The structure of the bacterial DNA segregation ATPase filament reveals the conformational plasticity of ParA upon DNA binding

AbstractThe efficient segregation of replicated genetic material is an essential step for cell division. Bacterial cells use several evolutionarily-distinct genome segregation systems, the most common of which is the type I Par system. It consists of an adapter protein, ParB, that binds to the DNA cargo via interaction with the parS DNA sequence; and an ATPase, ParA, that binds nonspecific DNA and mediates cargo transport. However, the molecular details of how this system functions are not well understood. Here, we report the cryo-EM structure of the Vibrio cholerae ParA2 filament bound to DNA, as well as the crystal structures of this protein in various nucleotide states. These structures show that ParA forms a left-handed filament on DNA, stabilized by nucleotide binding, and that ParA undergoes profound structural rearrangements upon DNA binding and filament assembly. Collectively, our data suggest the structural basis for ParA’s cooperative binding to DNA and the formation of high ParA density regions on the nucleoid.

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The cryo-EM structure of the bacterial type I DNA segregation ATPase filament reveals its conformational plasticity upon DNA binding.

10.1101/2021.03.22.436490 ◽

2021 ◽

Author(s):

Alexandra V Parker ◽

Daniel Mann ◽

Svetomir B Tzokov ◽

Ling Chin Hwang ◽

Julien RC Bergeron

Keyword(s):

Dna Binding ◽

Conformational Changes ◽

Genetic Material ◽

Structural Basis ◽

Type I ◽

Bacterial Cells ◽

Chromosome 2 ◽

Filament Formation ◽

Filament Assembly ◽

Conformational Plasticity

The efficient segregation of replicated genetic material is an essential step for cell division. In eukaryotic cells, sister chromatids are separated via the mitotic spindles. In contrast, bacterial cells use several evolutionarily-distinct genome segregation systems. The most common of these is the Type I Par system. It consists of an adapter protein, ParB, that binds to the DNA cargo via interaction with the parS DNA sequence; and an ATPase, ParA, that binds nonspecific DNA and mediates cargo transport. However, the molecular details of how this system functions are not well understood. Here, we report the cryo-EM structure of a ParA filament bound to its DNA template, using the chromosome 2 (Chr2) of Vibrio cholerae as a model system. We also report the crystal structures of this protein in various nucleotide states, which collectively offer insight into its conformational changes from dimerization through to DNA binding and filament assembly. Specifically, we show that the ParA dimer is stabilized by nucleotide binding, and forms a left-handed filament using DNA as a scaffold. Our structural analyses also reveal dramatic structural rearrangements upon DNA binding and filament assembly. Finally, we show that filament formation is controlled by nucleotide hydrolysis. Collectively, our data provide the structural basis for the cooperative binding of ParA to DNA and the formation of high ParA density regions on the nucleoid, and suggest a role for its filament formation.

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The Nucleoid-Associated Protein GapR Uses Conserved Structural Elements To Oligomerize and Bind DNA

mBio ◽

10.1128/mbio.00448-20 ◽

2020 ◽

Vol 11 (3) ◽

Author(s):

Rogério F. Lourenço ◽

Saumya Saurabh ◽

Jonathan Herrmann ◽

Soichi Wakatsuki ◽

Lucy Shapiro

Keyword(s):

Dna Binding ◽

Caulobacter Crescentus ◽

Genetic Material ◽

Bacterial Cells ◽

Structural Elements ◽

Content Type ◽

Time Dynamic ◽

Associated Proteins ◽

And Function

ABSTRACT Nucleoid-associated proteins (NAPs) are DNA binding proteins critical for the organization and function of the bacterial chromosome. A newly discovered NAP in Caulobacter crescentus, GapR, is thought to facilitate the movement of the replication and transcription machines along the chromosome by stimulating type II topoisomerases to remove positive supercoiling. Here, utilizing genetic, biochemical, and biophysical studies of GapR in light of a recently published DNA-bound crystal structure of GapR, we identified the structural elements involved in oligomerization and DNA binding. Moreover, we show that GapR is maintained as a tetramer upon its dissociation from DNA and that tetrameric GapR is capable of binding DNA molecules in vitro. Analysis of protein chimeras revealed that two helices of GapR are functionally conserved in H-NS, demonstrating that two evolutionarily distant NAPs with distinct mechanisms of action utilize conserved structural elements to oligomerize and bind DNA. IMPORTANCE Bacteria organize their genetic material in a structure called the nucleoid, which needs to be compact to fit inside the cell and, at the same time, dynamic to allow high rates of replication and transcription. Nucleoid-associated proteins (NAPs) play a pivotal role in this process, so their detailed characterization is crucial for our understanding of DNA organization into bacterial cells. Even though NAPs affect DNA-related processes differently, all of them have to oligomerize and bind DNA for their function. The significance of this study is the identification of structural elements involved in the oligomerization and DNA binding of a newly discovered NAP in C. crescentus and the demonstration that structural elements are conserved in evolutionarily distant and functionally distinct NAPs.

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Faculty Opinions recommendation of Structural basis of DNA recognition by PCG2 reveals a novel DNA binding mode for winged helix-turn-helix domains.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.725289371.793525937 ◽

2016 ◽

Author(s):

Nicolas Buchler

Keyword(s):

Dna Binding ◽

Dna Recognition ◽

Binding Mode ◽

Structural Basis ◽

Winged Helix

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Faculty Opinions recommendation of Structural basis for osmotic regulation of the DNA binding properties of H-NS proteins.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.737196844.793569585 ◽

2020 ◽

Author(s):

Charles Dorman

Keyword(s):

Dna Binding ◽

Structural Basis ◽

Osmotic Regulation ◽

Binding Properties ◽

Dna Binding Properties

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Rely on Each Other: DNA Binding Cooperativity Shapes p53 Functions in Tumor Suppression and Cancer Therapy

Cancers ◽

10.3390/cancers13102422 ◽

2021 ◽

Vol 13 (10) ◽

pp. 2422

Author(s):

Oleg Timofeev ◽

Thorsten Stiewe

Keyword(s):

Cancer Therapy ◽

Dna Binding ◽

Cell Fate ◽

Tumor Suppression ◽

Quaternary Structure ◽

Three Dimensional ◽

Structural Basis ◽

Sequence Specificity ◽

Cell Fate Decisions ◽

Cancer Mutations

p53 is a tumor suppressor that is mutated in half of all cancers. The high clinical relevance has made p53 a model transcription factor for delineating general mechanisms of transcriptional regulation. p53 forms tetramers that bind DNA in a highly cooperative manner. The DNA binding cooperativity of p53 has been studied by structural and molecular biologists as well as clinical oncologists. These experiments have revealed the structural basis for cooperative DNA binding and its impact on sequence specificity and target gene spectrum. Cooperativity was found to be critical for the control of p53-mediated cell fate decisions and tumor suppression. Importantly, an estimated number of 34,000 cancer patients per year world-wide have mutations of the amino acids mediating cooperativity, and knock-in mouse models have confirmed such mutations to be tumorigenic. While p53 cancer mutations are classically subdivided into “contact” and “structural” mutations, “cooperativity” mutations form a mechanistically distinct third class that affect the quaternary structure but leave DNA contacting residues and the three-dimensional folding of the DNA-binding domain intact. In this review we discuss the concept of DNA binding cooperativity and highlight the unique nature of cooperativity mutations and their clinical implications for cancer therapy.

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Structural basis of the p53 DNA binding domain and PUMA complex

Biochemical and Biophysical Research Communications ◽

10.1016/j.bbrc.2021.02.049 ◽

2021 ◽

Vol 548 ◽

pp. 39-46

Author(s):

Chang Woo Han ◽

Han Na Lee ◽

Mi Suk Jeong ◽

So Young Park ◽

Se Bok Jang

Keyword(s):

Dna Binding ◽

Binding Domain ◽

Structural Basis ◽

Dna Binding Domain

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Structural basis for ALK2/BMPR2 receptor complex signaling through kinase domain oligomerization

Nature Communications ◽

10.1038/s41467-021-25248-5 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Christopher Agnew ◽

Pelin Ayaz ◽

Risa Kashima ◽

Hanna S. Loving ◽

Prajakta Ghatpande ◽

...

Keyword(s):

Md Simulations ◽

Kinase Domain ◽

Structural Basis ◽

Type I ◽

Type Ii ◽

Exchange Mass Spectrometry ◽

Receptor Complexes ◽

Bmp Receptors ◽

Morphogenetic Protein ◽

Smad Proteins

AbstractUpon ligand binding, bone morphogenetic protein (BMP) receptors form active tetrameric complexes, comprised of two type I and two type II receptors, which then transmit signals to SMAD proteins. The link between receptor tetramerization and the mechanism of kinase activation, however, has not been elucidated. Here, using hydrogen deuterium exchange mass spectrometry (HDX-MS), small angle X-ray scattering (SAXS) and molecular dynamics (MD) simulations, combined with analysis of SMAD signaling, we show that the kinase domain of the type I receptor ALK2 and type II receptor BMPR2 form a heterodimeric complex via their C-terminal lobes. Formation of this dimer is essential for ligand-induced receptor signaling and is targeted by mutations in BMPR2 in patients with pulmonary arterial hypertension (PAH). We further show that the type I/type II kinase domain heterodimer serves as the scaffold for assembly of the active tetrameric receptor complexes to enable phosphorylation of the GS domain and activation of SMADs.

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A new model for packing of type-I collagen molecules in the native fibril

Bioscience Reports ◽

10.1007/bf01114803 ◽

1981 ◽

Vol 1 (10) ◽

pp. 801-810 ◽

Cited By ~ 49

Author(s):

Karl A. Piez ◽

Benes L. Trus

Keyword(s):

Type I Collagen ◽

Hexagonal Lattice ◽

Three Dimensional ◽

Equatorial Region ◽

Type I ◽

X Ray Diffraction ◽

X Ray ◽

Left Handed ◽

Crystal Model ◽

Collagen Molecules

A specific fibril model is presented consisting of bundles of five-stranded microfibrils, which are usually disordered (except axially) but under lateral compression become ordered. The features are as follows (where D = 234 residues or 67 nm): (1) D-staggered collagen molecules 4.5 D long in the helical microfibril have a left-handed supercoil with a pitch of 400–700 residues, but microfibrils need not have helical symmetry. (2) Straight-tilted 0.5-D overlap regions on a near-hexagonal lattice contribute the discrete x-ray diffraction reflections arising from lateral order, while the gap regions remain disordered. (3) The overlap regions are equivalent, but are crystallographically distinguished by systematic displacements from the near-hexagonal lattice. (4) The unit cell is the same as in a recently proposed three-dimensional crystal model, and calculated intensities in the equatorial region of the x-ray diffraction pattern agree with observed values.

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Crystallization of and selenomethionine phasing strategy for a SETMAR–DNA complex

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x16012723 ◽

2016 ◽

Vol 72 (9) ◽

pp. 713-719 ◽

Cited By ~ 2

Author(s):

Qiujia Chen ◽

Millie Georgiadis

Keyword(s):

Dna Binding ◽

Catalytic Domain ◽

Specific Binding ◽

Critical Role ◽

Binding Activity ◽

Structural Basis ◽

Unexpected Finding ◽

Terminal Inverted Repeat ◽

Dna Binding Activity ◽

New Genes

Transposable elements have played a critical role in the creation of new genes in all higher eukaryotes, including humans. Although the chimeric fusion protein SETMAR is no longer active as a transposase, it contains both the DNA-binding domain (DBD) and catalytic domain of theHsmar1transposase. The amino-acid sequence of the DBD has been virtually unchanged in 50 million years and, as a consequence, SETMAR retains its sequence-specific binding to the ancestralHsmar1terminal inverted repeat (TIR) sequence. Thus, the DNA-binding activity of SETMAR is likely to have an important biological function. To determine the structural basis for the recognition of TIR DNA by SETMAR, the design of TIR-containing oligonucleotides and SETMAR DBD variants, crystallization of DBD–DNA complexes, phasing strategies and initial phasing experiments are reported here. An unexpected finding was that oligonucleotides containing two BrdUs in place of thymidines produced better quality crystals in complex with SETMAR than their natural counterparts.

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