Markus A. Seeger

Nanobodies are the recombinant antigen binding fragments derived from camelid heavy chain-only antibodies (HCAbs). The serendipitous discovery of functional HCAbs in 19931 opened new avenues for si...

Eukaryotes inherited the core ribosome biogenesis apparatus from archaea. However, nucleocytoplasmic compartmentalisation and expansion to >200 assembly factors created the challenge of integrating this ancestral machinery into a complex maturation programme. One solution is the formation of transient modules in which newly acquired assembly factors support deeply conserved components. Here, we identify such a module, centred on the ancestral isoaspartylase Fap7, which couples the modification of the ribosomal protein uS11 to its incorporation into pre-ribosomes. Fap7 partners with Krr1 to capture uS11, forming an early checkpoint in which uS11 loading licenses Kri1 engagement and assembly progression. Loss of uS11 modification triggers a late checkpoint, preventing aberrant pre-ribosomes from acquiring translational competence. Integrative structure-function studies reveal how the ancestral isoaspartylase is embedded within a conserved eukaryotic assembly-factor network to safeguard the timing, order, and fidelity of ribosome production.

Synthetic nanobodies—also called sybodies—have proven valuable for stabilizing conformations of purified proteins, advancing structural and functional studies for example of transmembrane proteins. However, their utility in modulating protein function in living cells has remained less well explored. Structural Maintenance of Chromosomes (SMC) complexes facilitate chromosome organization, a fundamental process in all domains of life. In this study, we target the bacterial SMC complex, Smc-ScpAB, in Bacillus subtilis with synthetic nanobodies, aiming to identify key functional regions of the protein complex in a largely unbiased manner. We first isolate sybodies that specifically bind purified Smc-ScpAB and then express them in B. subtilis to select binders capable of disrupting Smc-ScpAB function, leading to chromosome segregation defects and cell death. Mapping and biochemical characterization show that the 14 disruptive sybodies belong to one of three library designs, target the Smc subunit near the same coiled coil arm interface and modulate its ATPase activity in two principal ways, highlighting the mid-region of the Smc coiled coil as critical feature of the SMC-DNA folding process. These findings underscore the potential of sybodies—and, by extension, designed binders—as versatile tools for probing dynamic protein function in living cells.

Synthetic nanobodies—also called sybodies—have proven valuable for stabilizing conformations of purified proteins, advancing structural and functional studies for example of transmembrane protein complexes. However, their utility in modulating protein function in living cells has remained less well explored. Structural Maintenance of Chromosomes (SMC) complexes facilitate chromosome organization by DNA loop extrusion, a fundamental process in all domains of life. In this study, we target the bacterial SMC complex, Smc-ScpAB, in Bacillus subtilis with synthetic nanobodies, aiming to identify key functional regions of the protein complex in a largely unbiased manner. We first isolate sybodies that specifically bind purified Smc-ScpAB and then express them in B. subtilis to select binders capable of disrupting Smc-ScpAB function, leading to chromosome segregation defects and cell death. Mapping and biochemical characterization show that the fourteen disruptive sybodies belong to one of three library designs, target the Smc subunit near the same coiled coil arm interface and modulate its ATPase activity in two principal ways, highlighting the mid-region of the Smc coiled coil as critical feature of the DNA folding process. These findings underscore the potential of sybodies—and, by extension, designed binders—as versatile tools for probing dynamic protein function in living cells, with potentially broad applications in cell and synthetic biology.

Mycobacteria, including Mycobacterium tuberculosis , possess a unique cell envelope containing arabinogalactan, a heteropolysaccharide critical for cell wall integrity and target of several tuberculosis drugs. The cytosolic precursor of arabinogalactan, lipid-linked galactan (LLG), is translocated across the plasma membrane by the essential ABC transporter Wzm-Wzt through a molecular mechanism that is poorly understood. Here, we present a series of cryo-EM structures of Wzm-Wzt from Mycobacterium abscessus , representing different conformations of the transport cycle. Conserved residues lining the proposed LLG translocation pathway were investigated by three orthologous functional assays, revealing that the cytosolic gate helix (GH) plays a key functional role in polysaccharide transport. Our data suggests that the hydrophobic polyprenyl-moiety is translocated first, followed by the galactan-polysaccharide, which requires Wzm-Wzt to open a continuous channel through which the sugar chain is ratcheted at the expense of ATP hydrolysis. Our results provide a rational basis for the development of drugs that inhibit mycobacterial cell wall biosynthesis.

Mycobacterium tuberculosis contains thirteen Mycobacterial membrane protein Large (MmpL) transporters, which belong to the family of secondary active RND transporters. MmpL4 and MmpL5, together with their operon partners MmpS4 and MmpS5, export the mycobacterial siderophore mycobactin and the last resort TB drug bedaquiline. Recently, we determined a structure of the MmpL4 monomer in complex with desferrated mycobactin, which lacked a functionally essential coiled-coil domain predicted to extend far into the periplasm. Here, we present a cryo-EM structure of the hexameric (MmpS4)3-(MmpL4)3 complex, which was enabled by rational disulfide cross-links based on AlphaFold predictions. We observed density for the coiled-coil domain, which protrudes into the periplasmic space at an angle of around 60° relative to the symmetry axis of the MmpL4 trimer. In the context of the hexameric complex, MmpL4’s conformation differs strikingly from the one observed for monomeric MmpL4, which includes formation of a large cavity in the periplasmic domain and rearrangements of conserved proton coupling residues at the transmembrane domain. Our work provides an experimental workflow to obtain single particle cryo-EM structures of labile multiprotein complexes by AlphaFold-informed stabilization of predicted protein interfaces.

Bacteria in surface-attached communities often engage in social interactions with neighbouring microbes. Spatial structure within these communities is thought to strongly influence these interactions, yet there is a significant lack of experimental platforms which allow for the tight spatial control of microbial interactions at the microscale, severely limiting our ability to investigate the relationship between spatial structure and community development. Here, we demonstrate a workflow for patterning and growing two bacterial species on a template with high throughput (∼10⁵ patterned cells per template) and micron-scale precision. We demonstrate a methodology for directional sequential capillary assembly of colloidal particles in combination with nanobody-functionalised particles that enable highly specific, bio-orthogonal binding reactions between bacteria and surface deposited particles. Using Staphylococcus aureus and Escherichia coli as model systems, we demonstrate how these organisms can be patterned in any desired spatial configuration where resulting communal growth can be monitored under the microscope. This technique enables careful investigations into the role of initial spatial structure on microbial interactions at low cell density, which is crucial to understanding and manipulating microbial community development.

Mycobacterium tuberculosis contains thirteen Mycobacterial membrane protein Large (MmpL) transporters, which belong to the family of secondary active RND transporters. MmpL4 and MmpL5, together with their operon partners MmpS4 and MmpS5, export the mycobacterial siderophore mycobactin and the last resort TB drug bedaquiline. Recently, we determined a structure of the MmpL4 monomer in complex with desferrated mycobactin, which lacked a functionally essential coiled-coil domain predicted to extend far into the periplasm. Here, we present a cryo-EM structure of the hexameric (MmpS4)3-(MmpL4)3 complex, which was enabled by rational disulfide cross-links based on AlphaFold predictions. We observed density for the coiled-coil domain, which protrudes into the periplasmic space at an angle of around 60° relative to the symmetry axis of the MmpL4 trimer. In the context of the hexameric complex, MmpL4’s conformation differs strikingly from the one observed for monomeric MmpL4, which includes formation of a large cavity in the periplasmic domain and rearrangements of conserved proton coupling residues at the transmembrane domain. Our work provides an experimental workflow to obtain single particle cryo-EM structures of labile multiprotein complexes by AlphaFold-informed stabilization of predicted protein interfaces.

Synthetic nanobodies—also called sybodies—have proven valuable for stabilizing conformations of purified proteins, advancing structural and functional studies for example of transmembrane protein complexes. However, their utility in modulating protein function in living cells has remained less well explored. Structural Maintenance of Chromosomes (SMC) complexes facilitate chromosome organization by DNA loop extrusion, a fundamental process in all domains of life. In this study, we target the bacterial SMC complex, Smc-ScpAB, in Bacillus subtilis with synthetic nanobodies, aiming to identify key functional regions of the protein complex in a largely unbiased manner. We first isolate sybodies that specifically bind purified Smc-ScpAB and then express them in B. subtilis to select binders capable of disrupting Smc-ScpAB function, leading to chromosome segregation defects and cell death. Mapping and biochemical characterization show that the fourteen disruptive sybodies belong to one of three library designs, target the Smc subunit near the same coiled coil arm interface and modulate its ATPase activity in two principal ways, highlighting the mid-region of the Smc coiled coil as critical feature of the DNA folding process. These findings underscore the potential of sybodies—and, by extension, designed binders—as versatile tools for probing dynamic protein function in living cells, with potentially broad applications in cell and synthetic biology.