Jakob Meier-Credo

Lipid acquisition and transport are fundamental processes in all organisms. Here, we investigate the lipid uptake and delivery mechanism of the minimal model organism Mycoplasma pneumoniae . We show that the essential protein P116 can transport lipids between liposomes independently and without adenosine 5′-triphosphate consumption. Our structural data and molecular dynamics simulations reveal the mechanism by which the amino-terminal region of P116 perturbs the membrane, the lipid transfer route, and the regulation of membrane binding by the cargo mass within P116’s large hydrophobic cavity. When adequately filled with cargo, P116 undergoes a rapid conformational change that modulates membrane binding. Together, our results show that Mycoplasma developed an integrated lipid uptake and delivery machinery that simplifies the complex multiprotein pathways used by higher developed organisms.

In situ cryo-electron tomography (cryo-ET) offers direct access to protein structures in their native state and environment. A central challenge for this technique is the identification and classification of imaged cells, as sample heterogeneity with respect to cell type and functional state can significantly impact the data. Currently, cells can be classified using subtomogram averaging or fluorescent labeling, which require sufficiently distinguishable macromolecules, genetic modifications, or the availability of suitable labels. In MALDI-MS Imaging (MSI) individual cells can be identified and classified reliably, making use of the distributions of phospholipid species, metabolites and small proteins with characteristic profiles for specific cell types. Here we present an integrated pipeline combining MSI and in situ cryo-ET for single-cell identification and classification. Our approach enables MSI data acquisition directly from EM grids and can be used on any eukaryotic single-cell cryo-ET sample without prior modification in less than 36 h. We apply this workflow to describe cell-to-cell heterogeneity in cultured HeLa cells, to distinguish between distantly related HeLa and Polytomella cells, and between primary cultured rat neurons and glial cells. Finally we present a data integration workflow combining cryo-ET, light microscopy and MSI aligned in the same coordinate system to facilitate efficient data visualization and interpretation. We anticipate that this approach will change the way that structural biologists can approach cellular heterogeneity in cryo-ET, as cells can be identified and classified reliably post acquisition.

Photosystem II (PSII) assembly is a stepwise process in which intermediate complexes with auxiliary proteins are transiently formed to allow efficient de novo biogenesis or repair of damaged PSII. In particular, the role of extrinsic PSII subunits (PsbO, PsbU, PsbV) and auxiliary proteins such as Psb27 for the formation and photoactivation of the Mn ₄ O ₅ Ca cluster, which catalyzes the unique water splitting reaction in mature PSII, remains unclear. Using cryo-electron microscopy, we have determined the structure of two novel late-stage PSII assembly intermediates from Thermosynechococcus vestitus BP-1. In contrast to previous studies, the resulting monomeric PSII complexes contain both PsbJ and Psb27 and exhibit a fully mature acceptor side, while the oxygen evolving complex (OEC) is still in an immature state. The second complex additionally associates with the late-acting assembly factor Psb32 and the extrinsic subunit PsbV. While Psb32 has received little attention, its proposed role in the complex challenges the previous assumption that all extrinsic subunits associate spontaneously, as well as the notion that PsbO initiates binding and solely drives OEC formation. Our structures of the Psb27-PSII and Psb32-PSII intermediates provide novel insights, how structural changes of C-termini of the D1 and D2 core proteins regulate maturation of the OEC and how the catalytic side is prepared for binding of the Mn ₄ O ₅ Ca cluster. The Psb32-PSII complex potentially represents the final PSII assembly intermediate that precedes the incorporation and photoactivation of the Mn ₄ O ₅ Ca cluster, allowing us to explain the final steps in the PSII biogenesis and assembly pipeline in great detail, as only the two extrinsic subunits PsbO and PsbU are missing.

Lipid acquisition and transport are fundamental processes in all organisms. Here, we investigate the lipid uptake and delivery mechanism of the minimal model organism Mycoplasma pneumoniae. We show that the essential protein P116 can transport lipids between liposomes independently and without ATP consumption. Our structural data and molecular dynamics simulations reveal the mechanism by which the N-terminal region of P116 perturbs the membrane, the lipid transfer route, and the regulation of membrane binding by the cargo mass within P116's large hydrophobic cavity. When adequately filled with cargo, P116 undergoes a rapid conformational change that modulates membrane binding. Taken together, our results show that Mycoplasma developed one integrated lipid uptake and delivery machinery that simplifies the complex multi-protein pathways used by higher developed organisms.

Small proteins of around 50 aa in length have been largely overlooked in genetic and biochemical assays due to the inherent challenges with detecting and characterizing them. Recent discoveries of their critical roles in many biological processes have led to an increased recognition of the importance of small proteins for basic research and as potential new drug targets. One example is CcoM, a 36 aa subunit of the cbb3-type oxidase that plays an essential role in adaptation to oxygen-limited conditions in Pseudomonas stutzeri (P. stutzeri), a model for the clinically relevant, opportunistic pathogen Pseudomonas aeruginosa. However, as no comprehensive data were available in P. stutzeri, we devised an integrated, generic approach to study small proteins more systematically. Using the first complete genome as basis, we conducted bottom-up proteomics analyses and established a digest-free, direct-sequencing proteomics approach to study cells grown under aerobic and oxygen-limiting conditions. Finally, we also applied a proteogenomics pipeline to identify missed protein-coding genes. Overall, we identified 2921 known and 29 novel proteins, many of which were differentially regulated. Among 176 small proteins 16 were novel. Direct sequencing, featuring a specialized precursor acquisition scheme, exhibited advantages in the detection of small proteins with higher (up to 100%) sequence coverage and more spectral counts, including sequences with high proline content. Three novel small proteins, uniquely identified by direct sequencing and not conserved beyond P. stutzeri, were predicted to form an operon with a conserved protein and may represent de novo genes. These data demonstrate the power of this combined approach to study small proteins in P. stutzeri and show its potential for other prokaryotes.

Mycoplasma pneumoniae, responsible for approximately 30% of community-acquired human pneumonia, needs to extract lipids from the host environment for survival and proliferation. Here, we report a comprehensive structural and functional analysis of the previously uncharacterized protein P116 (MPN_213). Single-particle cryo-electron microscopy of P116 reveals a homodimer presenting a previously unseen fold, forming a huge hydrophobic cavity, which is fully accessible to solvent. Lipidomics analysis shows that P116 specifically extracts lipids such as phosphatidylcholine, sphingomyelin and cholesterol. Structures of different conformational states reveal the mechanism by which lipids are extracted. This finding immediately suggests a way to control Mycoplasma infection by interfering with lipid uptake.

Mitochondrial complex I is a redox-driven proton pump that generates proton-motive force across the inner mitochondrial membrane, powering oxidative phosphorylation and ATP synthesis in eukaryotes. We report the structure of complex I from the thermophilic fungus Chaetomium thermophilum , determined by cryoEM up to 2.4-Å resolution. We show that the complex undergoes a transition between two conformations, which we refer to as state 1 and state 2. The conformational switch is manifest in a twisting movement of the peripheral arm relative to the membrane arm, but most notably in substantial rearrangements of the Q-binding cavity and the E-channel, resulting in a continuous aqueous passage from the E-channel to subunit ND5 at the far end of the membrane arm. The conformational changes in the complex interior resemble those reported for mammalian complex I, suggesting a highly conserved, universal mechanism of coupling electron transport to proton pumping.

Identification and sequence determination by mass spectrometry have become routine analyses for soluble proteins. Membrane proteins, however, remain challenging targets due to their hydrophobicity and poor annotation. In particular small membrane proteins often remain unnoticed as they are largely inaccessible to Bottom-Up proteomics. Recent advances in structural biology, though, have led to multiple membrane protein complex structures being determined at sufficiently high resolution to detect uncharacterized, small subunits. In this work we offer a guide for the mass spectrometric characterization of solvent extraction-based purifications of small membrane proteins isolated from protein complexes and cellular membranes. We first demonstrate our Top-Down MALDI-MS/MS approach on a Photosystem II preparation, analyzing target protein masses between 2.5 and 9 kDa with high accuracy and sensitivity. Then we apply our technique to purify and sequence the mycobacterial ATP synthase c subunit, the molecular target of the antibiotic drug bedaquiline. We show that our approach can be used to directly track and pinpoint single amino acid mutations that lead to antibiotic resistance in only 4 h. While not applicable as a high-throughput pipeline, our MALDI-MS/MS and ISD-based approach can identify and provide valuable sequence information on small membrane proteins, which are inaccessible to conventional Bottom-Up techniques. We show that our approach can be used to unambiguously identify single-point mutations leading to antibiotic resistance in mycobacteria.

The multi-subunit membrane protein complex Photosystem II (PSII) catalyzes the light-driven oxidation of water and with this the initial step of photosynthetic electron transport in plants, algae, and cyanobacteria. Its biogenesis is coordinated by a network of auxiliary proteins that facilitate the stepwise assembly of individual subunits and cofactors, forming various intermediate complexes until fully functional mature PSII is present at the end of the process. In the current study, we purified PSII complexes from a mutant line of the thermophilic cyanobacterium Thermosynechococcus vestitus BP-1 in which the extrinsic subunit PsbO, characteristic for active PSII, was fused with an N-terminal Twin-Strep-tag. Three distinct PSII complexes were separated by ion-exchange chromatography after the initial affinity purification. Two complexes differ in their oligomeric state (monomeric and dimeric) but share the typical subunit composition of mature PSII. They are characterized by the very high oxygen-evolving activity of approx. 6,000 µmol O ₂ · (mg Chl·h) ^-1 . Analysis of the third (heterodimeric) PSII complex revealed lower oxygen-evolving activity of approx. 3,000 µmol O ₂ · (mg Chl·h) ^-1 and manganese content of 2.7 (± 0.2) per reaction center compared to 3.7 (± 0.2) of fully active PSII. Mass spectrometry and time-resolved fluorescence spectroscopy further indicated that PsbO is partially replaced by Psb27 in this PSII fraction, thus implying a role in the repair of the complex.