InstructThe latest webinar in the Instruct-ERIC Structure Meets Function series will be hosted by Instruct Spain on 10th May 2022, 11:00 - 12:30 CET. Watch the webinar below.
Featuring expert speakers from Instruct Centres across Europe, Instruct-ERIC Webinar Series: Structure Meets Function highlights some of the latest developments in structural biology, demonstrating how integrative methods are enabling scientists to decipher the mechanisms that underpin health and disease.
Watch the previous webinars in the series here.
Moderator: Jose Maria Carazo
Talk 1: Characterisation of the Chikungunya virus nsP1 RNA capping pathway using the cryoEM facilities at Instruct CNB
Speaker: Rhian Jones
Abstract: Chikungunya virus (CHIKV) is an arbovirus that causes debilitating joint pain and fever in infected human hosts, where symptoms can persist for many years following infection. Currently, there are no available antivirals against CHIKV or members of the alphavirus family.
During infection, replication of the CHIKV single stranded positive sense RNA genome is orchestrated by a complex of four virally encoded non-structural proteins (nsPs) within a replication complex (RC). RCs are housed within invaginations (replication organelles or ROs) at the host plasma membrane that are generated during viral infection. We recently demonstrated that nsP1 forms membrane anchored dodecameric rings that gate the entrance to the RO and solved the structure by cryoEM in collaboration with the Instruct facility at the CNB.
NsP1 is also responsible for the addition of a 7-methylguanosine cap0 structure to the 5’ end of nascently synthesized CHIKV RNA, allowing for hijacking of the host translational machinery. NsP1 caps the RNA via a non-canonical pathway, possessing both methyltransferase and guanyltransferase activities and forming a covalent complex with the cap structure prior to transferal to the RNA. Using the Instruct cryoEM facilities at the CNB, we solved the structure of nsP1 in complex with SAM and GTP substrates at different stages of the pathway, providing insights into the specificity of the reactions. Although the SAM binding site is highly conserved with other methyltransferases, we demonstrate that the GTP pocket is slightly reconfigured to allow for transfer of a newly formed cap structure to the catalytic histidine residue. We show that simultaneous occupancy of the SAM and GTP substrate sites may be necessary for correct positioning of the substrates, possibly underlying the non-canonical order of the capping reactions. Finally, using complementary biochemical assays, we demonstrate that nsP1 exhibits sequence and structural specificity in capping of RNA substrates, and is also capable of cap removal from the viral RNA.
The results could provide a molecular rationale for the recent observations that many CHIKV virions contain uncapped RNAs and will also inform the design of novel therapeutics against CHIKV.
Talk 2: From Structure to Function and Back - identifying a medically crucial membrane protein by CryoEM
Speaker: Simone Weyand
Abstract: Neglected tropical diseases are a major burden on several continents and often difficult to treat or even untreatable. In this talk we deciphered a protein, which causes drug resistance in one of these fatal diseases and by solving the protein structure of the wildtype and drug bound states, we are now in a position to understand its molecular mechanism and new and highly effective drugs can be designed.
Talk 3: Visualizing key regulatory allosteric mechanisms in bacterial glycogen biosynthesis
Speaker: Javier Cifuente
Abstract: Glycogen is a classic glucose polymer that functions as a central carbon and energy reserve in bacteria and other organisms. In this regard, the evolution of the glycogenesis pathway resulted in the acquisition of mechanisms to control glucose storage according to the cell's energy status. In bacteria, the tetrameric enzyme ADP-glucose pyrophosphorylase (AGPase) catalyzes and regulates the production of the sugar donor ADP-glucose, the first step in glycogen biosynthesis. AGPase displays cooperativity and allosteric regulation by sensing metabolites from the cell energy flux.
Our work unveils snapshots of AGPase's allosteric signal transductions, a challenge we afford using a multidisciplinary approach centered on X-ray crystallography and cryoEM. Specifically, we disclosed the structures of E. coli AGPase in complex with either positive or negative physiological allosteric regulators, FBP and AMP, revealing the regulators' binding sites. Moreover, we show how regulators promote conformational changes at the active site, modulating substrates binding and activating the enzyme. Our results provide unique details laying the foundations for understanding the allosteric control of bacterial glycogen biosynthesis at the molecular level of detail.
