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| 2. |
- Wahlgren, Weixiao Yuan, 1970, et al.
(författare)
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Substrate-bound outward-open structure of a Na+-coupled sialic acid symporter reveals a new Na+ site.
- 2018
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Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 9:1
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Tidskriftsartikel (refereegranskat)abstract
- Many pathogenic bacteria utilise sialic acids as an energy source or use them as an external coating to evade immune detection. As such, bacteria that colonise sialylated environments deploy specific transporters to mediate import of scavenged sialic acids. Here, we report a substrate-bound 1.95Å resolution structure and subsequent characterisation of SiaT, a sialic acid transporter from Proteus mirabilis. SiaT is a secondary active transporter of the sodium solute symporter (SSS) family, which use Na+ gradients to drive the uptake of extracellular substrates. SiaT adopts the LeuT-fold and is in an outward-open conformation in complex with the sialic acid N-acetylneuraminic acid and two Na+ ions. One Na+ binds to the conserved Na2 site, while the second Na+ binds to a new position, termed Na3, which is conserved in many SSS family members. Functional and molecular dynamics studies validate the substrate-binding site and demonstrate that both Na+ sites regulate N-acetylneuraminic acid transport.
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| 3. |
- Andersson, Rebecka, 1988, et al.
(författare)
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Serial femtosecond crystallography structure of cytochrome c oxidase at room temperature.
- 2017
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Ingår i: Scientific reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 7:1
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Tidskriftsartikel (refereegranskat)abstract
- Cytochrome c oxidase catalyses the reduction of molecular oxygen to water while the energy released in this process is used to pump protons across a biological membrane. Although an extremely well-studied biological system, the molecular mechanism of proton pumping by cytochrome c oxidase is still not understood. Here we report a method to produce large quantities of highly diffracting microcrystals of ba 3-type cytochrome c oxidase from Thermus thermophilus suitable for serial femtosecond crystallography. The room-temperature structure of cytochrome c oxidase is solved to 2.3Å resolution from data collected at an X-ray Free Electron Laser. We find overall agreement with earlier X-ray structures solved from diffraction data collected at cryogenic temperature. Previous structures solved from synchrotron radiation data, however, have shown conflicting results regarding the identity of the active-site ligand. Our room-temperature structure, which is free from the effects of radiation damage, reveals that a single-oxygen species in the form of a water molecule or hydroxide ion is bound in the active site. Structural differences between the ba 3-type and aa 3-type cytochrome c oxidases around the proton-loading site are also described.
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| 4. |
- Dunevall, Elin, 1986
(författare)
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Structural and Functional Studies of Membrane Proteins For Future Development of Antimicrobial Drugs
- 2019
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Antibiotic resistance is a world-wide occurring problem which threatens human health. Without development of any new and effective antibiotics, the rapid growth of antibiotic-resistant bacterial infections could put society in a situation resembling the pre-antibiotic era when a simple lung infection could kill a human being. This thesis presents two venues for targeting antibiotic resistance. Pathogenic bacteria present in mucus rich environments are able to utilize host-derived sialic acid either as an alternative food source or by incorporating sialic acid to their surface glycoconjugates as a way to evade the host´s immune system. Hence, molecular mimicry enables bacteria to secure an ecological niche for survival. Transport of scavenged sialic acid into the cytoplasm of bacteria occurs through specific membrane bound sialic acid transporters. The cell wall is an essential protective barrier for bacteria. The membrane bound enzyme MraY catalyzes the synthesis of lipid I, an intermediate step in the biosynthesis of peptidoglycan, the cell wall of bacteria. This thesis presents work aimed to structurally and functionally characterize sialic acid transporters and MraY for future development of antibacterial drugs. Starting with a broad approach for expression and purification of sialic acid transporters resulted in low-resolution diffracting crystals of the Pasteurella multocida sialic acid TRAP transporter. In addition the X-ray crystallography structure of the sialic acid transporter SiaT from Proteus mirabilis was determined at 1.95 Å resolution in a substrate-bound outward-open conformation revealing a new sodium site. Furthermore, SiaT transporters have been characterized in vivo and the sialic acid specificity has been characterized for SiaT from Staphylococcus aureus. Structural comparison between MraY and the human homologue GPT have highlighted regions where to modify the natural product inhibitor tunicamycin to selectively target MraY. Further characterization of tunicamycin analogues identified potent inhibitors with reduced eukaryotic toxicity.
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| 5. |
- Hering, Jenny, et al.
(författare)
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Exploring the Active Site of the Antibacterial Target MraY by Modified Tunicamycins.
- 2020
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Ingår i: ACS chemical biology. - : American Chemical Society (ACS). - 1554-8937 .- 1554-8929. ; 15:11, s. 2885-2895
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Tidskriftsartikel (refereegranskat)abstract
- The alarming growth of antibiotic resistance that is currently ongoing is a serious threat to human health. One of the most promising novel antibiotic targets is MraY (phospho-MurNAc-pentapeptide-transferase), an essential enzyme in bacterial cell wall synthesis. Through recent advances in biochemical research, there is now structural information available for MraY, and for its human homologue GPT (GlcNAc-1-P-transferase), that opens up exciting possibilities for structure-based drug design. The antibiotic compound tunicamycin is a natural product inhibitor of MraY that is also toxic to eukaryotes through its binding to GPT. In this work, we have used tunicamycin and modified versions of tunicamycin as tool compounds to explore the active site of MraY and to gain further insight into what determines inhibitor potency. We have investigated tunicamycin variants where the following motifs have been modified: the length and branching of the tunicamycin fatty acyl chain, the saturation of the fatty acyl chain, the 6″-hydroxyl group of the GlcNAc ring, and the ring structure of the uracil motif. The compounds are analyzed in terms of how potently they bind to MraY, inhibit the activity of the enzyme, and affect the protein thermal stability. Finally, we rationalize these results in the context of the protein structures of MraY and GPT.
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| 6. |
- Hering, Jenny, et al.
(författare)
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Structural basis for selective inhibition of antibacterial target MraY, a membrane-bound enzyme involved in peptidoglycan synthesis
- 2018
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Ingår i: Drug Discovery Today. - : Elsevier BV. - 1359-6446 .- 1878-5832. ; 23:7, s. 1426-1435
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Forskningsöversikt (refereegranskat)abstract
- The rapid growth of antibiotic-resistant bacterial infections is of major concern for human health. Therefore, it is of great importance to characterize novel targets for the development of antibacterial drugs. One promising protein target is MraY (UDP-N-acetylmuramyl-pentapeptide: undecaprenyl phosphate N-acetylmuramyl-pentapeptide-1-phosphate transferase or MurNAc-1-P-transferase), which is essential for bacterial cell wall synthesis. Here, we summarize recent breakthroughs in structural studies of bacterial MraYs and the closely related human GPT (UDP-N-acetylglucosamine: dolichyl phosphate N-acetylglucosamine-1-phosphate transferase or GlcNAc-1-P-transferase). We present a detailed comparison of interaction modes with the natural product inhibitors tunicamycin and muraymycin D2. Finally, we speculate on possible routes to design an antibacterial agent in the form of a potent and selective inhibitor against MraY.
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| 7. |
- Safari, Cecilia, 1989, et al.
(författare)
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Time-resolved serial crystallography to track the dynamics of carbon monoxide in the active site of cytochrome c oxidase
- 2023
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Ingår i: Science advances. - 2375-2548. ; 9:49
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Tidskriftsartikel (refereegranskat)abstract
- Cytochrome c oxidase (CcO) is part of the respiratory chain and contributes to the electrochemical membrane gradient in mitochondria as well as in many bacteria, as it uses the energy released in the reduction of oxygen to pump protons across an energy-transducing biological membrane. Here, we use time-resolved serial femtosecond crystallography to study the structural response of the active site upon flash photolysis of carbon monoxide (CO) from the reduced heme a3 of ba3-type CcO. In contrast with the aa3-type enzyme, our data show how CO is stabilized on CuB through interactions with a transiently ordered water molecule. These results offer a structural explanation for the extended lifetime of the CuB-CO complex in ba3-type CcO and, by extension, the extremely high oxygen affinity of the enzyme.
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| 8. |
- Sharma, Amit, et al.
(författare)
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Asymmetry in serial femtosecond crystallography data
- 2017
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Ingår i: Acta Crystallographica a-Foundation and Advances. - : International Union of Crystallography (IUCr). - 2053-2733. ; 73, s. 93-101
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Tidskriftsartikel (refereegranskat)abstract
- Serial crystallography is an increasingly important approach to protein crystallography that exploits both X-ray free-electron laser (XFEL) and synchrotron radiation. Serial crystallography recovers complete X-ray diffraction data by processing and merging diffraction images from thousands of randomly oriented non-uniform microcrystals, of which all observations are partial Bragg reflections. Random fluctuations in the XFEL pulse energy spectrum, variations in the size and shape of microcrystals, integrating over millions of weak partial observations and instabilities in the XFEL beam position lead to new types of experimental errors. The quality of Bragg intensity estimates deriving from serial crystallography is therefore contingent upon assumptions made while modeling these data. Here it is observed that serial femtosecond crystallography (SFX) Bragg reflections do not follow a unimodal Gaussian distribution and it is recommended that an idealized assumption of single Gaussian peak profiles be relaxed to incorporate apparent asymmetries when processing SFX data. The phenomenon is illustrated by re-analyzing data collected from microcrystals of the Blastochloris viridis photosynthetic reaction center and comparing these intensity observations with conventional synchrotron data. The results show that skewness in the SFX observations captures the essence of the Wilson plot and an empirical treatment is suggested that can help to separate the diffraction Bragg intensity from the background.
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