Periodic Reporting for period 3 - Orgasome (Protein synthesis in organelles)
Periodo di rendicontazione: 2022-05-01 al 2024-11-30
Building on our accumulated knowledge of functional mechanisms of bioenergetic complexes and their synthesis, we take the structural investigation of mitochondria to the next step by dissecting their architecture and macromolecular networks also on the systemic level. Particularly, we will bring the emerging fields of mitochondrial biology, and membrane formation together with the most recent advances in cryo-ET and cryo-FIB. At the same time, on the molecular level, will continue the progress towards characterisation of less stable intermediates using the single-particle approach with a computational input to expand the insight to the modality of the RNA folding and produce models of evolution. We already found several new cofactors, which need to be incorporated into the nucleic acid environment. Those questions also involve a capacity of stepping away from model organisms into natural environment to generate new observations, and our preliminary data shows how it would effectively enhance the understanding.
For membrane proteins and membrane architecture, we have been specialised in multisubunit assemblies, i.e ATP synthases and respiratory complexes, their macro-organisation, and roles in membrane formation. This approach is currently being applied in our lab to a variety of organisms that have noticeable functional and compositional distinctions. In addition, such microorganisms are of particular interest because they permit insight into a mode of life that is extremely different. For example, we reported that the infectious Apicomplexa parasites differ substantially in their mitochondria and cristae morphology that is shaped by the ATP synthase cyclic hexamers localised to the curved apical membrane regions. Recent proteomic characterisation of the respiratory complexes also suggested substantial compositional differences, and most of the detected subunits were not previously known, hinting at a possible unique protein organisation. This leads us to examine the structures of a mitochondrial supercomplexes, their remodelling, and associated membrane changes during life cycle stages. Those targets are particularly attractive for structural biology techniques.
For membrane proteins and membrane architecture, we have been specialised in multisubunit assemblies, i.e ATP synthases and respiratory complexes, their macro-organisation, and roles in membrane formation. This approach is currently being applied in our lab to a variety of organisms that have noticeable functional and compositional distinctions. In addition, such microorganisms are of particular interest because they permit insight into a mode of life that is extremely different. For example, we reported that the infectious Apicomplexa parasites differ substantially in their mitochondria and cristae morphology that is shaped by the ATP synthase cyclic hexamers localised to the curved apical membrane regions. Recent proteomic characterisation of the respiratory complexes also suggested substantial compositional differences, and most of the detected subunits were not previously known, hinting at a possible unique protein organisation. This leads us to examine the structures of a mitochondrial supercomplexes, their remodelling, and associated membrane changes during life cycle stages. Those targets are particularly attractive for structural biology techniques.
One of the directions in our research is evolution of bioenergetics, where we apply the molecular and cellular approaches to non-model organisms. Through this exploration, not only that we engage in the biodiversity research, but also show that characterising bioenergetic molecules from different environments provides key insights into previously unknown fundamental mechanisms. For example, we obtained permission to work with a marine plant Posidonia, which is critical for ecosystem due to its ability to form multi-kilometre underwater meadows. Those represent clones of a single organism that is more than 10,000 years old, and its carbon absorption capacity is 15 times greater than that of rainforests. Because of the temperature rise in coastal waters, those plants are damaged, which leads to release of the carbon into the atmosphere. We found that photosystems of Posidonia have an architecture that includes additional light-harvesting proteins not reported for any other plant species. Then, by establishing AlphaFold pipeline and using RosetaDock, we’ve shown that the association is likely to be universal, but more pronounced in Posidonia due to an extra stabilization that is probably induced by a relatively low light and high salt environment.
Another example is a photosynthetic unicellular eukaryote Chromera that was discovered in corals of Sydney Harbor in 2008 and represents a transition form between symbiotic dinoflagellates and parasitic apicomplexan. Here, we discovered that a heterodimer of superoxide dismutase is associated with photosystem I. The mechanistic insight is that photosystem I is the major producer of reactive oxygen species, and thus superoxide dismutase is colocalised to act on those to reduce potential damage. As in the previous example, the finding has led to a series of computational and experimental analyses in the lab to show that the detected supercomplex is likely universally conserved in all species. In addition, in the cryo-EM map we identified a pigment density for which none of the known model library fits, and by isolating the pigment and obtaining its NMR structure, as well as of a reconstituted sample, we discovered a previously unknown type of pigment modification.
Those examples illustrate that investigating the processes of life in their natural context can be surprisingly informative.
Another example is a photosynthetic unicellular eukaryote Chromera that was discovered in corals of Sydney Harbor in 2008 and represents a transition form between symbiotic dinoflagellates and parasitic apicomplexan. Here, we discovered that a heterodimer of superoxide dismutase is associated with photosystem I. The mechanistic insight is that photosystem I is the major producer of reactive oxygen species, and thus superoxide dismutase is colocalised to act on those to reduce potential damage. As in the previous example, the finding has led to a series of computational and experimental analyses in the lab to show that the detected supercomplex is likely universally conserved in all species. In addition, in the cryo-EM map we identified a pigment density for which none of the known model library fits, and by isolating the pigment and obtaining its NMR structure, as well as of a reconstituted sample, we discovered a previously unknown type of pigment modification.
Those examples illustrate that investigating the processes of life in their natural context can be surprisingly informative.
With respect to the aspect of membrane proteins and cristae architecture, we currently apply the structural approach to parasites, which is a new venue. Parasites presumably have noticeable functional and compositional distinctions, which have not been investigated and might be related to disease. Such microorganisms and their relatives are of a special interest because they permit insight into a mode of life that is extremely different. Particularly, we find that the infectious Apicomplexa parasites differ substantially in their mitochondria and cristae morphology that is shaped by the ATP synthase cyclic hexamers localised to the curved apical membrane regions, which we reported last year. More recent proteomic characterisation of the apicomplexan respiratory complexes suggested substantial compositional differences, and most of the detected subunits were not previously known, hinting at a possible unique protein organisation. This leads us now to examine mitochondrial supercomplexes from parasites, their remodelling, and associated membrane changes during life cycle stages.
Among specific cases we’ve recently identified are: 1) 5.8-MDa assembly of 150 different subunits and 311 lipids that collectively form a CI-CII-CIII2-CIV2 megacomplex that is not only the largest of its kind, but also illustrates how the architecture reflects the functional specialization of bioenergetics by bending the membrane by 90 degrees (bioRxiv 2022). This organisation is coordinated with the ATP synthase tetramers to fuel the host cell. 2) CII2-CIII2-CIV2 supercomplex from Apicomplexa-related organism that has been solved to 2.1 Å resolution and allowed to detect some new biological phenomena. For example, a membrane protein splitting that reduces hydrophobicity to allow transfer of the corresponding encoding genes from mitochondria to the nucleolus representing a previously unknown evolutionary mechanism. Another example is direct evidence for +2 programmed translational frameshift we found in the structure of a protein synthesised on the mitoribosome of a parasite, and this occurrence is unprecedented in the studied biology, and has probably evolved via imbalance of available tRNAs in mitochondria, although the precise molecular mechanism remains to be investigated.
Among specific cases we’ve recently identified are: 1) 5.8-MDa assembly of 150 different subunits and 311 lipids that collectively form a CI-CII-CIII2-CIV2 megacomplex that is not only the largest of its kind, but also illustrates how the architecture reflects the functional specialization of bioenergetics by bending the membrane by 90 degrees (bioRxiv 2022). This organisation is coordinated with the ATP synthase tetramers to fuel the host cell. 2) CII2-CIII2-CIV2 supercomplex from Apicomplexa-related organism that has been solved to 2.1 Å resolution and allowed to detect some new biological phenomena. For example, a membrane protein splitting that reduces hydrophobicity to allow transfer of the corresponding encoding genes from mitochondria to the nucleolus representing a previously unknown evolutionary mechanism. Another example is direct evidence for +2 programmed translational frameshift we found in the structure of a protein synthesised on the mitoribosome of a parasite, and this occurrence is unprecedented in the studied biology, and has probably evolved via imbalance of available tRNAs in mitochondria, although the precise molecular mechanism remains to be investigated.