| 1. |
- Agback, Peter, et al.
(författare)
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Direct evidence of a low barrier hydrogen bond in the catalytic triad of a Serine protease
- 2018
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 8
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Tidskriftsartikel (refereegranskat)abstract
- Serine proteases are one of the largest groups of enzymes, found in both eukaryotes and prokaryotes, and are responsible for many different functions. The detailed information about the hydrogen-bonds in the catalytic triad (Asp. His. Ser) of these enzymes is of importance in order to fully understand the mechanism of action. The aspartate of the triad is hydrogen bonded to the histidine but the exact nature of this bond has been under discussion for some time. It is either a common short ionic hydrogen bond (SIHB) or a delocalized low barrier hydrogen bond (LBHB) were the hydrogen bond is shorter. So far, the evidence for LBHB in proteins have not been conclusive. Here we show clear NMR evidence that LBHB does exist in NS3, a serine protease from Dengue. The one bond coupling constant between the hydrogen and nitrogen was shown to be only 52 Hz instead of the usual 90 Hz. This together with a 1H chemical shift of 19.93 ppm is evidence that the hydrogen bond distance between His and Asp is shorter than for SIHB. Our result clearly shows the existence of LBHB and will help in understanding the mechanism of the catalytic triad in the important group of serine proteases.
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| 2. |
- Agback, Peter, et al.
(författare)
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Multiple Host Factors Interact with the Hypervariable Domain of Chikungunya Virus nsP3 and Determine Viral Replication in Cell-Specific Mode
- 2018
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Ingår i: Journal of Virology. - 0022-538X. ; 92
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Tidskriftsartikel (refereegranskat)abstract
- Alphaviruses are widely distributed in both hemispheres and circulate between mosquitoes and amplifying vertebrate hosts. Geographically separated alphaviruses have adapted to replication in particular organisms. The accumulating data suggest that this adaptation is determined not only by changes in their glycoproteins but also by the amino acid sequence of the hypervariable domain (HVD) of the alphavirus nsP3 protein. We performed a detailed investigation of chikungunya virus (CHIKV) nsP3 HVD interactions with host factors and their roles in viral replication in vertebrate and mosquito cells. The results demonstrate that CHIKV HVD is intrinsically disordered and binds several distinctive cellular proteins. These host factors include two members of the G3BP family and their mosquito homolog Rin, two members of the NAP1 family, and several SH3 domain-containing proteins. Interaction with G3BP proteins or Rin is an absolute requirement for CHIKV replication, although it is insufficient to solely drive it in either vertebrate or mosquito cells. To achieve a detectable level of virus replication, HVD needs to bind members of at least one more protein family in addition to G3BPs. Interaction with NAP1L1 and NAP1L4 plays a more proviral role in vertebrate cells, while binding of SH3 domain-containing proteins to a proline-rich fragment of HVD is more critical for virus replication in the cells of mosquito origin. Modifications of binding sites in CHIKV HVD allow manipulation of the cell specificity of CHIKV replication. Similar changes may be introduced into HVDs of other alphaviruses to alter their replication in particular cells or tissues.IMPORTANCE Alphaviruses utilize a broad spectrum of cellular factors for efficient formation and function of replication complexes (RCs). Our data demonstrate for the first time that the hypervariable domain (HVD) of chikungunya virus nonstructural protein 3 (nsP3) is intrinsically disordered. It binds at least 3 families of cellular proteins, which play an indispensable role in viral RNA replication. The proteins of each family demonstrate functional redundancy. We provide a detailed map of the binding sites on CHIKV nsP3 HVD and show that mutations in these sites or the replacement of CHIKV HVD by heterologous HVD change cell specificity of viral replication. Such manipulations with alphavirus HVDs open an opportunity for development of new irreversibly attenuated vaccine candidates. To date, the disordered protein fragments have been identified in the nonstructural proteins of many other viruses. They may also interact with a variety of cellular factors that determine critical aspects of virus-host interactions.
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| 3. |
- Agback, Tatiana, et al.
(författare)
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Co-refolding of a functional complex of Dengue NS3 protease and NS2B co-factor domain and backbone resonance assignment by solution NMR
- 2017
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Ingår i: Protein Expression and Purification. - : Elsevier BV. - 1046-5928 .- 1096-0279. ; 140, s. 16-27
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Tidskriftsartikel (refereegranskat)abstract
- A novel approach for separate expression of dengue virus NS3 protease and its NS2B cofactor domain is described in this paper. The two proteins are expressed in E.coli and purified separately and subsequently efficiently co-refolded to form a stable complex. This straightforward and robust method allows for separate isotope labeling of the two proteins, facilitating analysis by nuclear magnetic resonance (NMR) spectroscopy. Unlinked NS2B-NS3pro behaves better in NMR spectroscopy than linked NS2B-NS3pro, which has resulted in the backbone resonance assignment of the unlinked NS2B-NS3 complex bound to a peptidic boronic acid inhibitor. (C) 2017 Elsevier Inc. All rights reserved.
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| 4. |
- Agback, Tatiana
(författare)
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The optimal DFT approach in DP4 NMR structure analysis - pushing the limits of relative configuration elucidation
- 2019
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Ingår i: Organic and Biomolecular Chemistry. - : Royal Society of Chemistry (RSC). - 1477-0520 .- 1477-0539. ; 17, s. 5886-5890
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Tidskriftsartikel (refereegranskat)abstract
- What computational methods should be used to achieve the most reliable result in computational structure elucidation? A study on the effect of quality and quantity of geometries on computational NMR structure elucidation performance is reported. Semi-empirical, HF and DFT methods were explored, and B3LYP optimized geometries in combination with mPW1PW91 shifts and M06-2X conformer energies was found to be best. The required number of conformers considered has also been investigated, as well as several methods for the reduction of this number. Clear guidelines for the best computational NMR structure elucidation methods for different levels of available computing power are provided.
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| 5. |
- Lundquist, Per-Olof, et al.
(författare)
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Root traits of seabuckthorn
- 2016
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Ingår i: RPD Abstracts. - 2255-8624 .- 2500-9788. ; 2, s. 87-87
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)
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