Explore the vast range of titles available.

Numerous proteins are involved in the multiple pathways of the DNA damage response network and play a key role to protect the genome from the wide variety of damages that can occur to DNA. An example of this is the structure-specific endonuclease ERCC1-XPF. This heterodimeric complex is in particular involved in nucleotide excision repair (NER), but also in double strand break repair and interstrand cross-link repair pathways. Here we review the function of ERCC1-XPF in various DNA repair pathways and discuss human disorders associated with ERCC1-XPF deficiency. We also overview our molecular and structural understanding of XPF-ERCC1.

In ubiquitin conjugation, different combinations of E2 and E3 enzymes catalyse either monoubiquitination or ubiquitin chain formation. The E2/E3 complex Rad6/Rad18 exclusively monoubiquitinates the proliferating cell nuclear antigen (PCNA) to signal for \"error prone\" DNA damage tolerance, whereas a different set of conjugation enzymes is required for ubiquitin chain formation on PCNA. Here we show that human E2 enzyme Rad6b is intrinsically capable of catalyzing ubiquitin chain formation. This activity is prevented during PCNA ubiquitination by the interaction of Rad6 with E3 enzyme Rad18. Using NMR and X-ray crystallography we show that the R6BD of Rad18 inhibits this activity by competing with ubiquitin for a noncovalent \"backside\" binding site on Rad6. Our findings provide mechanistic insights into how E3 enzymes can regulate the ubiquitin conjugation process.

Interaction of regulatory DNA binding proteins with their target sites is usually preceded by binding to nonspecific DNA. This speeds up the search for the target site by several orders of magnitude. We report the solution structure and dynamics of the complex of a dimeric lac repressor DNA binding domain with nonspecific DNA. The same set of residues can switch roles from a purely electrostatic interaction with the DNA backbone in the nonspecific complex to a highly specific binding mode with the base pairs of the cognate operator sequence. The protein-DNA interface of the nonspecific complex is flexible on biologically relevant time scales that may assist in the rapid and efficient finding of the target site.

Background Recognition of histone modifications by specialized protein domains is a key step in the regulation of DNA-mediated processes like gene transcription. The structural basis of these interactions is usually studied using histone peptide models, neglecting the nucleosomal context. Here, we provide the structural and thermodynamic basis for the recognition of H3K36-methylated (H3K36me) nucleosomes by the PSIP1-PWWP domain, based on extensive mutational analysis, advanced nuclear magnetic resonance (NMR), and computational approaches. Results The PSIP1-PWWP domain binds H3K36me3 peptide and DNA with low affinity, through distinct, adjacent binding surfaces. PWWP binding to H3K36me nucleosomes is enhanced approximately 10,000-fold compared to a methylated peptide. Based on mutational analyses and NMR data, we derive a structure of the complex showing that the PWWP domain is bound to H3K36me nucleosomes through simultaneous interactions with both methylated histone tail and nucleosomal DNA. Conclusion Concerted binding to the methylated histone tail and nucleosomal DNA underlies the high- affinity, specific recognition of H3K36me nucleosomes by the PSIP1-PWWP domain. We propose that this bipartite binding mechanism is a distinctive and general property in the recognition of histone modifications close to the nucleosome core.

The molecular chaperone Hsp90 is a protein folding machine that is conserved from bacteria to man. Human, cytosolic Hsp90 is dedicated to folding of chiefly signal transduction components. The chaperoning mechanism of Hsp90 is controlled by ATP and various cochaperones, but is poorly understood and controversial. Here, we characterized the Apo and ATP states of the 170-kDa human Hsp90 full-length protein by NMR spectroscopy in solution, and we elucidated the mechanism of the inhibition of its ATPase by its cochaperone p23. We assigned isoleucine side chains of Hsp90 via specific isotope labeling of their δ-methyl groups, which allowed the NMR analysis of the full-length protein. We found that ATP caused exclusively local changes in Hsp90's N-terminal nucleotide-binding domain. Native mass spectrometry showed that Hsp90 and p23 form a 2:2 complex via a positively cooperative mechanism. Despite this stoichiometry, NMR data indicated that the complex was not fully symmetric. The p23-dependent NMR shifts mapped to both the lid and the adenine end of Hsp90's ATP binding pocket, but also to large parts of the middle domain. Shifts distant from the p23 binding site reflect p23-induced conformational changes in Hsp90. Together, we conclude that it is Hsp90's nucleotide-binding domain that triggers the formation of the Hsp90₂p23₂ complex. We anticipate that our NMR approach has significant impact on future studies of full-length Hsp90 with cofactors and substrates, but also for the development of Hsp90 inhibiting anticancer drugs.

Covalent attachment of ubiquitin to substrates is crucial to protein degradation, transcription regulation and cell signalling. Highly specific interactions between ubiquitin‐conjugating enzymes (E2) and ubiquitin protein E3 ligases fulfil essential roles in this process. We performed a global yeast‐two hybrid screen to study the specificity of interactions between catalytic domains of the 35 human E2s with 250 RING‐type E3s. Our analysis showed over 300 high‐quality interactions, uncovering a large fraction of new E2–E3 pairs. Both within the E2 and the E3 cohorts, several members were identified that are more versatile in their interaction behaviour than others. We also found that the physical interactions of our screen compare well with reported functional E2–E3 pairs in in vitro ubiquitination experiments. For validation we confirmed the interaction of several versatile E2s with E3s in in vitro protein interaction assays and we used mutagenesis to alter the E3 interactions of the E2 specific for K63 linkages, UBE2N(Ubc13), towards the K48‐specific UBE2D2(UbcH5B). Our data provide a detailed, genome‐wide overview of binary E2–E3 interactions of the human ubiquitination system. Synopsis Initially identified as the main process for protein degradation, ubiquitination is believed nowadays to be crucial for a wider range of cellular processes. The outcome of the ubiquitin‐conjugation reaction, and thereby the fate of the substrate, is heavily dependent on the number of ubiquitin molecules attached and how these ubiquitin molecules are inter‐connected. For example, attachment of a single ubiquitin moiety (mono‐ubiquitination) to substrates, serves mainly for signalling and transcriptional regulatory functions. On the other hand, substrates marked with a chain of ubiquitin linked through lysine‐48 are targeted for proteasomal degradation, whereas modification with ubiquitin linked through any of the other six lysines (like K63), serves multiple, often non‐proteolytical, goals like DNA repair and endocytosis. To deal with this complexity and to allow adequate ubiquitination in time and space, a highly sophisticated conjugation machinery has been developed. In a sequential manner, ubiquitin becomes activated by an ubiquitin‐activating enzyme (E1), which then transfers the ubiquitin to a group of ubiquitin‐conjugating enzymes (E2s). Next, ubiquitin‐loaded E2s are interacting with ubiquitin protein ligases (E3s) and ubiquitin is conjugated to substrates on recruitment by the E3. These three key enzymes are operating in a hierarchical system, wherein two E1s and 35 E2s have been found and hundreds of E3s have been identified in humans. Within the ubiquitin‐conjugating system, the E3s are the main determinants for substrate selection, whereas the E2s are determining which lysine within the ubiquitin molecule is used to construct chains. The pyramid‐like shape of the system allows ordered one‐on‐one interactions between the E1 and the complete family of E2s, to provide E2 with activated ubiquitin. In contrast, interactions between E2s and E3s indicate a high selectivity and specificity in E2–E3 complex formation. This is despite the observation that both E2 and E3 enzymes display high levels of sequence homology within their proposed interaction interface surfaces and that their three‐dimensional structure is conserved. To address this on a systems‐wide scale, we initiated a Y2H screen to study binary interactions between the catalytic domains of all human E2s and 250 human RING‐type E3 ligase domains. In an array‐based set‐up, ∼9000 binary E2–E3 combinations were systematically screened and interactions were quantified on colorimetric and auxotrophic selection. In total, the screen identified 346 high‐confidence interactions. For 20 E2s, one or more E3 interaction could be found, whereas one‐third of the E3s showed one or more interactions. Both interaction patterns of E2 and E3s display domains are more connected than the average number of domains. This indicates the existence of high‐connected E2s and E3s. Among the 346 binary interactions found many novel interactions were identified as compared with literature‐curated, low‐throughput interaction studies. Additional pull‐down analyses showed that many E2–E3 interactions could be independently reproduced indicating the quality of the found interactions. Finally, the interaction specificity of UBE2N(Ubc13), which is specific for K63 poly‐ubiquitin chains, was altered to that of the K48‐specific UBE2D2(UbcH5B). It is predicted that the mutant UBE2N(Ubc13) now directs K63‐chains on substrates that normally decorated with K48‐chains, opening intriguing possibilities for future research. Taken together, this network centred on human E2–E3 enzymes provides a high resolution, global overview of how the complexity of the ubiquitin system is organized at the level of individual enzymes. The organization of key players in this pathway reveal new insights into how the system functions as a whole and provide useful links between ubiquitin activation and the covalent conjugation to downstream targets. A high performance, genome‐wide yeast two‐hybrid screen is reported to identify binary interactions between the catalytic domains of all human ubiquitin‐conjugating enzymes (E2s) and RING‐type ubiquitin protein (E3s) ligases. The screen identified 346 high‐confidence E2‐E3 interactions. The E2‐E3 network, compared with reported biochemically functional literature‐curated interactions and additional pull‐down analysis, represents a comprehensive high quality network of interactions in the human ubiquitin‐proteasome system. In a first attempt, the RING interaction specificity of UBE2N(Ubc13) was altered to mimic that of UBE2D2(UbcH5B) and the UBE2N mutant is expected to modify certain substrates with K63 ubiquitin chains which otherwise become modified with K48 polyubiquitin chains.

Translational initiation factor 2 (IF2) is a guanine nucleotidebinding protein that can bind guanosine 3´,5´-(bis) diphosphate (ppGpp), an alarmone involved in stringent response in bacteria. In cells growing under optimal conditions, the GTP concentration is very high, and that of ppGpp very low. However, under stress conditions, the GTP concentration may decline by as much as 50%, and that of ppGpp can attain levels comparable to those of GTP. Here we show that IF2 binds ppGpp at the same nucleotide-binding site and with similar affinity as GTP. Thus, GTP and the alarmone ppGpp can be considered two alternative physiologically relevant IF2 ligands. ppGpp interferes with IF2-dependent initiation complex formation, severely inhibits initiation dipeptide formation, and blocks the initiation step of translation. Our data suggest that IF2 has the properties of a cellular metabolic sensor and regulator that oscillates between an active GTP-bound form under conditions allowing active protein syntheses and an inactive ppGpp-bound form when shortage of nutrients would be detrimental, if not accompanied by slackening of this synthesis.

Three-dimensional domain swapping is a common phenomenon in pancreatic-like ribonucleases. In the aggregated state, these proteins acquire new biological functions, including selective cytotoxicity against tumour cells. RNase A is able to dislocate both N- and C-termini, but usually this process requires denaturing conditions. In contrast, bovine seminal ribonuclease (BS-RNase), which is a homo-dimeric protein sharing 80% of sequence identity with RNase A, occurs natively as a mixture of swapped and unswapped isoforms. The presence of two disulfides bridging the subunits, indeed, ensures a dimeric structure also to the unswapped molecule. In vitro, the two BS-RNase isoforms interconvert under physiological conditions. Since the tendency to swap is often related to the instability of the monomeric proteins, in these paper we have analysed in detail the stability in solution of the monomeric derivative of BS-RNase (mBS) by a combination of NMR studies and Molecular Dynamics Simulations. The refinement of NMR structure and relaxation data indicate a close similarity with RNase A, without any evidence of aggregation or partial opening. The high compactness of mBS structure is confirmed also by H/D exchange, urea denaturation, and TEMPOL mapping of the protein surface. The present extensive structural and dynamic investigation of (monomeric) mBS did not show any experimental evidence that could explain the known differences in swapping between BS-RNase and RNase A. Hence, we conclude that the swapping in BS-RNase must be influenced by the distinct features of the dimers, suggesting a prominent role for the interchain disulfide bridges.

Background The activity of proteins within the cell is characterized by their motions, flexibility, interactions or even the particularly intriguing case of partially unfolded states. In the last two cases, a part of the protein is affected either by binding or unfolding and the detection of the respective perturbed and unperturbed region(s) is a fundamental part of the structural characterization of these states. This can be achieved by comparing experimental data of the same protein in two different states (bound/unbound, folded/unfolded). For instance, measurements of chemical shift perturbations (CSPs) from NMR 1 H- 15 N HSQC experiments gives an excellent opportunity to discriminate both moieties. Results We describe an innovative, automatic and unbiased method to distinguish perturbed and unperturbed regions in a protein existing in two distinct states (folded/partially unfolded, bound/unbound). The SAMPLEX program takes as input a set of data and the corresponding three-dimensional structure and returns the confidence for each residue to be in a perturbed or unperturbed state. Its performance is demonstrated for different applications including the prediction of disordered regions in partially unfolded proteins and of interacting regions in protein complexes. Conclusions The proposed approach is suitable for partially unfolded states of proteins, local perturbations due to small ligands and protein-protein interfaces. The method is not restricted to NMR data, but is generic and can be applied to a wide variety of information.