1. |
- Freitag-Pohl, Stefanie, et al.
(author)
-
Crystal structures of the Bacillus subtilis prophage lytic cassette proteins XepA and YomS.
- 2019
-
In: Acta Crystallographica Section D: Structural Biology. - 2059-7983. ; 75, s. 1028-1039
-
Journal article (peer-reviewed)abstract
- As part of the Virus-X Consortium that aims to identify and characterize novel proteins and enzymes from bacteriophages and archaeal viruses, the genes of the putative lytic proteins XepA from Bacillus subtilis prophage PBSX and YomS from prophage SPβ were cloned and the proteins were subsequently produced and functionally characterized. In order to elucidate the role and the molecular mechanism of XepA and YomS, the crystal structures of these proteins were solved at resolutions of 1.9 and 1.3 Å, respectively. XepA consists of two antiparallel β-sandwich domains connected by a 30-amino-acid linker region. A pentamer of this protein adopts a unique dumbbell-shaped architecture consisting of two discs and a central tunnel. YomS (12.9 kDa per monomer), which is less than half the size of XepA (30.3 kDa), shows homology to the C-terminal part of XepA and exhibits a similar pentameric disc arrangement. Each β-sandwich entity resembles the fold of typical cytoplasmic membrane-binding C2 domains. Only XepA exhibits distinct cytotoxic activity in vivo, suggesting that the N-terminal pentameric domain is essential for this biological activity. The biological and structural data presented here suggest that XepA disrupts the proton motive force of the cytoplasmatic membrane, thus supporting cell lysis.
|
|
2. |
- Jasilionis, Andrius, et al.
(author)
-
AmiP from hyperthermophilic Thermus parvatiensis prophage is a thermoactive and ultrathermostable peptidoglycan lytic amidase
- 2023
-
In: Protein Science. - : Wiley. - 1469-896X .- 0961-8368. ; 32:3, s. 4585-4585
-
Journal article (peer-reviewed)abstract
- Bacteriophages encode a wide variety of cell wall disrupting enzymes that aid the viral escape in the final stages of infection. These lytic enzymes have accumulated notable interest due to their potential as novel antibacterials for infection treatment caused by multiple-drug resistant bacteria. Here, the detailed functional and structural characterization of Thermus parvatiensis prophage peptidoglycan lytic amidase AmiP, a globular Amidase_3 type lytic enzyme adapted to high temperatures is presented. The sequence and structure comparison with homologous lytic amidases reveals the key adaptation traits that ensure the activity and stability of AmiP at high temperatures. The crystal structure determined at a resolution of 1.8 Å displays a compact α/β-fold with multiple secondary structure elements omitted or shortened compared with protein structures of similar proteins. The functional characterization of AmiP demonstrates high efficiency of catalytic activity and broad substrate specificity toward thermophilic and mesophilic bacteria strains containing Orn-type or DAP-type peptidoglycan. The here presented AmiP constitutes the most thermoactive and ultrathermostable Amidase_3 type lytic enzyme biochemically characterized with a temperature optimum at 85°C. The extraordinary high melting temperature Tm 102.6°C confirms fold stability up to approximately 100°C. Furthermore, AmiP is shown to be more active over the alkaline pH range with pH optimum at pH 8.5 and tolerates NaCl up to 300 mM with the activity optimum at 25 mM NaCl. This set of beneficial characteristics suggests that AmiP can be further exploited in biotechnology.
|
|
3. |
- Lüking, Malin, 1993-
(author)
-
Modelling the Protein-DNA Interface
- 2020
-
Licentiate thesis (other academic/artistic)abstract
- Protein-DNA interactions are crucial to life. Several millions of DNA base pair steps are organ- ised, read and protected by proteins in every cell. Protein-DNA interactions must be specific, controllable and reasonably fast. Understanding how these features coexist is one of the great challenges for biochemists and molecular biologists. Great interest has been directed towards the fast association rates measured for DNA-binding proteins such as bacterial transcription factors. These interactions have been described as proceeding by ‘facilitated diffusion’, which means that the non-specific interaction of a protein with DNA guides its way towards the target site. This can be studied using fluorescent labels and high resolution microscopes. This tech- niques can record traces of proteins, that diffuse along DNA until they bind their target sites and stop diffusion. But, which role the conformation of the protein or the DNA play a during the pro- cess of non-specific binding and recognition cannot be revealed. It is still unclear how proteins recognise their target sites. It is likely that conformational changes in both, the protein and in the DNA, play important roles. We can solve structures of protein-DNA complexes in molecular detail using crystallography or nuclear magnetic resonance. With all-atom molecular dynamic simulations we can elucidating their dynamics and obtain insights about conformational vari- ability. But some, large conformational changes and especially diffusion are processes that can be out of reach for the time-scales of normal all-atom simulations. Simplified representations of large biomolecules, so called coarse-grained models, can be used to study diffusion instead. The repressor of the lac operon is a well studied model system for understanding protein-DNA interactions. In this study, we applied coarse-grained simulations to define the search confor- mation of the protein, answering how it can sample the DNA effectively and how it recognises the target site sequence. Additionally we applied all-atom molecular dynamics to understand the stabilisation of the specific complex.
|
|
4. |
- Tatum, Natalie J., et al.
(author)
-
Relative Binding Energies Predict Crystallographic Binding Modes of Ethionamide Booster Lead Compounds
- 2019
-
In: The Journal of Physical Chemistry Letters. - : AMER CHEMICAL SOC. - 1948-7185. ; 10:9, s. 2244-2249
-
Journal article (peer-reviewed)abstract
- Transcriptional repressor EthR from Mycobacterium tuberculosis is a valuable target for antibiotic booster drugs. We previously reported a virtual screening campaign to identify EthR inhibitors for development. Two ligand binding orientations were often proposed, though only the top scoring pose was utilized for filtering of the large data set. We obtained biophysically validated hits, some of which yielded complex crystal structures. In some cases, the crystallized binding mode and top scoring mode agree, while for others an alternate ligand binding orientation was found. In this contribution, we combine rigid docking, molecular dynamics simulations, and the linear interaction energy method to calculate binding free energies and derive relative binding energies for a number of EthR inhibitors in both modes. This strategy allowed us to correctly predict the most favorable orientation. Therefore, this widely applicable approach will be suitable to triage multiple binding modes within EthR and other potential drug targets with similar characteristics.
|
|