| 1. |
- Bairy, Sneha, et al.
(författare)
-
Automation aided optimization of cloning, expression and purification of enzymes of the bacterial sialic acid catabolic and sialylation pathways enzymes for structural studies.
- 2018
-
Ingår i: Microbial biotechnology. - : Wiley. - 1751-7915. ; 11:2, s. 420-428
-
Tidskriftsartikel (refereegranskat)abstract
- The process of obtaining a well-expressing, soluble and correctly folded constructs can be made easier and quicker by automating the optimization of cloning, expression and purification. While there are many semiautomated pipelines available for cloning, expression and purification, there is hardly any pipeline that involves complete automation. Here, we achieve complete automation of all the steps involved in cloning and invivo expression screening. This is demonstrated using 18 genes involved in sialic acid catabolism and the surface sialylation pathway. Our main objective was to clone these genes into a His-tagged Gateway vector, followed by their small-scale expression optimization invivo. The constructs that showed best soluble expression were then selected for purification studies and scaled up for crystallization studies. Our technique allowed us to quickly find conditions for producing significant quantities of soluble proteins in Escherichia coli, their large-scale purification and successful crystallization of a number of these proteins. The method can be implemented in other cases where one needs to screen a large number of constructs, clones and expression vectors for successful recombinant production of functional proteins.
|
|
| 2. |
- Bodor, Andrea, et al.
(författare)
-
DYNLL2 Dynein Light Chain Binds to an Extended Linear Motif of Myosin 5a Tail That Has Structural Plasticity.
- 2014
-
Ingår i: Biochemistry. - : American Chemical Society (ACS). - 1520-4995 .- 0006-2960. ; 53:45, s. 7107-7122
-
Tidskriftsartikel (refereegranskat)abstract
- LC8 dynein light chains (DYNLL) are conserved homodimeric eukaryotic hub proteins that participate in diverse cellular processes. Among the binding partners of DYNLL2, myosin 5a (myo5a) is a motor protein involved in cargo transport. Here we provide a profound characterization of the DYNLL2 binding motif of myo5a in free and DYNLL2-bound form by using nuclear magnetic resonance spectroscopy, X-ray crystallography, and molecular dynamics simulations. In the free form, the DYNLL2 binding region, located in an intrinsically disordered domain of the myo5a tail, has a nascent helical character. The motif becomes structured and folds into a β-strand upon binding to DYNLL2. Despite differences of the myo5a sequence from the consensus binding motif, one peptide is accommodated in each of the parallel DYNLL2 binding grooves, as for all other known partners. Interestingly, while the core motif shows a similar interaction pattern in the binding groove as seen in other complexes, the flanking residues make several additional contacts, thereby lengthening the binding motif. The N-terminal extension folds back and partially blocks the free edge of the β-sheet formed by the binding motif itself. The C-terminal extension contacts the dimer interface and interacts with symmetry-related residues of the second myo5a peptide. The involvement of flanking residues of the core binding site of myo5a could modify the quaternary structure of the full-length myo5a and affect its biological functions. Our results deepen the knowledge of the diverse partner recognition of DYNLL proteins and provide an example of a Janus-faced linear motif.
|
|
| 3. |
- Bountra, K., et al.
(författare)
-
Structural basis for antibacterial peptide self-immunity by the bacterial ABC transporter McjD
- 2017
-
Ingår i: Embo Journal. - : EMBO. - 0261-4189 .- 1460-2075. ; 36:20, s. 3062-3079
-
Tidskriftsartikel (refereegranskat)abstract
- Certain pathogenic bacteria produce and release toxic peptides to ensure either nutrient availability or evasion from the immune system. These peptides are also toxic to the producing bacteria that utilize dedicated ABC transporters to provide self-immunity. The ABC transporter McjD exports the antibacterial peptide MccJ25 in Escherichia coli. Our previously determined McjD structure provided some mechanistic insights into antibacterial peptide efflux. In this study, we have determined its structure in a novel conformation, apo inward-occluded and a new nucleotide-bound state, high-energy outward-occluded intermediate state, with a defined ligand binding cavity. Predictive cysteine cross-linking in E.coli membranes and PELDOR measurements along the transport cycle indicate that McjD does not undergo major conformational changes as previously proposed for multi-drug ABC exporters. Combined with transport assays and molecular dynamics simulations, we propose a novel mechanism for toxic peptide ABC exporters that only requires the transient opening of the cavity for release of the peptide. We propose that shielding of the cavity ensures that the transporter is available to export the newly synthesized peptides, preventing toxic-level build-up.
|
|
| 4. |
- Bozzola, Tiago, et al.
(författare)
-
Sialic Acid Derivatives Inhibit SiaT Transporters and Delay Bacterial Growth
- 2022
-
Ingår i: Acs Chemical Biology. - : American Chemical Society (ACS). - 1554-8929 .- 1554-8937. ; 17:7, s. 1890-1900
-
Tidskriftsartikel (refereegranskat)abstract
- Antibiotic resistance is a major worldwide concern, and new drugs with mechanistically novel modes of action are urgently needed. Here, we report the structure-based drug design, synthesis, and evaluation in vitro and in cellular systems of sialic acid derivatives able to inhibit the bacterial sialic acid symporter SiaT. We designed and synthesized 21 sialic acid derivatives and screened their affinity for SiaT by a thermal shift assay and elucidated the inhibitory mechanism through binding thermodynamics, computational methods, and inhibitory kinetic studies. The most potent compounds, which have a 180-fold higher affinity compared to the natural substrate, were tested in bacterial growth assays and indicate bacterial growth delay in methicillin-resistant Staphylococcus aureus. This study represents the first example and a promising lead in developing sialic acid uptake inhibitors as novel antibacterial agents.
|
|
| 5. |
- Carrillo, M., et al.
(författare)
-
High-resolution crystal structures of transient intermediates in the phytochrome photocycle
- 2021
-
Ingår i: Structure. - : Elsevier BV. - 0969-2126. ; 29:7
-
Tidskriftsartikel (refereegranskat)abstract
- Phytochromes are red/far-red light photoreceptors in bacteria to plants, which elicit a variety of important physiological responses. They display a reversible photocycle between the resting Pr state and the light-activated Pfr state. Light signals are transduced as structural change through the entire protein to modulate its activity. It is unknown how the Pr-to-Pfr interconversion occurs, as the structure of intermediates remains notoriously elusive. Here, we present short-lived crystal structures of the photosensory core modules of the bacteriophytochrome from myxobacterium Stigmatella aurantiaca captured by an X-ray free electron laser 5 ns and 33 ms after light illumination of the Pr state. We observe large structural displacements of the covalently bound bilin chromophore, which trigger a bifurcated signaling pathway that extends through the entire protein. The snapshots show with atomic precision how the signal progresses from the chromophore, explaining how plants, bacteria, and fungi sense red light.
|
|
| 6. |
- Cellini, Andrea, 1991, et al.
(författare)
-
Directed ultrafast conformational changes accompany electron transfer in a photolyase as resolved by serial crystallography.
- 2024
-
Ingår i: Nature chemistry. - : Springer Nature. - 1755-4349 .- 1755-4330.
-
Tidskriftsartikel (refereegranskat)abstract
- Charge-transfer reactions in proteins are important for life, such as in photolyases which repair DNA, but the role of structural dynamics remains unclear. Here, using femtosecond X-ray crystallography, we report the structural changes that take place while electrons transfer along a chain of four conserved tryptophans in the Drosophila melanogaster (6-4) photolyase. At femto- and picosecond delays, photoreduction of the flavin by the first tryptophan causes directed structural responses at a key asparagine, at a conserved salt bridge, and by rearrangements of nearby water molecules. We detect charge-induced structural changes close to the second tryptophan from 1 ps to 20 ps, identifying a nearby methionine as an active participant in the redox chain, and from 20 ps around the fourth tryptophan. The photolyase undergoes highly directed and carefully timed adaptations of its structure. This questions the validity of the linear solvent response approximation in Marcus theory and indicates that evolution has optimized fast protein fluctuations for optimal charge transfer.
|
|
| 7. |
- Cellini, Andrea, 1991, et al.
(författare)
-
Structural basis of the radical pair state in photolyases and cryptochromes
- 2022
-
Ingår i: Chemical Communications. - : Royal Society of Chemistry (RSC). - 1359-7345 .- 1364-548X. ; , s. 4889-4892
-
Tidskriftsartikel (refereegranskat)abstract
- We present the structure of a photoactivated animal (6-4) photolyase in its radical pair state, captured by serial crystallography. We observe how a conserved asparigine moves towards the semiquinone FAD chromophore and stabilizes it by hydrogen bonding. Several amino acids around the final tryptophan radical rearrange, opening it up to the solvent. The structure explains how the protein environment stabilizes the radical pair state, which is crucial for function of (6-4) photolyases and cryptochromes.
|
|
| 8. |
- Cellini, Andrea, 1991, et al.
(författare)
-
The three-dimensional structure of Drosophila melanogaster (6-4) photolyase at room temperature
- 2021
-
Ingår i: Acta Crystallographica Section D-Structural Biology. - : International Union of Crystallography (IUCr). - 2059-7983. ; 77, s. 1001-1009
-
Tidskriftsartikel (refereegranskat)abstract
- (6-4) photolyases are flavoproteins that belong to the photolyase/cryptochrome family. Their function is to repair DNA lesions using visible light. Here, crystal structures of Drosophila melanogaster (6-4) photolyase [Dm(6-4)photolyase] at room and cryogenic temperatures are reported. The room-temperature structure was solved to 2.27 angstrom resolution and was obtained by serial femtosecond crystallography (SFX) using an X-ray free-electron laser. The crystallization and preparation conditions are also reported. The cryogenic structure was solved to 1.79 angstrom resolution using conventional X-ray crystallography. The structures agree with each other, indicating that the structural information obtained from crystallography at cryogenic temperature also applies at room temperature. Furthermore, UV-Vis absorption spectroscopy confirms that Dm(6-4)photolyase is photoactive in the crystals, giving a green light to time-resolved SFX studies on the protein, which can reveal the structural mechanism of the photoactivated protein in DNA repair.
|
|
| 9. |
- Claesson, Elin, 1989, et al.
(författare)
-
The primary structural photoresponse of phytochrome proteins captured by a femtosecond X-ray laser
- 2020
-
Ingår i: eLife. - 2050-084X. ; 9
-
Tidskriftsartikel (refereegranskat)abstract
- Phytochrome proteins control the growth, reproduction, and photosynthesis of plants, fungi, and bacteria. Light is detected by a bilin cofactor, but it remains elusive how this leads to activation of the protein through structural changes. We present serial femtosecond X-ray crystallographic data of the chromophore-binding domains of a bacterial phytochrome at delay times of 1 ps and 10 ps after photoexcitation. The data reveal a twist of the D-ring, which leads to partial detachment of the chromophore from the protein. Unexpectedly, the conserved so-called pyrrole water is photodissociated from the chromophore, concomitant with movement of the A-ring and a key signaling aspartate. The changes are wired together by ultrafast backbone and water movements around the chromophore, channeling them into signal transduction towards the output domains. We suggest that the observed collective changes are important for the phytochrome photoresponse, explaining the earliest steps of how plants, fungi and bacteria sense red light.
|
|
| 10. |
- Currie, Michael J., et al.
(författare)
-
Structural and biophysical analysis of a Haemophilus influenzae tripartite ATP-independent periplasmic (TRAP) transporter
- 2024
-
Ingår i: ELIFE. - 2050-084X. ; 12
-
Tidskriftsartikel (refereegranskat)abstract
- Tripartite ATP-independent periplasmic (TRAP) transporters are secondary-active transporters that receive their substrates via a soluble-binding protein to move bioorganic acids across bacterial or archaeal cell membranes. Recent cryo-electron microscopy (cryo-EM) structures of TRAP transporters provide a broad framework to understand how they work, but the mechanistic details of transport are not yet defined. Here we report the cryo-EM structure of the Haemophilus influenzae N-acetylneuraminate TRAP transporter (HiSiaQM) at 2.99 & Aring; resolution (extending to 2.2 & Aring; at the core), revealing new features. The improved resolution (the previous HiSiaQM structure is 4.7 & Aring; resolution) permits accurate assignment of two Na+ sites and the architecture of the substrate-binding site, consistent with mutagenic and functional data. Moreover, rather than a monomer, the HiSiaQM structure is a homodimer. We observe lipids at the dimer interface, as well as a lipid trapped within the fusion that links the SiaQ and SiaM subunits. We show that the affinity (K-D) for the complex between the soluble HiSiaP protein and HiSiaQM is in the micromolar range and that a related SiaP can bind HiSiaQM. This work provides key data that enhances our understanding of the 'elevator-with-an-operator' mechanism of TRAP transporters.
|
|
