Abstract Septins are conserved cytoskeletal GTP-binding proteins that form higher-order structures critical for cytokinesis and polarized growth across opisthokonts. In budding yeast and humans, four homologous septins assemble into hetero-octameric protofilaments through alternating interactions between adjacent G-domains (G-interface) or N- and C-termini (NC-interface), which then polymerize end-to-end into filaments. How nucleotide binding and hydrolysis control filament assembly has remained elusive. We uncover a septin-specific mechanism in which nucleotide binding stabilizes the monomeric G-interface and primes it for protofilament formation. Cryo-EM and DEER spectroscopy reveal nucleotide-induced conformational changes that engage the G-interface and enable an NC-compatible conformation absent in the nucleotide-free state. In vitro binding and reconstitution assays confirm that G-interface formation is strictly nucleotide-dependent and required for subsequent NC-interface assembly. These findings establish unidirectional allosteric signaling from the G-to the NC-interface, revealing the molecular basis for controlled septin protofilament assembly.

Energy-converting NADH:ubiquinone oxidoreductase, respiratory complex I, is central to energy metabolism by coupling NADH oxidation and quinone reduction with proton translocation across the membrane. Electrons are transferred from the primary acceptor flavin mononucleotide via a chain of iron-sulfur clusters to quinone. The enigmatic cluster N1a is conserved, but not part of this electron transfer chain. We reported on variants of the complex in which N1a is not detectable by EPR spectroscopy. This was tentatively attributed to the lower redox potential of the variant N1a. However, it remained an open question, whether the variants contain this cluster at all. Here, we determined the structures of these variants by X-ray crystallography and cryogenic-electron microscopy. Cluster N1a is present in all variants and the shift of its redox potential is explained by nearby structural changes. A role of the cluster for the mechanism of the complex is discussed.

It has been recently realized that some Gram-negative organisms such as Escherichia coli produce a multiheme cytochrome c to serve as a quinol peroxidase that couples electrons from the quinol pool directly to H2O2. The E. coli version of this enzyme, termed YhjA, has been predicted to be a member of the bacterial cytochrome c peroxidase (bCCP) superfamily, where a novel N-terminal single-heme binding domain is fused to the canonical bCCP diheme domain found widely in Gram-negative bacteria. Here, we present an X-ray crystal structure of YhjA, revealing the triheme architecture that nature has employed to couple the quinol pool to the reduction of H2O2. We also show kinetic, spectroscopic, and electrochemical data that detail the differences between the three hemes that are observed in the structure, where two of the heme irons are both six-coordinate, ligated by Met and His residues, and the third peroxidatic heme is found to be five-coordinate. Electrocatalytic voltammetry of YhjA illustrates how the high-potential hemes serve as relays to the peroxidatic active site. Together, these data suggest a model of the catalytic chemistry of YhjA, illustrating how this member of the bCCP family may react with substrates and engage in multielectron redox reactions.

The septins are conserved, filament-forming, guanine nucleotide binding cytoskeletal proteins. They assemble into palindromic protofilaments which polymerize further into higher-ordered structures that participate in essential intracellular processes such as cytokinesis or polarity establishment. Septins belong structurally to the P-Loop NTPases but, unlike their relatives Ras or Rho, do not mediate signals to effectors through GTP binding and hydrolysis. Biochemical approaches addressing how and why septins utilize nucleotides are hampered by the lack of nucleotide free complexes. Using molecular dynamics simulations, we determined structural alterations and inter-subunit binding free energies in human and yeast septin dimer structures and in their in silico generated apo forms. An interchain salt bridge network around the septin unique β-meander, conserved across all kingdoms of septin containing species, is destabilized upon nucleotide removal, concomitant with disruption of the entire G-interface. Within this network, we confirmed a conserved arginine residue, which coordinates the guanine base of the nucleotide, as the central interaction hub. The essential role of this arginine for interface integrity was experimentally confirmed to be conserved in septins from yeast to human.

This study explores the potential of small light-oxygen-voltage (LOV) domains for utilization as protein spin labels in different dipolar EPR spectroscopy methods. The distinctive photochemical properties of selected LOV domain variants are exploited to generate a variety of (meta)stable flavin mononucleotide (FMN) radicals upon blue light absorption. Three different radicals, FMN·-, FMNH·, and an FMN-methionine radical, and an excited FMN triplet species, were generated. The FMN radicals were generated in LOV single domains and two model LOV1-LOV2 fusion proteins, and the latter proteins demonstrated that simple and effective orthogonal spin labeling can be performed. Subsequently, dipolar EPR experiments were conducted in aqueous solution and in cells with and without additional light excitation, in order to measure the distances between the FMN cofactor radicals, and to infer the structure and dynamics of the LOV domain proteins. Interestingly, all LOV1-LOV2 fusion proteins exhibit defined but largely distinct distances. This can be attributed to two factors: the respective LOV domains have different interactions with each other, and the presence of neutral FMN radicals leads to dimerization of the LOV1 domains. Nevertheless, using LOV domains as genetically encoded spin labels could offer numerous advantages. As a true molecular biology concept, labeling and measurements can be performed in any accessible cell type using light as the only stimulus. Additionally, the various paramagnetic FMN states enable the measurement of distances between two radicals, as well as between a radical and a triplet state.

Polyphosphate kinases (PPK) play crucial roles in various biological processes, including energy storage and stress responses, through their interaction with inorganic polyphosphate (polyP) and the intracellular nucleotide pool. Members of the PPK family 2 (PPK2s) catalyse polyP-consuming phosphorylation of nucleotides. In this study, we characterised two PPK2 enzymes from Bacillus cereus (BcPPK2) and Lysinibacillus fusiformis (LfPPK2) to investigate their substrate specificity and potential for selective nucleotide synthesis. Both enzymes exhibited a broad substrate scope, selectively converting over 85 % of pyrimidine nucleoside monophosphates (NMPs) to nucleoside diphosphates (NDPs), while nucleoside triphosphate (NTP) formation was observed only with purine NMPs. Preparative enzymatic synthesis of cytidine diphosphate (CDP) was applied to achieve an yield of 49 %. Finally, structural analysis of five crystal structures of BcPPK2 and LfPPK2 provided insights into their active sites and substrate interactions. This study highlights PPK2-II enzymes as promising biocatalysts for the efficient and selective synthesis of pyrimidine NDPs.

The septins of the yeast Saccharomyces cerevisiae assemble into hetero-octameric rods by alternating interactions between neighboring G-domains or N- and C-termini, respectively. These rods polymerize end to end into apolar filaments, forming a ring beneath the prospective new bud that expands during the cell cycle into an hourglass structure. The hourglass finally splits during cytokinesis into a double ring. Understanding these transitions as well as the plasticity of the higher order assemblies requires a detailed knowledge of the underlying structures. Here we present the first X-ray crystal structure of a tetrameric Shs1-Cdc12-Cdc3-Cdc10 complex at a resolution of 3.2 Å. Close inspection of the NC-interfaces of this and other septin structures reveals a conserved contact motif that is essential for NC-interface integrity of yeast and human septins in vivo and in vitro. Using the tetrameric structure in combination with AlphaFold-Multimer allowed us to propose a model of the octameric septin rod.

S-adenosyl-l-methionine-dependent methyltransferases (MTs) are involved in the C-methylation of a variety of natural products. The MTs SgvM from Streptomyces griseoviridis and MrsA from Pseudomonas syringae pv. syringae catalyze the methylation of the β-carbon atom of α-keto acids in the biosynthesis of the antibiotic natural products viridogrisein and 3-methylarginine, respectively. MrsA shows high substrate selectivity for 5-guanidino-2-oxovalerate, while other α-keto acids, such as the SgvM substrates 4-methyl-2-oxovalerate, 2-oxovalerate, and phenylpyruvate, are not accepted. Here we report the crystal structures of SgvM and MrsA in the apo form and bound with substrate or S-adenosyl-l-methionine. By investigating key residues for substrate recognition in the active sites of both enzymes and engineering MrsA by site-directed mutagenesis, the substrate range of MrsA was extended to accept α-keto acid substrates of SgvM with uncharged and lipophilic β-residues. Our results showcase the transfer of the substrate scope of α-keto acid MTs from different biosynthetic pathways by rational design.

NADH:ubiquinone oxidoreductase, respiratory complex I, plays a central role in cellular energy metabolism. As a major source of reactive oxygen species (ROS) it affects ageing and mitochondrial dysfunction. The novel inhibitor NADH-OH specifically blocks NADH oxidation and ROS production by complex I in nanomolar concentrations. Attempts to elucidate its structure by NMR spectroscopy have failed. Here, by using X-ray crystallographic analysis, we report the structure of NADH-OH bound in the active site of a soluble fragment of complex I at 2.0 Å resolution. We have identified key amino acid residues that are specific and essential for binding NADH-OH. Furthermore, the structure sheds light on the specificity of NADH-OH towards the unique Rossmann-fold of complex I and indicates a regulatory role in mitochondrial ROS generation. In addition, NADH-OH acts as a lead-structure for the synthesis of a novel class of ROS suppressors.

The S-adenosyl-L-methionine-dependent methyltransferase Rv0560c of Mycobacterium tuberculosis belongs to an orthologous group of heterocyclic toxin methyltransferases (Htm) which likely contribute to resistance of mycobacteria towards antimicrobial natural compounds as well as drugs. HtmM.t. catalyzes the methylation of the Pseudomonas aeruginosa toxin 2-heptyl-1-hydroxyquinolin-4(1H)-one (also known as 2-heptyl-4-hydroxyquinoline N-oxide), a potent inhibitor of respiratory electron transfer, its 1-hydroxyquinolin-4(1H)-one core (QNO), structurally related (iso)quinolones, and some mycobactericidal compounds. In this study, crystal structures of HtmM.t. in complex with S-adenosyl-L-homocysteine (SAH) and the methyl-accepting substrates QNO or 4hydroxyisoquinoline-1(2H)-one, or the methylated product 1-methoxyquinolin-4(1H)-one, were determined at < 1.9 angstrom resolution. The monomeric protein exhibits the typical Rossmann fold topology and conserved residues of class I methyltransferases. Its SAH binding pocket is connected via a short tunnel to a large solvent-accessible cavity, which accommodates the methyl-accepting substrate. Residues W44, F168, and F208 in connection with F212 form a hydrophobic clamp around the heteroaromatic ring of the methyl-accepting substrate and likely play a major role in substrate positioning. Structural and biochemical data suggest that H139 and T136 are key active site residues, with H139 acting as general base that activates the methyl-accepting hydroxy group. Our structural data may contribute to the design of Htm inhibitors or of antimycobacterial drugs unamenable for methylation.

Respiratory complex I plays a central role in cellular energy metabolism coupling NADH oxidation to proton translocation. In humans its dysfunction is associated with degenerative diseases. Here we report the structure of the electron input part of Aquifex aeolicus complex I at up to 1.8 Å resolution with bound substrates in the reduced and oxidized states. The redox states differ by the flip of a peptide bond close to the NADH binding site. The orientation of this peptide bond is determined by the reduction state of the nearby [Fe-S] cluster N1a. Fixation of the peptide bond by site-directed mutagenesis led to an inactivation of electron transfer and a decreased reactive oxygen species (ROS) production. We suggest the redox-gated peptide flip to represent a previously unrecognized molecular switch synchronizing NADH oxidation in response to the redox state of the complex as part of an intramolecular feed-back mechanism to prevent ROS production.

Inorganic polyphosphate is a ubiquitous, linear biopolymer built of up to thousands of phosphate residues that are linked by energy-rich phosphoanhydride bonds. Polyphosphate kinases of the family 2 (PPK2) use polyphosphate to catalyze the reversible phosphorylation of nucleotide phosphates and are highly relevant as targets for new pharmaceutical compounds and as biocatalysts for cofactor regeneration. PPK2s can be classified based on their preference for nucleoside mono-or diphosphates or both. The detailedmechanism of PPK2s and the molecular basis for their substrate preference is unclear, which is mainly due to the lack of high-resolution structures with substrates or substrate analogs. Here, we report the structural analysis and comparison of a class I PPK2 (ADP-phosphorylating) and a class III PPK2 (AMP- and ADP-phosphorylating), both complexed with polyphosphate and/or nucleotide substrates. Together with complementary biochemical analyses, these define the molecular basis of nucleotide specificity and are consistent with a Mg2+ catalyzed in-line phosphoryl transfer mechanism. This mechanistic insight will guide the development of PPK2 inhibitors as potential antibacterials or genetically modified PPK2s that phosphorylate alternative substrates.

Nitrogenases catalyze the biological fixation of inert N2 into bioavailable ammonium. They are bipartite systems consisting of the catalytic dinitrogenase and a complementary reductase, the Fe protein that is also the site where ATP is hydrolyzed to drive the reaction forward. Three different subclasses of dinitrogenases are known, employing either molybdenum, vanadium or only iron at their active site cofactor. Although in all these classes the mode and mechanism of interaction with Fe protein is conserved, each one encodes its own orthologue of the reductase in the corresponding gene cluster. Here we present the 2.2 Å resolution structure of VnfH from Azotobacter vinelandii, the Fe protein of the alternative, vanadium-dependent nitrogenase system, in its ADP-bound state. VnfH adopts the same conformation that was observed for NifH, the Fe protein of molybdenum nitrogenase, in complex with ADP, representing a state of the functional cycle that is ready for reduction and subsequent nucleotide exchange. The overall similarity of NifH and VnfH confirms the experimentally determined cross-reactivity of both ATP-hydrolyzing reductases.

Bromodomain and extra-terminal domain (BET) inhibitors are widely used both as chemical tools to study the biological role of their targets in living organisms and as candidates for drug development against several cancer variants and human disorders. However, non-BET bromodomains such as those in p300 and CBP are less studied. XDM-CBP is a highly potent and selective inhibitor for the bromodomains of CBP and p300 derived from a pan-selective BET BRD-binding fragment. Along with X-ray crystal-structure analysis and thermodynamic profiling, XDM-CBP was used in screenings of several cancer cell lines in vitro to study its inhibitory potential on cancer cell proliferation. XDM-CBP is demonstrated to be a potent and selective CBP/p300 inhibitor that acts on specific cancer cell lines, in particular malignant melanoma, breast cancer, and leukemia.

Mg2+‐dependent catechol‐O‐methyltransferases occur in animals as well as in bacteria, fungi and plants, often with a pronounced selectivity towards one of the substrate's hydroxyl groups. Here, we show that the bacterial MxSafC exhibits excellent regioselectivity for para as well as for meta methylation, depending on the substrate's characteristics. The crystal structure of MxSafC was solved in apo and in holo form. The structure complexed with a full set of substrates clearly illustrates the plasticity of the active site region. The awareness that a wide range of factors influences the regioselectivity will aid the further development of catechol‐O‐methyltransferases as well as other methyltransferases as selective and efficient biocatalysts for chemical synthesis.

Sirtuins are NAD(+)-dependent protein deacylases that cleave off acetyl but also other acyl groups from the ε-amino group of lysines in histones and other substrate proteins. Dysregulation of human Sirt2 (hSirt2) activity has been associated with the pathogenesis of cancer, inflammation, and neurodegeneration, which makes the modulation of hSirt2 activity a promising strategy for pharmaceutical intervention. The sirtuin rearranging ligands (SirReals) have recently been discovered by us as highly potent and isotype-selective hSirt2 inhibitors. Here, we present a well-defined structure-activity relationship study, which rationalizes the unique features of the SirReals and probes the limits of modifications on this scaffold regarding inhibitor potency. Moreover, we present a crystal structure of hSirt2 in complex with an optimized SirReal derivative that exhibits an improved in vitro activity. Lastly, we show cellular hyperacetylation of the hSirt2 targeted tubulin caused by our improved lead structure.

The two-component metalloprotein nitrogenase catalyzes the reductive fixation of atmospheric dinitrogen into bioavailable ammonium in diazotrophic prokaryotes. The process requires an efficient energy metabolism, so that although the metal clusters of nitrogenase rapidly decompose in the presence of dioxygen, many free-living diazotrophs are obligate aerobes. In order to retain the functionality of the nitrogen-fixing enzyme, some of these are able to rapidly "switch-off" nitrogenase, by shifting the enzyme into an inactive but oxygen-tolerant state. Under these conditions the two components of nitrogenase form a stable, ternary complex with a small [2Fe:2S] ferredoxin termed FeSII or the "Shethna protein II". Here we have produced and isolated Azotobacter vinelandii FeS II and have determined its three-dimensional structure to 2.1 Å resolution by X-ray diffraction. In the crystals, the dimeric protein was present in two distinct states that differ in the conformation of an extended loop in close proximity to the iron-sulfur cluster. We show that this rearrangement is redox-dependent and forms the molecular basis for oxygen-dependent conformational protection of nitrogenase. Protection assays highlight that FeSII binds to a preformed complex of MoFe and Fe protein upon activation, primarily through electrostatic interactions. The surface properties and known complexes of nitrogenase component proteins allow us to propose a model of the conformationally protected ternary complex of nitrogenase.