Biology subjects

Vollrath, R.

Publications and source records attributed to Vollrath, R..

4 recordsLinked to original sources

bioRxiv2023

Structural snapshots along K48-linked ubiquitin chain formation by the HECT E3 UBR5

Ubiquitin chain formation by HECT catalytic domain-containing E3 ligases regulates vast biology, yet the structural mechanisms remain unknown. We employed chemistry and cryo-EM to visualize stable mimics of the intermediates along K48-linked ubiquitin chain formation by the human E3, UBR5. The structural data reveal a {approx}620 kDa UBR5 dimer as the functional unit, comprising a scaffold with flexibly-tethered ubiquitin-binding UBA domains, and elaborately arranged HECT domains. Chains are forged by a UBA domain capturing an acceptor ubiquitin, with its K48 lured into the active site by numerous interactions between the acceptor ubiquitin, manifold UBR5 elements, and the donor ubiquitin. The cryo-EM reconstructions allow defining conserved HECT domain conformations catalyzing ubiquitin transfer from E2 to E3, and from E3. Our data show how a full-length E3, ubiquitins to be adjoined, E2, and intermediary products guide a feed-forward HECT domain conformational cycle establishing a highly efficient, broadly targeting, K48-linked ubiquitin chain forging machine.

biochemistry↗

bioRxiv2023

Insights into the ISG15 transfer cascade by the UBE1L activating enzyme

The attachment of the ubiquitin-like protein ISG15 to substrates is a well-established antiviral signalling mechanism of the innate immune response. However, despite the identification of thousands of substrates and clear roles in antiviral immunity, a molecular understanding of ISG15 selection and transfer through its cognate E1-E2- E3 enzyme cascade is largely unknown. Here, we present a 3.45 [A] cryo-EM structure of a chemically trapped UBE1L-UBE2L6 complex bound to activated ISG15. This structure reveals the details of the first steps of ISG15 recognition and UBE2L6 recruitment by UBE1L. Taking advantage of viral effector proteins from severe acute respiratory coronavirus 2 (SARS-CoV-2) and influenza B virus (IBV), we validated the structure and confirmed the importance of the ISG15 C-terminal ubiquitin-like domain in the adenylation reaction. Moreover, biochemical characterization of the UBE1L-ISG15 and UBE1L-UBE2L6 interactions enabled the design of ISG15 and UBE2L6 mutants with altered selectively for the ISG15 and ubiquitin conjugation pathways. Together, our study provides much needed insight into the specificity determinants that ensure the fidelity of ISG15 signalling during the antiviral response.

biochemistry↗

bioRxiv2019

A Protein Engineering Approach for Uncovering Cryptic Ubiquitin-binding Sites: from a Ubiquitin-Variant Inhibitor of APC/C to K48 Chain Binding

Ubiquitin-mediated proteolysis is a fundamental mechanism used by eukaryotic cells to maintain homeostasis and protein quality, and to control timing in biological processes. Two essential aspects of ubiquitin regulation are conjugation through E1-E2-E3 enzymatic cascades, and recognition by ubiquitin-binding domains. An emerging theme in the ubiquitin field is that these two properties are often amalgamated in conjugation enzymes. In addition to covalent thioester linkage to ubiquitins C-terminus for ubiquitin transfer reactions, conjugation enzymes often bind non-covalently and weakly to ubiquitin at \"exosites\". However, identification of such sites is typically empirical and particularly challenging in large molecular machines. Here, studying the 1.2 MDa E3 ligase Anaphase-Promoting Complex/Cyclosome (APC/C), which controls cell division and many aspects of neurobiology, we discover a method for identifying unexpected ubiquitin-binding sites. Using a panel of ubiquitin variants (UbVs) we identify a protein-based inhibitor that blocks ubiquitin ligation to APC/C substrates in vitro and ex vivo. Biochemistry, NMR, and cryo EM structurally define the UbV interaction, explain its inhibitory activity through binding the surface on the APC2 subunit that recruits the E2 enzyme UBE2C, and ultimately reveal that this APC2 surface is also a ubiquitin-binding exosite with preference for K48-linked chains. The results provide a new tool for probing APC/C activity, have implications for the coordination of K48-linked Ub chain binding by APC/C with the multistep process of substrate polyubiquitylation, and demonstrate the power of UbV technology for identifying cryptic ubiquitin binding sites within large multiprotein complexes.\n\nSIGNIFICANCE STATEMENTUbiquitin-mediated interactions influence numerous biological processes. These are often transient or a part of multivalent interactions. Therefore, unmasking these interactions remains a significant challenge for large, complicated enzymes such as the Anaphase-Promoting Complex/Cyclosome (APC/C), a multisubunit RING E3 ubiquitin (Ub) ligase. APC/C activity regulates numerous facets of biology by targeting key regulatory proteins for Ub-mediated degradation. Using a series of Ub variants (UbVs), we identified a new Ub-binding site on the APC/C that preferentially binds to K48-linked Ub chains. More broadly, we demonstrate a workflow that can be exploited to uncover Ub-binding sites within ubiquitylation machinery and other associated regulatory proteins to interrogate the complexity of the Ub code in biology.

biochemistry↗

bioRxiv2018

Molecular mechanisms in fungal fatty acid synthase (FAS) assembly

The fungal fatty acid synthase (fFAS) multienzyme is a barrel-shaped 2.6 MDa complex comprising six times eight catalytic domains. Upon barrel-formation, up to several hundred kDa large polypeptides intertwine to bury about 170,000 [A]2 of protein surface. Functional, regulatory and structural data as well as evolutionary aspects of fFAS have been elucidated during the last decades. Notwithstanding a profound knowledge of this protein family, the biogenesis of the elaborate structure remained elusive. Remarkably, experimental data have recently demonstrated that fFAS self-assembles without the assistance of specific factors. Considering the infinitesimal probability that the barrel-shaped complex forms simply by domains approaching in the correct orientation, we were interested in understanding the sequence of events that have to orchestrate fFAS assembly. Here, we show that fFAS attains its quaternary structure along a pathway of successive domain-domain interactions, which is strongly related to the evolutionary development of this protein family. The knowledge on fFAS assembly may pave the way towards antifungal therapy, and further develops fFAS as biofactory in technological applications.

molecular biology↗