C-terminal extensions beyond residue 219 yielded a stable, soluble human XPA_98–239 construct that binds to a Y-shaped ssDNA–dsDNA junction and other substrates with the same affinity as the full-length protein. Two-dimensional ¹⁵N–¹H NMR suggested XPA_98–239 contains the same globular core as XPA_98–219 and likely undergoes a conformational change upon binding DNA. Together, our results demonstrate that the XPA DBD should be redefined and that XPA_98–239 is a suitable model to examine the DNA binding activity of human XPA

Biochemical and Proteomic Analysis of Ubiquitination of Hsc70 and Hsp70 by the E3 Ligase CHIP

<div><p>The E3 ubiquitin ligase CHIP is involved in protein triage, serving as a co-chaperone for refolding as well as catalyzing ubiquitination of substrates. CHIP functions with both the stress induced Hsp70 and constitutive Hsc70 chaperones, and also plays a role in maintaining their balance in the cell. When the chaperones carry no client proteins, CHIP catalyzes their polyubiquitination and subsequent proteasomal degradation. Although Hsp70 and Hsc70 are highly homologous in sequence and similar in structure, CHIP mediated ubiquitination promotes degradation of Hsp70 with a higher efficiency than for Hsc70. Here we report a detailed and systematic investigation to characterize if there are significant differences in the CHIP in vitro ubiquitination of human Hsp70 and Hsc70. Proteomic analysis by mass spectrometry revealed that only 12 of 39 detectable lysine residues were ubiquitinated by UbcH5a in Hsp70 and only 16 of 45 in Hsc70. The only conserved lysine identified as ubiquitinated in one but not the other heat shock protein was K159 in Hsc70. Ubiquitination assays with K-R ubiquitin mutants showed that multiple Ub chain types are formed and that the distribution is different for Hsp70 versus Hsc70. CHIP ubiquitination with the E2 enzyme Ube2W is predominantly directed to the N-terminal amine of the substrate; however, some internal lysine modifications were also detected. Together, our results provide a detailed view of the differences in CHIP ubiquitination of these two very similar proteins, and show a clear example where substantial differences in ubiquitination can be generated by a single E3 ligase in response to not only different E2 enzymes but subtle differences in the substrate.</p></div

Hsp70 is more rapidly degraded than Hsc70 by 26S in vitro.

<p>Concurrent ubiquitination and degradation reactions were incubated for 2 hours at 37°C before separating the products on the gel. Key bands are highlighted and quantification of both the Coomassie stained and immunoblot analyses confirm that only Hsp70 ubiquitinated by UbcH5a is significantly degraded under these conditions.</p

Summary of results from mass spectrometry proteomics analysis of +1 or +2 Ub samples from Fig 1A.

<p>Summary of results from mass spectrometry proteomics analysis of +1 or +2 Ub samples from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128240#pone.0128240.g001" target="_blank">Fig 1A</a>.</p

CHIP ubiquitination of Hsc70 (A) and Hsp70 (B) is wide-spread but largely focused on the C-terminal half of the protein.

<p>Homology models based on the structure of DnaK (PDB ID 2KHO) were created using SWISS-MODEL, and are here used to map the sequence coverage and ubiquitination sites found by mass spectrometry proteomics. Observed regions are colored in red, with unobserved regions in grey. All lysine side-chains are explicitly shown with confirmed ubiquitination sites highlighted in yellow.</p

Ubiquitin is predominantly attached to the N-terminus of His-GST-Hsp70 by Ube2W.

<p>In vitro ubiquitination reactions with the fusion protein and various Ub mutants were incubated as above for 30 min (A). To isolate N-terminal ubiquitination, 0.2 μM H3C protease (to cleave His-GST from Hsp70) and 50 mM L-Cys (to quench E1 activity) were then added to a portion of the reaction and allowed to incubate for 30 min at room temperature prior to separation by SDS-PAGE (B). The cleaved reaction products in B were also investigated by western blots for C) Hsp70 and D) GST. The majority of the ubiquitination products in all reactions containing Ube2W (lanes 4–15) follow His-GST and indicate ubiquitination of the N-terminus.</p

Ubiquitin mutants alter the rate of reaction in vitro.

<p>All Ub species were incubated for 30 minutes with UbcH5a or Ube2W+ UbcH13/Uev1a as the E2 as labeled. Both the Coomassie stain, left, and immunoblot, right, are shown for each set of reactions. A) CHIP autoubiquitination, B) Hsc70 ubiquitination by CHIP, and C) Hsp70 ubiquitination by CHIP. The * indicates a weak signal in the western blot that is more clearly seen by Coomassie stain. Note that K11R Ub retains a fusion tag that results in a larger protein and thus larger ladder spacing.</p