scholarly journals Structural insights into the interaction of helicase and primase in Mycobacterium tuberculosis

2018 ◽  
Vol 475 (21) ◽  
pp. 3493-3509 ◽  
Author(s):  
Dhakaram Pangeni Sharma ◽  
Ramachandran Vijayan ◽  
Syed Arif Abdul Rehman ◽  
Samudrala Gourinath
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Replication Initiation ◽  
Binding Studies ◽  
Functional Conservation ◽  
Terminal Domain ◽  
Binding Domains ◽  
Initiation System ◽  
Helical Hairpin ◽  
Helicase Dnab ◽  
Species Specific

The helicase–primase interaction is an essential event in DNA replication and is mediated by the highly variable C-terminal domain of primase (DnaG) and N-terminal domain of helicase (DnaB). To understand the functional conservation despite the low sequence homology of the DnaB-binding domains of DnaGs of eubacteria, we determined the crystal structure of the helicase-binding domain of DnaG from Mycobacterium tuberculosis (MtDnaG-CTD) and did so to a resolution of 1.58 Å. We observed the overall structure of MtDnaG-CTD to consist of two subdomains, the N-terminal globular region (GR) and the C-terminal helical hairpin region (HHR), connected by a small loop. Despite differences in some of its helices, the globular region was found to have broadly similar arrangements across the species, whereas the helical hairpins showed different orientations. To gain insights into the crucial helicase–primase interaction in M. tuberculosis, a complex was modeled using the MtDnaG-CTD and MtDnaB-NTD crystal structures. Two nonconserved hydrophobic residues (Ile605 and Phe615) of MtDnaG were identified as potential key residues interacting with MtDnaB. Biosensor-binding studies showed a significant decrease in the binding affinity of MtDnaB-NTD with the Ile605Ala mutant of MtDnaG-CTD compared with native MtDnaG-CTD. The loop, connecting the two helices of the HHR, was concluded to be largely responsible for the stability of the DnaB–DnaG complex. Also, MtDnaB-NTD showed micromolar affinity with DnaG-CTDs from Escherichia coli and Helicobacter pylori and unstable binding with DnaG-CTD from Vibrio cholerae. The interacting domains of both DnaG and DnaB demonstrate the species-specific evolution of the replication initiation system.

scholarly journals Structural insights into DNA replication initiation in Helicobacter pylori

2014 ◽  
Vol 70 (a1) ◽  
pp. C1632-C1632
Author(s):  
Alexandre Bazin ◽  
Mickaël Cherrier ◽  
Laurent Terradot
Keyword(s):  
Helicobacter Pylori ◽  
Dna Replication ◽  
Bacterial Genome ◽  
Coli Strain ◽  
Replication Initiation ◽  
Replicative Helicase ◽  
Dna Replication Initiation ◽  
Terminal Domain ◽  
Helicase Dnab ◽  
H Pylori

In Gram-negative bacteria, opening of DNA double strand during replication is performed by the replicative helicase DnaB. This protein allows for replication fork elongation by unwinding DNA and interacting with DnaG primase. DnaB is composed of two domains: an N-terminal domain (NTD) and a C-terminal domain (CTD) connected by a flexible linker. The protein forms two-tiered hexamers composed of a NTD-ring and a CTD-ring. In Escherichia coli, the initiator protein DnaA binds to the origin of replication oriC and induces the opening of a AT-rich region. The replicative helicase DnaB is then loaded onto single stranded DNA by interacting with DnaA and with the AAA+ helicase loader DnaC. However, AAA+ loaders are absent in 80% of the bacterial genome, raising the question of how helicases are loaded in these bacteria [1]. In the genome of human pathogen Helicobacter pylori, no AAA+ loader has been identified. Moreover H. pylori DnaB (HpDnaB) has the ability to support replication of an otherwise unviable E. coli strain that bears a defective copy of DnaC by complementation [2]. In order to better understand the properties of HpDnaB we have first shown that HpDnaB forms double hexamers by negative stain electron microscopy [3]. Then, we have then solved the crystal structure of HpDnaB at a resolution of 6.7Å by X-ray crystallography with Rfree/Rfactor of 0.29/0.25. The structure reveals that the protein adopts a new dodecameric arrangement generated by crystallographic three fold symmetry. When compared to hexameric DnaBs, the hexamer of HpDnaB displays an original combination of NTD-ring and CTD-ring symmetries, intermediate between apo and ADP-bound structure. Biochemistry studies of HpDnaB interaction with HpDnaG-CTD and ssDNA provides mechanistic insights into the initial steps of DNA replication in H. pylori. Our results offer an alternative solution of helicase loading and DNA replication initiation in H. pylori and possibly other bacteria that do not employ helicase loaders.

scholarly journals Structural Basis for the C-Terminal Domain of Mycobacterium tuberculosis Ribosome Maturation Factor RimM to Bind Ribosomal Protein S19

Biomolecules ◽  
2021 ◽  
Vol 11 (4) ◽  
pp. 597
Author(s):  
Haoran Zhang ◽  
Qiuxiang Zhou ◽  
Chenyun Guo ◽  
Liubin Feng ◽  
Huilin Wang ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Ribosomal Protein ◽  
Solution Structure ◽  
Molecular Mechanisms ◽  
Structural Model ◽  
Ribosomal Subunit ◽  
Structural Basis ◽  
Hydrophobic Core ◽  
Terminal Domain ◽  
Ribosomal Protein S19

Multidrug-resistant tuberculosis (TB) is a serious threat to public health, calling for the development of new anti-TB drugs. Chaperon protein RimM, involved in the assembly of ribosomal protein S19 into 30S ribosomal subunit during ribosome maturation, is a potential drug target for TB treatment. The C-terminal domain (CTD) of RimM is primarily responsible for binding S19. However, both the CTD structure of RimM from Mycobacterium tuberculosis (MtbRimMCTD) and the molecular mechanisms underlying MtbRimMCTD binding S19 remain elusive. Here, we report the solution structure, dynamics features of MtbRimMCTD, and its interaction with S19. MtbRimMCTD has a rigid hydrophobic core comprised of a relatively conservative six-strand β-barrel, tailed with a short α-helix and interspersed with flexible loops. Using several biophysical techniques including surface plasmon resonance (SPR) affinity assays, nuclear magnetic resonance (NMR) assays, and molecular docking, we established a structural model of the MtbRimMCTD–S19 complex and indicated that the β4-β5 loop and two nonconserved key residues (D105 and H129) significantly contributed to the unique pattern of MtbRimMCTD binding S19, which might be implicated in a form of orthogonality for species-dependent RimM–S19 interaction. Our study provides the structural basis for MtbRimMCTD binding S19 and is beneficial to the further exploration of MtbRimM as a potential target for the development of new anti-TB drugs.

scholarly journals Transcription Activation Domains of the Yeast Factors Met4 and Ino2: Tandem Activation Domains with Properties Similar to the Yeast Gcn4 Activator

2018 ◽  
Vol 38 (10) ◽  
Author(s):  
Derek Pacheco ◽  
Linda Warfield ◽  
Michelle Brajcich ◽  
Hannah Robbins ◽  
Jie Luo ◽  
...  
Keyword(s):  
Transcription Initiation ◽  
Transcription Activation ◽  
Binding Studies ◽  
Eukaryotic Transcription ◽  
Activation Domains ◽  
Conserved Sequence ◽  
Functional Studies ◽  
Intrinsically Disordered ◽  
Binding Domains ◽  
Optimal Function

ABSTRACTEukaryotic transcription activation domains (ADs) are intrinsically disordered polypeptides that typically interact with coactivator complexes, leading to stimulation of transcription initiation, elongation, and chromatin modifications. Here we examined the properties of two strong and conserved yeast ADs: Met4 and Ino2. Both factors have tandem ADs that were identified by conserved sequence and functional studies. While the AD function of both factors depended on hydrophobic residues, Ino2 further required key conserved acidic and polar residues for optimal function. Binding studies showed that the ADs bound multiple Med15 activator-binding domains (ABDs) with similar orders of micromolar affinity and similar but distinct thermodynamic properties. Protein cross-linking data show that no unique complex was formed upon Met4-Med15 binding. Rather, we observed heterogeneous AD-ABD contacts with nearly every possible AD-ABD combination. Many of these properties are similar to those observed with yeast activator Gcn4, which forms a large heterogeneous, dynamic, and fuzzy complex with Med15. We suggest that this molecular behavior is common among eukaryotic activators.

The N-terminal domain of Sxl protein disrupts Sxl autoregulation in females and promotes female-specific splicing of tra in males

Development ◽  
1999 ◽  
Vol 126 (13) ◽  
pp. 2841-2853 ◽  
Author(s):  
G. Deshpande ◽  
G. Calhoun ◽  
P.D. Schedl
Keyword(s):  
Fusion Protein ◽  
Rna Binding ◽  
Chimeric Protein ◽  
Dominant Negative ◽  
Terminal Domain ◽  
Binding Domains ◽  
Transcriptional Regulatory ◽  
Dominant Negative Activity ◽  
Female Specific

Sex determination in Drosophila depends upon the post-transcriptional regulatory activities of the Sex-lethal (Sxl) gene. Sxl maintains the female determined state and activates female differentiation pathways by directing the female-specific splicing of Sxl and tra pre-mRNAs. While there is compelling evidence that Sxl proteins regulate splicing by directly binding to target RNAs, previous studies indicate that the two Sxl RNA-binding domains are not in themselves sufficient for biological activity and that an intact N-terminal domain is also critical for splicing function. To further investigate the functions of the Sxl N terminus, we ectopically expressed a chimeric protein consisting of the N-terminal 99 amino acids fused to ss-galactosidase. The Nss-gal fusion protein behaves like a dominant negative, interfering with the Sxl autoregulatory feedback loop and killing females. This dominant negative activity can be attributed to the recruitment of the fusion protein into the large Sxl:Snf splicing complexes that are found in vivo and the consequent disruption of these complexes. In addition to the dominant negative activity, the Nss-gal fusion protein has a novel gain-of-function activity in males: it promotes the female-specific processing of tra pre-mRNAs. This novel activity is discussed in light of the blockage model for the tra splicing regulation.

scholarly journals Cell surface-mediated activation of progelatinase A: demonstration of the involvement of the C-terminal domain of progelatinase A in cell surface binding and activation of progelatinase A by primary fibroblasts

1994 ◽  
Vol 304 (1) ◽  
pp. 263-269 ◽  
Author(s):  
R V Ward ◽  
S J Atkinson ◽  
J J Reynolds ◽  
G Murphy
Keyword(s):  
Cell Surface ◽  
Concanavalin A ◽  
Plasma Membranes ◽  
Scatchard Analysis ◽  
Gelatinase A ◽  
Surface Binding ◽  
Binding Studies ◽  
Cell Surface Binding ◽  
Terminal Domain ◽  
Primary Fibroblasts

We report that the isolated C-terminal domain of progelatinase A is inhibitory to the activation of this proenzyme by primary skin fibroblast plasma membranes but is unable to inhibit organomercurial-induced self-cleavage and activation. Ligand binding studies demonstrate that fibroblasts stimulated with concanavalin A to activate progelatinase A have a significantly enhanced level of cell surface-associated progelatinase A. Tissue inhibitor of metalloproteinases-2 (TIMP-2), an effective inhibitor of membrane-mediated progelatinase A activation, is able to abolish the enhanced level of cell surface-associated progelatinase A that occurs following stimulation. TIMP-1, a poor inhibitor of membrane activation, is unable to inhibit the cell surface binding of progelatinase A. The enhancement in the binding of 125I-progelatinase A to fibroblasts following concanavalin A stimulation can be blocked by the inclusion of excess C-terminal gelatinase A but not by a truncated form of gelatinase A lacking the C-terminal domain. Scatchard analysis of the binding of 125I-progelatinase A to concanavalin A-stimulated fibroblasts has identified 950,000 gelatinase binding sites per cell with a Kd of 1.3 x 10(-8) M. Analysis of non-stimulated fibroblasts has identified 500,000 sites per cell with a Kd of 2.6 x 10(-8) M. We propose that membrane-mediated activation of progelatinase A involves binding of the proenzyme through its C-terminal domain to the cell surface and that TIMP-2 can inhibit activation by interaction with progelatinase A through the C-terminal domain, thus preventing binding of the proenzyme.

scholarly journals Identification of Novel Efflux Proteins Rv0191, Rv3756c, Rv3008, and Rv1667c Involved in Pyrazinamide Resistance in Mycobacterium tuberculosis

2017 ◽  
Vol 61 (8) ◽  
Author(s):  
Yumeng Zhang ◽  
Jia Zhang ◽  
Peng Cui ◽  
Ying Zhang ◽  
Wenhong Zhang
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Efflux Pump ◽  
Binding Studies ◽  
Content Type ◽  
Clinical Strains ◽  
Efflux Pump Inhibitors ◽  
Level Resistance ◽  
Pyrazinoic Acid ◽  
Increased Susceptibility ◽  
Efflux Proteins

ABSTRACT Pyrazinamide (PZA) is a critical drug used for the treatment of tuberculosis (TB). PZA is a prodrug that requires conversion to the active component pyrazinoic acid (POA) by pyrazinamidase (PZase) encoded by the pncA gene. Although resistance to PZA is mostly caused by pncA mutations and less commonly by rpsA, panD, and clpC1 mutations, clinical strains without these mutations are known to exist. While efflux of POA was demonstrated in Mycobacterium tuberculosis previously, the efflux proteins involved have not been identified. Here we performed POA binding studies with an M. tuberculosis proteome microarray and identified four efflux proteins (Rv0191, Rv3756c, Rv3008, and Rv1667c) that bind POA. Overexpression of the four efflux pump genes in M. tuberculosis caused low-level resistance to PZA and POA but not to other drugs. Furthermore, addition of efflux pump inhibitors such as reserpine, piperine, and verapamil caused increased susceptibility to PZA in M. tuberculosis strains overexpressing the efflux proteins Rv0191, Rv3756c, Rv3008, and Rv1667c. Our studies indicate that these four efflux proteins may be responsible for PZA/POA efflux and cause PZA resistance in M. tuberculosis. Future studies are needed to assess their roles in PZA resistance in clinical strains.

Abstract 42: Differential Contribution Of The N- And C-terminal Domains To The Folding And Function Of Tandem Calponin-homology Domains

2014 ◽  
Vol 115 (suppl_1) ◽  
Author(s):  
Krishna Mallela ◽  
Swati Bandi ◽  
Surinder Singh ◽  
Geoffrey Armstrong
Keyword(s):  
Full Length ◽  
Actin Binding ◽  
Main Function ◽  
Calponin Homology ◽  
Terminal Domain ◽  
Binding Domains ◽  
Binding Function ◽  
Ch2 Domain ◽  
The Stability ◽  
And Function

Tandem calponin-homology (CH) domains constitute a major class of actin-binding domains that include dystrophin and utrophin, the two key proteins involved in muscular dystrophy. Despite their importance, how their structure controls their function is not understood. Here, we study the contribution of individual CH domains to the actin-binding function and thermodynamic stability of utrophin’s tandem CH domain. Traditional actin co-sedimentation assays indicate that the isolated C-terminal CH2 domain binds weakly to F-actin when compared with the full-length tandem CH domain. In contrast, isolated CH1 binds to F-actin with a similar efficiency as that of the full-length tandem CH domain. Thus, the obvious question that arises is why tandem CH domains require CH2, when their actin-binding efficiency is originating primarily from CH1. To answer, we probed the thermodynamic stabilities of individual CH domains. Isolated CH1 domain is unstable and is prone to serious aggregation. Isolated CH2 is very stable, even appears to be more stable than the full-length tandem CH domain. In addition, the CH2 domain, which is more stable, is less functional. These results indicate that the main function of CH2 is to stabilize CH1. Consistently, the proposed structure of utrophin’s tandem CH domain based on earlier X-ray studies indicates a close proximity between the C-terminal helix of CH2 and the N-terminal helix of CH1, and this helix in CH2 is more dynamic in the full-length protein when compared with that in the absence of CH1, suggesting the mechanism by which CH2 stabilizes CH1. These observations indicate that the two CH domains contribute differentially to the folding and function of tandem CH domains, although both domains essentially have the same native structure in the tandem CH domain. The N-terminal domain determines the function, whereas the C-terminal domain determines the stability. This work was funded by the AHA Grant 11SDG4880046.

THE ROLE OF TRANSPOSABLE ELEMENTS IN ENDOCRINE CHANGES DURING AGING

2020 ◽  
pp. 418-428
Author(s):  
Р. Н. Мустафин ◽  
Э. К. Хуснутдинова
Keyword(s):  
Hormonal Regulation ◽  
Endocrine System ◽  
Cell Types ◽  
Key Factors ◽  
Dna Binding Domains ◽  
Binding Domains ◽  
Stage Of Development ◽  
Expression Of Genes ◽  
Transposon Activity ◽  
Species Specific

Одним из ключевых механизмов старения является изменение гормональной регуляции, для эффективного воздействия на которую с целью продления жизни необходимо определение первопричины данных процессов. В качестве молекулярных драйверов, управляющих динамикой уровня гормонов, могут служить транспозоны. Это связано с их использованием в качестве источников нуклеотидных последовательностей, воспринимающих специфические сигналы рибозимов, транскрипционных факторов, гормонов и их мессенджеров. В то же время, в эволюции транспозоны являются источниками рибозимов и белков, обладающих ДНК-связывающими доменами. Начиная с деления зиготы, видоспецифический состав и распределение транспозонов в геноме могут использоваться как биологическая кодировка, необходимая для последовательной и специфической для типов клеток экспрессии генов. Сделано предположение, что гормональная регуляция является одним из компонентов сложной системы управления онтогенезом под влиянием мобильных элементов. В качестве подтверждения приведены работы о роли транспозонов в управлении генами эндокринной системы, а также о влиянии гормонов на активность транспозонов. Исследование этих взаимосвязей может иметь перспективы для разработки методов продления жизни, так как эпигенетические изменения под влиянием транспозонов носят обратимый характер. Species-specific changes in the endocrine system are key factors in aging. Therefore, to prolong life, it is necessary to find regulators of the highest level, the changes of which lead to physiological aging. The molecular drivers that control dynamics of hormone levels can be transposons. This is due to the use of nucleotide sequences of transposons as binding sites that perceive specific signals of ribozymes, transcription factors, hormones and their messengers. At the same time, transposons are evolutionary sources of ribozymes and proteins that have DNA-binding domains. Starting from zygote division, the species-specific composition and distribution of transposons in the genome serves as a biological coding, which is necessary for the sequential expression of genes specific to cell types and stage of development. We suggest that hormonal regulation is one of the components of this complex system of regulation of ontogenesis under the control of transposons. To confirm our hypothesis, this review contains articles that prove the importance of transposons for species-specific control of endocrine system genes, as well as the effect of hormones on transposon activity. The research of these relationships is promising for the development of methods for the effective prolongation of life, since epigenetic changes under the influence of transposons are reversible.

Crystal structure of the complex of the interaction domains of Escherichia coli DnaB helicase and DnaC helicase loader: structural basis implying a distortion-accumulation mechanism for the DnaB ring opening caused by DnaC binding

2019 ◽  
Vol 167 (1) ◽  
pp. 1-14
Author(s):  
Koji Nagata ◽  
Akitoshi Okada ◽  
Jun Ohtsuka ◽  
Takatoshi Ohkuri ◽  
Yusuke Akama ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Escherichia Coli ◽  
Molecular Model ◽  
Structural Basis ◽  
Terminal Domain ◽  
Helicase Dnab ◽  
Accumulation Mechanism ◽  
Interaction Domains ◽  
Α Helix ◽  
Available Information

Abstract Loading the bacterial replicative helicase DnaB onto DNA requires a specific loader protein, DnaC/DnaI, which creates the loading-competent state by opening the DnaB hexameric ring. To understand the molecular mechanism by which DnaC/DnaI opens the DnaB ring, we solved 3.1-Å co-crystal structure of the interaction domains of Escherichia coli DnaB–DnaC. The structure reveals that one N-terminal domain (NTD) of DnaC interacts with both the linker helix of a DnaB molecule and the C-terminal domain (CTD) of the adjacent DnaB molecule by forming a three α-helix bundle, which fixes the relative orientation of the two adjacent DnaB CTDs. The importance of the intermolecular interface in the crystal structure was supported by the mutational data of DnaB and DnaC. Based on the crystal structure and other available information on DnaB–DnaC structures, we constructed a molecular model of the hexameric DnaB CTDs bound by six DnaC NTDs. This model suggested that the binding of a DnaC would cause a distortion in the hexameric ring of DnaB. This distortion of the DnaB ring might accumulate by the binding of up to six DnaC molecules, resulting in the DnaB ring to open.

Sign in / Sign up

Export Citation Format

Share Document