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.     

Two Mms2 residues cooperatively interact with ubiquitin and are critical for Lys63 polyubiquitination in vitro and in vivo

Recent structural analyses support a model whereby Mms2 interacts with and orientates Ub to promote Ubc13-mediated Lys63 chain formation. However, residues of the hMms2-Ub interface have not been addressed. We found two hMms2 residues to be critical for binding and polyUb conjugation. Surprisingly, while each single mutation reduces the binding affinity, the double mutation causes significant reduction of Ub binding and abolishes polyUb chain formation. Furthermore, the corresponding yeast mms2 double mutant exhibited an additive phenotype that caused a complete loss of MMS2 function. Taken together, this study identifies key residues of the Mms2-Ub interface and provides direct experimental evidence that Mms2 physical association with Ub is correlated with its ability to promote Lys63-linked Ub chain assembly.     

Development of concepts for human labour accounting in Emergy Assessment and other Environmental Sustainability Assessment methods

Human labour is central to the functioning of any human-influenced process. Nevertheless, Environmental Sustainability Assessments (ESAs) do not systematically include human labour as an input. Systematic omission of labour inputs in ESAs may constitute an unfortunate, significant bias in favour of labour intensive processes and a systematic underestimation of environmental impacts has implications for decision-making. A brief review of the evaluation of human labour in ESAs reveals that only Emergy Assessment (EmA) accounts for labour as standard. Focussing on EmA, we find, however, that there is no agreement on the calculation method for labour. We formalise the calculation of human labour unit emergy values (UEVs) as being the ratio between the emergy resource basis of the labour system and a proxy for labour, with or without allocation to account for different qualities of labour. The formalised calculation approach is demonstrated using examples from the literature (USA, with allocation based on educational level; Ghana, with allocation based on income level; the World, with no allocation). We elaborate on how labour may be considered as endogenous or exogenous to the studied system, and how inputs can be categorised as direct labour taking place in the system under study and indirect labour occurring upstream in the supply chain associated with the studied system. With appropriate modifications, the formalised calculation approach and the distinction between direct and indirect labour may be transferred to other ESA methodologies. Concerning EmA, we recommend that product UEVs should systematically be calculated with and without labour, and that working hours rather than salary should be used when accounting for labour inputs. We recognise that there is a risk of double counting of environmental impacts when including labour. We conclude, however, that it can be ignored for most production systems, since only a negligible fraction of emergy already accounted for is likely to be included in the emergy flow from labour inputs.     

Proline-rich regions and motifs in trafficking: from ESCRT interaction to viral exploitation

Most membrane-enveloped viruses bud from infected cells by hijacking the host ESCRT machinery. The ESCRTs are recruited to the budding sites by viral proteins that contain short proline (Pro)-rich motifs (PRMs) known as late domains. The late domains probably evolved by co-opting host PRMs involved in the normal functions of ESCRTs in endosomal sorting and cytokinesis. The solution and crystal structures of PRMs bound to their interaction partners explain the conserved roles of Pro and other residues that predominate in these sequences. PRMs are often grouped together in much larger Pro-rich regions (PRRs) of as many as 150 residues. The PRR of the ESCRT-associated protein, ALIX, autoregulates its conformation and activity. The robustness of different viral budding and host pathways to impairments in Pro-based interactions varies considerably. The known biology of PRM recognition in the ESCRT pathway seems, in principle, compatible with antiviral development, given our increasingly nuanced understanding of the relative weakness and robustness of the host and viral processes.     

A Mechanism for Protein Monoubiquitination Dependent on a trans-Acting Ubiquitin-binding Domain

The length of the ubiquitin chain on a substrate dictates various functional outcomes, yet little is known about its regulation in vivo. The yeast arrestin-related protein Rim8/Art9 is monoubiquitinated in vivo by the Rsp5 ubiquitin ligase. This also requires Vps23, a protein that displays an ubiquitin-E2 variant (UEV) domain. Here, we report that binding of the UEV domain to Rim8 interferes with ubiquitin chain elongation and directs Rim8 monoubiquitination. We propose that Vps23 UEV competes with Rsp5 HECT N-lobe for binding to the first conjugated ubiquitin, thereby preventing polyubiquitination. These findings reveal a novel mechanism to control ubiquitin chain length on substrates in vivo.