| 1. |
- Gulen, Burak, et al.
(författare)
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Identification of targets of AMPylating Fic enzymes by co-substrate-mediated covalent capture
- 2020
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Ingår i: Nature Chemistry. - : Nature Publishing Group. - 1755-4330 .- 1755-4349. ; 12:8, s. 732-739
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Tidskriftsartikel (refereegranskat)abstract
- Various pathogenic bacteria use post-translational modifications to manipulate the central components of host cell functions. Many of the enzymes released by these bacteria belong to the large Fic family, which modify targets with nucleotide monophosphates. The lack of a generic method for identifying the cellular targets of Fic family enzymes hinders investigation of their role and the effect of the post-translational modification. Here, we establish an approach that uses reactive co-substrate-linked enzymes for proteome profiling. We combine synthetic thiol-reactive nucleotide derivatives with recombinantly produced Fic enzymes containing strategically placed cysteines in their active sites to yield reactive binary probes for covalent substrate capture. The binary complexes capture their targets from cell lysates and permit subsequent identification. Furthermore, we determined the structures of low-affinity ternary enzyme–nucleotide–substrate complexes by applying a covalent-linking strategy. This approach thus allows target identification of the Fic enzymes from both bacteria and eukarya.
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| 2. |
- Rogne, Per, et al.
(författare)
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Molecular mechanism of ATP versus GTP selectivity of adenylate kinase
- 2018
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 115:12, s. 3012-3017
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Tidskriftsartikel (refereegranskat)abstract
- Enzymatic substrate selectivity is critical for the precise control of metabolic pathways. In cases where chemically related substrates are present inside cells, robust mechanisms of substrate selectivity are required. Here, we report the mechanism utilized for catalytic ATP versus GTP selectivity during adenylate kinase (Adk) -mediated phosphorylation of AMP. Using NMR spectroscopy we found that while Adk adopts a catalytically competent and closed structural state in complex with ATP, the enzyme is arrested in a catalytically inhibited and open state in complex with GTP. X-ray crystallography experiments revealed that the interaction interfaces supporting ATP and GTP recognition, in part, are mediated by coinciding residues. The mechanism provides an atomic view on how the cellular GTP pool is protected from Adk turnover, which is important because GTP has many specialized cellular functions. In further support of this mechanism, a structure-function analysis enabled by synthesis of ATP analogs suggests that a hydrogen bond between the adenine moiety and the backbone of the enzyme is vital for ATP selectivity. The importance of the hydrogen bond for substrate selectivity is likely general given the conservation of its location and orientation across the family of eukaryotic protein kinases.
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| 3. |
- Rogne, Per, et al.
(författare)
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Structural Basis for GTP versus ATP Selectivity in the NMP Kinase AK3
- 2020
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 59:38, s. 3570-3581
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Tidskriftsartikel (refereegranskat)abstract
- ATP and GTP are exceptionally important molecules in biology with multiple, and often discrete, functions. Therefore, enzymes that bind to either of them must develop robust mechanisms to selectively utilize one or the other. Here, this specific problem is addressed by molecular studies of the human NMP kinase AK3, which uses GTP to phosphorylate AMP. AK3 plays an important role in the citric acid cycle, where it is responsible for GTP/GDP recycling. By combining a structural biology approach with functional experiments, we present a comprehensive structural and mechanistic understanding of the enzyme. We discovered that AK3 functions by recruitment of GTP to the active site, while ATP is rejected and nonproductively bound to the AMP binding site. Consequently, ATP acts as an inhibitor with respect to GTP and AMP. The overall features with specific recognition of the correct substrate and nonproductive binding by the incorrect substrate bear a strong similarity to previous findings for the ATP specific NMP kinase adenylate kinase. Taken together, we are now able to provide the fundamental principles for GTP and ATP selectivity in the large NMP kinase family. As a side-result originating from nonlinearity of chemical shifts in GTP and ATP titrations, we find that protein surfaces offer a general and weak binding affinity for both GTP and ATP. These nonspecific interactions likely act to lower the available intracellular GTP and ATP concentrations and may have driven evolution of the Michaelis constants of NMP kinases accordingly.
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| 4. |
- Vartak, Nachiket, et al.
(författare)
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Intravital Dynamic and Correlative Imaging of Mouse Livers Reveals Diffusion-Dominated Canalicular and Flow-Augmented Ductular Bile Flux
- 2021
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Ingår i: Hepatology. - : John Wiley & Sons. - 0270-9139 .- 1527-3350. ; 73:4, s. 1531-1550
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Tidskriftsartikel (refereegranskat)abstract
- Background and Aims: Small-molecule flux in tissue microdomains is essential for organ function, but knowledge of this process is scant due to the lack of suitable methods. We developed two independent techniques that allow the quantification of advection (flow) and diffusion in individual bile canaliculi and in interlobular bile ducts of intact livers in living mice, namely fluorescence loss after photoactivation and intravital arbitrary region image correlation spectroscopy.Approach and Results: The results challenge the prevailing "mechano-osmotic" theory of canalicular bile flow. After active transport across hepatocyte membranes, bile acids are transported in the canaliculi primarily by diffusion. Only in the interlobular ducts is diffusion augmented by regulatable advection. Photoactivation of fluorescein bis-(5-carboxymethoxy-2-nitrobenzyl)-ether in entire lobules demonstrated the establishment of diffusive gradients in the bile canalicular network and the sink function of interlobular ducts. In contrast to the bile canalicular network, vectorial transport was detected and quantified in the mesh of interlobular bile ducts.Conclusions: The liver consists of a diffusion-dominated canalicular domain, where hepatocytes secrete small molecules and generate a concentration gradient and a flow-augmented ductular domain, where regulated water influx creates unidirectional advection that augments the diffusive flux.
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