Biomedical subjects

Jan Löwe

Publications and source records attributed to Jan Löwe.

At least 19 recordsLinked to original sources

PubMed2006

A bacterial dynamin-like protein.

Dynamins form a superfamily of large mechano-chemical GTPases that includes the classical dynamins and dynamin-like proteins (DLPs). They are found throughout the Eukarya, functioning in core cellular processes such as endocytosis and organelle division. Many bacteria are predicted by sequence to possess large GTPases with the same multidomain architecture that is found in DLPs. Mechanistic dissection of dynamin family members has been impeded by a lack of high-resolution structural data currently restricted to the GTPase and pleckstrin homology domains, and the dynamin-related human guanylate-binding protein. Here we present the crystal structure of a cyanobacterial DLP in both nucleotide-free and GDP-associated conformation. The bacterial DLP shows dynamin-like qualities, such as helical self-assembly and tubulation of a lipid bilayer. In vivo, it localizes to the membrane in a manner reminiscent of FZL, a chloroplast-specific dynamin-related protein with which it shares sequence similarity. Our results provide structural and mechanistic insight that may be relevant across the dynamin superfamily. Concurrently, we show compelling similarity between a cyanobacterial and chloroplast DLP that, given the endosymbiotic ancestry of chloroplasts, questions the evolutionary origins of dynamins.

Animals↗

PubMed2006

The FtsK gamma domain directs oriented DNA translocation by interacting with KOPS.

The bacterial septum-located DNA translocase FtsK coordinates circular chromosome segregation with cell division. Rapid translocation of DNA by FtsK is directed by 8-base-pair DNA motifs (KOPS), so that newly replicated termini are brought together at the developing septum, thereby facilitating completion of chromosome segregation. Translocase functions reside in three domains, alpha, beta and gamma. FtsKalphabeta are necessary and sufficient for ATP hydrolysis-dependent DNA translocation, which is modulated by FtsKgamma through its interaction with KOPS. By solving the FtsKgamma structure by NMR, we show that gamma is a winged-helix domain. NMR chemical shift mapping localizes the DNA-binding site on the gamma domain. Mutated proteins with substitutions in the FtsKgamma DNA-recognition helix are impaired in DNA binding and KOPS recognition, yet remain competent in DNA translocation and XerCD-dif site-specific recombination, which facilitates the late stages of chromosome segregation.

Base Sequence↗

PubMed2006

RF cloning: a restriction-free method for inserting target genes into plasmids.

Restriction-free (RF) cloning provides a simple, universal method to precisely insert a DNA fragment into any desired location within a circular plasmid, independent of restriction sites, ligation, or alterations in either the vector or the gene of interest. The technique uses a PCR fragment encoding a gene of interest as a pair of primers in a linear amplification reaction around a circular plasmid. In contrast to QuickChange site-directed mutagenesis, which introduces single mutations or small insertions/deletions, RF cloning inserts complete genes without the introduction of unwanted extra residues. The absence of any alterations to the protein as well as the simplicity of both the primer design and the procedure itself makes it suitable for high-throughput expression and ideal for structural genomics.

Cell Line↗

PubMed2006

Double-stranded DNA translocation: structure and mechanism of hexameric FtsK.

FtsK is a DNA translocase that coordinates chromosome segregation and cell division in bacteria. In addition to its role as activator of XerCD site-specific recombination, FtsK can translocate double-stranded DNA (dsDNA) rapidly and directionally and reverse direction. We present crystal structures of the FtsK motor domain monomer, showing that it has a RecA-like core, the FtsK hexamer, and also showing that it is a ring with a large central annulus and a dodecamer consisting of two hexamers, head to head. Electron microscopy (EM) demonstrates the DNA-dependent existence of hexamers in solution and shows that duplex DNA passes through the middle of each ring. Comparison of FtsK monomer structures from two different crystal forms highlights a conformational change that we propose is the structural basis for a rotary inchworm mechanism of DNA translocation.

Adenosine Triphosphate↗

PubMed2006

Dynamic filaments of the bacterial cytoskeleton.

Bacterial cells contain a variety of structural filamentous proteins necessary for the spatial regulation of cell shape, cell division, and chromosome segregation, analogous to the eukaryotic cytoskeletal proteins. The molecular mechanisms by which these proteins function are beginning to be revealed, and these proteins show numerous three-dimensional structural features and biochemical properties similar to those of eukaryotic actin and tubulin, revealing their evolutionary relationship. Recent technological advances have illuminated links between cell division and chromosome segregation, suggesting a higher complexity and organization of the bacterial cell than was previously thought.

Actins↗

PubMed2005

Crystal structure of the ubiquitin-like protein YukD from Bacillus subtilis.

The YukD protein in Bacillus subtilis was identified in a hidden Markov model (HMM) search as being related in sequence to ubiquitin. By solving the crystal structure we show that YukD adopts a fold that is most closely related to ubiquitin, yet has the shortest C-terminal tail of all known ubiquitin-like proteins. The endogenous gene of yukD in B. subtilis was disrupted without an obvious phenotypic effect and an inducible copy encoding a C-Myc and His-tagged version of the protein was introduced at the ectopic locus amyE. Conjugation assays performed both in vitro and in vivo indicate that YukD lacks the capacity for covalent bond formation with other proteins.

Amino Acid Sequence↗

PubMed2005

Structure of bacterial tubulin BtubA/B: evidence for horizontal gene transfer.

alphabeta-Tubulin heterodimers, from which the microtubules of the cytoskeleton are built, have a complex chaperone-dependent folding pathway. They are thought to be unique to eukaryotes, whereas the homologue FtsZ can be found in bacteria. The exceptions are BtubA and BtubB from Prosthecobacter, which have higher sequence homology to eukaryotic tubulin than to FtsZ. Here we show that some of their properties are different from tubulin, such as weak dimerization and chaperone-independent folding. However, their structure is strikingly similar to tubulin including surface loops, and BtubA/B form tubulin-like protofilaments. Presumably, BtubA/B were transferred from a eukaryotic cell by horizontal gene transfer because their high degree of similarity to eukaryotic genes is unique within the Prosthecobacter genome.

Bacterial Proteins↗

PubMed2005

Towards understanding the molecular basis of bacterial DNA segregation.

Bacteria ensure the fidelity of genetic inheritance by the coordinated control of chromosome segregation and cell division. Here, we review the molecules and mechanisms that govern the correct subcellular positioning and rapid separation of newly replicated chromosomes and plasmids towards the cell poles and, significantly, the emergence of mitotic-like machineries capable of segregating plasmid DNA. We further describe surprising similarities between proteins involved in DNA partitioning (ParA/ParB) and control of cell division (MinD/MinE), suggesting a mechanism for intracellular positioning common to the two processes. Finally, we discuss the role that the bacterial cytoskeleton plays in DNA partitioning and the missing link between prokaryotes and eukaryotes that is bacterial mechano-chemical motor proteins.

Bacterial Physiological Phenomena↗

PubMed2005

Bacterial chromosome segregation: structure and DNA binding of the Soj dimer--a conserved biological switch.

Soj and Spo0J of the Gram-negative hyperthermophile Thermus thermophilus belong to the conserved ParAB family of bacterial proteins implicated in plasmid and chromosome partitioning. Spo0J binds to DNA near the replication origin and localises at the poles following initiation of replication. Soj oscillates in the nucleoid region in an ATP- and Spo0J-dependent fashion. Here, we show that Soj undergoes ATP-dependent dimerisation in solution and forms nucleoprotein filaments with DNA. Crystal structures of Soj in three nucleotide states demonstrate that the empty and ADP-bound states are monomeric, while a hydrolysis-deficient mutant, D44A, is capable of forming a nucleotide 'sandwich' dimer. Soj ATPase activity is stimulated by Spo0J or the N-terminal 20 amino-acid peptide of Spo0J. Our analysis shows that dimerisation and activation involving a peptide containing a Lys/Arg is conserved for Soj, ParA and MinD and their modulators Spo0J, ParB and MinE, respectively. By homology to the nitrogenase iron protein and the GTPases Ffh/FtsY, we suggest that Soj dimerisation and regulation represent a conserved biological switch.

Adenosine Triphosphate↗

PubMed2005

Increasing complexity of the bacterial cytoskeleton.

Bacteria contain cytoskeletal elements involved in major cellular processes including DNA segregation and cell morphogenesis and division. Distant bacterial homologues of tubulin (FtsZ) and actin (MreB and ParM) not only resemble their eukaryotic counterparts structurally but also show similar functional characteristics, assembling into filamentous structures in a nucleotide-dependent fashion. Recent advances in fluorescence microscopic imaging have revealed that FtsZ and MreB form highly dynamic helical structures that encircle the cells along the inside of the cell membrane. With the discovery of crescentin, a cell-shape-determining protein that resembles eukaryotic intermediate filament proteins, the third major cytoskeletal element has now been identified in bacteria as well.

Actins↗

PubMed2004

Structural insights into FtsZ protofilament formation.

The prokaryotic tubulin homolog FtsZ polymerizes into a ring structure essential for bacterial cell division. We have used refolded FtsZ to crystallize a tubulin-like protofilament. The N- and C-terminal domains of two consecutive subunits in the filament assemble to form the GTPase site, with the C-terminal domain providing water-polarizing residues. A domain-swapped structure of FtsZ and biochemical data on purified N- and C-terminal domains show that they are independent. This leads to a model of how FtsZ and tubulin polymerization evolved by fusing two domains. In polymerized tubulin, the nucleotide-binding pocket is occluded, which leads to nucleotide exchange being the rate-limiting step and to dynamic instability. In our FtsZ filament structure the nucleotide is exchangeable, explaining why, in this filament, nucleotide hydrolysis is the rate-limiting step during FtsZ polymerization. Furthermore, crystal structures of FtsZ in different nucleotide states reveal notably few differences.

Bacterial Proteins↗

PubMed2004

Robotic nanolitre protein crystallisation at the MRC Laboratory of Molecular Biology.

We have set up high-throughput robotic systems to screen and optimise crystallisation conditions of biological macromolecules with the aim to make difficult structural biology projects easier. The initial screening involves two robots. A Tecan Genesis liquid handler is used to transfer commercially available crystallisation reagents from 15 ml test tubes into the reservoirs of 96-well crystallisation plates. This step is fully automated and includes a carousel for intermediate plate storage, a Beckman plate sealer and a robotic arm, which transfers plates in between steps. For adding the sample, we use a second robot, a 17-tip Cartesian Technologies PixSys 4200 SynQuad liquid handler, which uses a syringe/solenoid valve combination to dispense small quantities of liquid (typically 100 nl) without touching the surface of the plate. Sixteen of the tips are used to transfer the reservoir solution to the crystallisation wells, while the 17th tip is used to dispense the protein. The screening of our standard set of 1440 conditions takes about 3 h and requires 300 microl of protein solution. Once crystallisation conditions have been found, they are optimised using a second Tecan Genesis liquid handler, which is programmed to pipette gradients from four different corner solutions into a wide range of crystallisation plate formats. For 96-well plates, the Cartesian robot can be used to add the sample. The methods described are now used almost exclusively for obtaining diffraction quality crystals in our laboratory with a throughput of several thousand plates per year. Our set-up has been copied in many institutions worldwide.

Combinatorial Chemistry Techniques↗

PubMed2004

Structure and stability of cohesin's Smc1-kleisin interaction.

A multisubunit complex called cohesin forms a huge ring structure that mediates sister chromatid cohesion, possibly by entrapping sister DNAs following replication. Cohesin's kleisin subunit Scc1 completes the ring, connecting the ABC-like ATPase heads of a V-shaped Smc1/3 heterodimer. Proteolytic cleavage of Scc1 by separase triggers sister chromatid disjunction, presumably by breaking the Scc1 bridge. One half of the SMC-kleisin bridge is revealed here by a crystal structure of Smc1's ATPase complexed with Scc1's C-terminal domain. The latter forms a winged helix that binds a pair of beta strands in Smc1's ATPase head. Mutation of conserved residues within the contact interface destroys Scc1's interaction with Smc1/3 heterodimers and eliminates cohesin function. Interaction of Scc1's N terminus with Smc3 depends on prior C terminus connection with Smc1. There is little or no turnover of Smc1-Scc1 interactions within cohesin complexes in vivo because expression of noncleavable Scc1 after DNA replication does not hinder anaphase.

Adenosine Triphosphatases↗

PubMed2004

The crystal structure of ZapA and its modulation of FtsZ polymerisation.

FtsZ is part of a mid-cell cytokinetic structure termed the Z-ring that recruits a hierarchy of fission related proteins early in the bacterial cell cycle. The widely conserved ZapA has been shown to interact with FtsZ, to drive its polymerisation and to promote FtsZ filament bundling thereby contributing to the spatio-temporal tuning of the Z-ring. Here, we show the crystal structure of ZapA (11.6 kDa) from Pseudomonas aeruginosa at 2.8 A resolution. The electron density reveals two dimers associating via an extensive C-terminal coiled-coil protrusion to form an elongated anti-parallel tetramer. In solution, ZapA exists in a dimer-tetramer equilibrium that is strongly correlated with concentration. An increase in concentration promotes formation of the higher oligomeric state. The dimer is postulated to be the predominant physiological species although the tetramer could become significant if, as FtsZ is integrated into the Z-ring and is cross-linked, the local concentration of the dimer becomes sufficiently high. We also show that ZapA binds FtsZ with an approximate 1:1 molar stoichiometry and that this interaction provokes dramatic FtsZ polymerisation and inter-filament association as well as yielding filaments, single or bundled, more stable and resistant to collapse. Whilst in vitro dynamics of FtsZ are well characterised, its in vivo arrangement within the ultra-structural architecture of the Z-ring is yet to be determined despite being fundamental to cell division. The ZapA dimer has single 2-fold symmetry whilst the bipolar tetramer displays triple 2-fold symmetry. Given the symmetry of these ZapA oligomers and the polar nature of FtsZ filaments, the structure of ZapA carries novel implications for the inherent architecture of the Z-ring in vivo.

Amino Acid Sequence↗

PubMed2004

Structural/functional homology between the bacterial and eukaryotic cytoskeletons.

Structural proteins are now known to be as necessary for controlling cell division and cell shape in prokaryotes as they are in eukaryotes. Bacterial ParM and MreB not only have atomic structures that resemble eukaryotic actin and form similar filaments, but they are also equivalent in function: the assembly of ParM drives intracellular motility and MreB maintains the shape of the cell. FtsZ resembles tubulin in structure and in its dynamic assembly, and is similarly controlled by accessory proteins. Bacterial MinD and eukaryotic dynamin appear to have similar functions in membrane control. In dividing eukaryotic organelles of bacterial origin, bacterial and eukaryotic proteins work together.

Actins↗

PubMed2004

Solution structure and domain architecture of the divisome protein FtsN.

Prokaryotic cell division occurs through the formation of a septum, which in Escherichia coli requires coordination of the invagination of the inner membrane, biosynthesis of peptidoglycan and constriction of the outer membrane. FtsN is an essential cell division protein and forms part of the divisome, a putative complex of proteins located in the cytoplasmic membrane. Structural analyses of FtsN by nuclear magnetic resonance (NMR) reveals an RNP-like fold at the C-terminus (comprising residues 243-319), which has significant sequence homology to a peptidoglycan-binding domain. Sequential deletion mutagenesis in combination with NMR shows that the remaining of the periplasmic region of FtsN is unfolded, with the exception of three short, only partially formed helices following the trans-membrane helix. Based on these findings we propose a model in which FtsN, anchored in the inner membrane, bridges over to the peptidoglycan layer, thereby enabling the coordination of the divisome and the murein-shaping machinery in the periplasm.

Amino Acid Sequence↗

PubMed2004

Structural analysis of the chromosome segregation protein Spo0J from Thermus thermophilus.

Prokaryotic chromosomes and plasmids encode partitioning systems that are required for DNA segregation at cell division. The plasmid partitioning loci encode two proteins, ParA and ParB, and a cis-acting centromere-like site denoted parS. The chromosomally encoded homologues of ParA and ParB, Soj and Spo0J, play an active role in chromosome segregation during bacterial cell division and sporulation. Spo0J is a DNA-binding protein that binds to parS sites in vivo. We have solved the X-ray crystal structure of a C-terminally truncated Spo0J (amino acids 1-222) from Thermus thermophilus to 2.3 A resolution by multiwavelength anomalous dispersion. It is a DNA-binding protein with structural similarity to the helix-turn-helix (HTH) motif of the lambda repressor DNA-binding domain. The crystal structure is an antiparallel dimer with the recognition alpha-helices of the HTH motifs of each monomer separated by a distance of 34 A corresponding to the length of the helical repeat of B-DNA. Sedimentation velocity and equilibrium ultracentrifugation studies show that full-length Spo0J exists in a monomer-dimer equilibrium in solution and that Spo0J1-222 is exclusively monomeric. Sedimentation of the C-terminal domain of Spo0J shows it to be exclusively dimeric, confirming that the C-terminus is the primary dimerization domain. We hypothesize that the C-terminus mediates dimerization of Spo0J, thereby effectively increasing the local concentration of the N-termini, which most probably dimerize, as shown by our structure, upon binding to a cognate parS site.

Amino Acid Sequence↗

PubMed2004

Murein (peptidoglycan) binding property of the essential cell division protein FtsN from Escherichia coli.

The binding of the essential cell division protein FtsN of Escherichia coli to the murein (peptidoglycan) sacculus was studied. Soluble truncated variants of FtsN, including the complete periplasmic part of the protein as well as a variant containing only the C-terminal 77 amino acids, did bind to purified murein sacculi isolated from wild-type cells. FtsN variants lacking this C-terminal region showed reduced or no binding to murein. Binding of FtsN was severely reduced when tested against sacculi isolated either from filamentous cells with blocked cell division or from chain-forming cells of a triple amidase mutant. Binding experiments with radioactively labeled murein digestion products revealed that the longer murein glycan strands (>25 disaccharide units) showed a specific affinity to FtsN, but neither muropeptides, peptides, nor short glycan fragments bound to FtsN. In vivo FtsN could be cross-linked to murein with the soluble disulfide bridge containing cross-linker DTSSP. Less FtsN, but similar amounts of OmpA, was cross-linked to murein of filamentous or of chain-forming cells compared to levels in wild-type cells. Expression of truncated FtsN variants in cells depleted in full-length FtsN revealed that the presence of the C-terminal murein-binding domain was not required for cell division under laboratory conditions. FtsN was present in 3,000 to 6,000 copies per cell in exponentially growing wild-type E. coli MC1061. We discuss the possibilities that the binding of FtsN to murein during cell division might either stabilize the septal region or might have a function unrelated to cell division.

Bacterial Proteins↗