| 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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H-1, C-13 and N-15 resonance assignment of backbone and IVL-methyl side chain of the S135A mutant NS3pro/NS2B protein of Dengue II virus reveals unique secondary structure features in solution
- 2022
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Ingår i: Biomolecular Nmr Assignments. - : Springer Science and Business Media LLC. - 1874-2718 .- 1874-270X. ; 16, s. 135-145
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Tidskriftsartikel (refereegranskat)abstract
- The serotype II Dengue (DENV 2) virus is the most prevalent of all four known serotypes. Herein, we present nearly complete H-1, N-15, and C-13 backbone and H-1, C-13 isoleucine, valine, and leucine methyl resonance assignment of the apo S135A catalytically inactive variant of the DENV 2 protease enzyme folded as a tandem formed between the serine protease domain NS3pro and the cofactor NS2B, as well as the secondary structure prediction of this complex based on the assigned chemical shifts using the TALOS-N software. Our results provide a solid ground for future elucidation of the structure and dynamic of the apo NS3pro/NS2B complex, key for adequate development of inhibitors, and a thorough molecular understanding of their function(s).
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| 3. |
- 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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| 4. |
- Agback, Peter, et al.
(författare)
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NAP1L1 and NAP1L4 Binding to Hypervariable Domain of Chikungunya Virus nsP3 Protein Is Bivalent and Requires Phosphorylation
- 2021
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Ingår i: Journal of Virology. - 0022-538X .- 1098-5514. ; 95
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Tidskriftsartikel (refereegranskat)abstract
- Chikungunya virus (CHIKV) is one of the most pathogenic members of the Alphavirus genus in the Togaviridae family. Within the last 2 decades, CHIKV has expanded its presence to both hemispheres and is currently circulating in both Old and New Worlds. Despite the severity and persistence of the arthritis it causes in humans, no approved vaccines or therapeutic means have been developed for CHIKV infection. Replication of alphaviruses, including CHIKV, is determined not only by their nonstructural proteins but also by a wide range of host factors, which are indispensable components of viral replication complexes (vRCs). Alphavirus nsP3s contain hypervariable domains (HVDs), which encode multiple motifs that drive recruitment of cell- and virus-specific host proteins into vRCs. Our previous data suggested that NAP1 family members are a group of host factors that may interact with CHIKV nsP3 HVD. In this study, we performed a detailed investigation of the NAP1 function in CHIKV replication in vertebrate cells. Our data demonstrate that (i) the NAP1-HVD interactions have strong stimulatory effects on CHIKV replication, (ii) both NAP1L1 and NAP1L4 interact with the CHIKV HVD, (iii) NAP1 family members interact with two motifs, which are located upstream and downstream of the G3BP-binding motifs of CHIKV HVD, (iv) NAP1 proteins interact only with a phosphorylated form of CHIKV HVD, and HVD phosphorylation is mediated by CK2 kinase, and (v) NAP1 and other families of host factors redundantly promote CHIKV replication and their bindings have additive stimulatory effects on viral replication.IMPORTANCE Cellular proteins play critical roles in the assembly of alphavirus replication complexes (vRCs). Their recruitment is determined by the viral nonstructural protein 3 (nsP3). This protein contains a long, disordered hypervariable domain (HVD), which encodes virus-specific combinations of short linear motifs interacting with host factors during vRC assembly. Our study defined the binding mechanism of NAP1 family members to CHIKV HVD and demonstrated a stimulatory effect of this interaction on viral replication. We show that interaction with NAP1L1 is mediated by two HVD motifs and requires phosphorylation of HVD by CK2 kinase. Based on the accumulated data, we present a map of the binding motifs of the critical host factors currently known to interact with CHIKV HVD. It can be used to manipulate cell specificity of viral replication and pathogenesis, and to develop a new generation of vaccine candidates.
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| 5. |
- Agback, Peter, et al.
(författare)
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Novel NMR Assignment Strategy Reveals Structural Heterogeneity in Solution of the nsP3 HVD Domain of Venezuelan Equine Encephalitis Virus
- 2020
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Ingår i: Molecules (Basel, Switzerland). - : MDPI AG. - 1420-3049. ; 25
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Tidskriftsartikel (refereegranskat)abstract
- In recent years, intrinsically disordered proteins (IDPs) and disordered domains have attracted great attention. Many of them contain linear motifs that mediate interactions with other factors during formation of multicomponent protein complexes. NMR spectrometry is a valuable tool for characterizing this type of interactions on both amino acid (aa) and atomic levels. Alphaviruses encode a nonstructural protein nsP3, which drives viral replication complex assembly. nsP3 proteins contain over 200-aa-long hypervariable domains (HVDs), which exhibits no homology between different alphavirus species, are predicted to be intrinsically disordered and appear to be critical for alphavirus adaptation to different cells. Previously, we have shown that nsP3 HVD of chikungunya virus (CHIKV) is completely disordered with low tendency to form secondary structures in free form. In this new study, we used novel NMR approaches to assign the spectra for the nsP3 HVD of Venezuelan equine encephalitis virus (VEEV). The HVDs of CHIKV and VEEV have no homology but are both involved in replication complex assembly and function. We have found that VEEV nsP3 HVD is also mostly disordered but contains a short stable alpha-helix in its C-terminal fragment, which mediates interaction with the members of cellular Fragile X syndrome protein family. Our NMR data also suggest that VEEV HVD has several regions with tendency to form secondary structures.
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| 6. |
- Agback, Peter, et al.
(författare)
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Probing contacts of inhibitor locked in transition states in the catalytic triad of DENV2 type serine protease and its mutants by 1H, 19F and 15 N NMR spectroscopy
- 2020
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Ingår i: BMC molecular and cell biology. - : Springer Science and Business Media LLC. - 2661-8850. ; 21
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Tidskriftsartikel (refereegranskat)abstract
- Background Detailed structural knowledge of enzyme-inhibitor complexes trapped in intermediate state is the key for a fundamental understanding of reaction mechanisms taking place in enzymes and is indispensable as a structure-guided drug design tool. Solution state NMR uniquely allows the study of active sites of enzymes in equilibrium between different tautomeric forms. In this study 1H, 19F and 15 N NMR spectroscopy has been used to probe the interaction contacts of inhibitors locked in transition states of the catalytic triad of a serine protease. It was demonstrated on the serotype II Dengue virus NS2B:NS3pro serine protease and its mutants, H51N and S135A, in complex with high-affinity ligands containing trifluoromethyl ketone (tfk) and boronic groups in the C-terminal of tetra-peptides. Results Monitoring 19F resonances, shows that only one of the two isomers of the tfk tetra-peptide binds with NS2B:NS3pro and that access to the bulk of the active site is limited. Moreover, there were no bound water found in proximity of the active site for any of the ligands manifesting in a favorable condition for formation of low barrier hydrogen bonds (LBHB) in the catalytic triad. Based on this data we were able to identify a locked conformation of the protein active site. The data also indicates that the different parts of the binding site most likely act independently of each other. Conclusions Our reported findings increases the knowledge of the detailed function of the catalytic triad in serine proteases and could facilitate the development of rational structure based inhibitors that can selectively target the NS3 protease of Dengue type II (DENV2) virus. In addition the results shows the usefulness of probing active sites using F-19 NMR spectroscopy.
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| 7. |
- Agback, Peter, et al.
(författare)
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Site-specific recognition of SARS-CoV-2 nsp1 protein with a tailored titanium dioxide nanoparticle - elucidation of the complex structure using NMR data and theoretical calculation
- 2022
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Ingår i: Nanoscale advances. - : Royal Society of Chemistry (RSC). - 2516-0230. ; 4, s. 1527-1532
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Tidskriftsartikel (refereegranskat)abstract
- The ongoing world-wide Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) pandemic shows the need for new potential sensing and therapeutic means against the CoV viruses. The SARS-CoV-2 nsp1 protein is important, both for replication and pathogenesis, making it an attractive target for intervention. In this study we investigated the interaction of this protein with two types of titania nanoparticles by NMR and discovered that while lactate capped particles essentially did not interact with the protein chain, the aminoalcohol-capped ones showed strong complexation with a distinct part of an ordered alpha-helix fragment. The structure of the forming complex was elucidated based on NMR data and theoretical calculation. To the best of our knowledge, this is the first time that a tailored titanium oxide nanoparticle was shown to interact specifically with a unique site of the full-length SARS-CoV-2 nsp1 protein, possibly interfering with its functionality.
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| 8. |
- Agback, Peter, et al.
(författare)
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Structural characterization and biological function of bivalent binding of CD2AP to intrinsically disordered domain of chikungunya virus nsP3 protein
- 2019
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Ingår i: Virology. - : Elsevier BV. - 0042-6822 .- 1178-122X. ; 537, s. 130-142
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Tidskriftsartikel (refereegranskat)abstract
- Alphavirus nsP3 proteins contain long, intrinsically disordered, hypervariable domains, HVD, which serve as hubs for interaction with many cellular proteins. Here, we have deciphered the mechanism and function of HVD interaction with host factors in alphavirus replication. Using NMR spectroscopy, we show that CHIKV HVD contains two SH3 domain-binding sites. Using an innovative chemical shift perturbation signature approach, we demonstrate that CD2AP interaction with HVD is mediated by its SH3-A and SH3–C domains, and this leaves the SH3–B domain available for interaction with other cellular factor(s). This cooperative interaction with two SH3 domains increases binding affinity to CD2AP and possibly induces long-range allosteric effects in HVD. Our data demonstrate that BIN1, CD2AP and SH3KBP1 play redundant roles in initiation of CHIKV replication. Point mutations in both CHIKV HVD binding sites abolish its interaction with all three proteins, CD2AP, BIN1 and SH3KBP1. This results in strong inhibition of viral replication initiation. © 2019 Elsevier Inc.
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| 9. |
- 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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| 10. |
- Agback, Tatiana, et al.
(författare)
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Combined NMR and molecular dynamics conformational filter identifies unambiguously dynamic ensembles of Dengue protease NS2B/NS3pro
- 2023
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Ingår i: COMMUNICATIONS BIOLOGY. - 2399-3642. ; 6:1
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Tidskriftsartikel (refereegranskat)abstract
- The dengue protease NS2B/NS3pro has been reported to adopt either an 'open' or a 'closed' conformation. We have developed a conformational filter that combines NMR with MD simulations to identify conformational ensembles that dominate in solution. Experimental values derived from relaxation parameters for the backbone and methyl side chains were compared with the corresponding back-calculated relaxation parameters of different conformational ensembles obtained from free MD simulations. Our results demonstrate a high prevalence for the 'closed' conformational ensemble while the 'open' conformation is absent, indicating that the latter conformation is most probably due to crystal contacts. Conversely, conformational ensembles in which the positioning of the co-factor NS2B results in a 'partially' open conformation, previously described in both MD simulations and X-ray studies, were identified by our conformational filter. Altogether, we believe that our approach allows for unambiguous identification of true conformational ensembles, an essential step for reliable drug discovery. A conformational filter that combines NMR with MD simulations to identify conformational ensembles that dominate in solution suggests dengue protease NS2B/NS3pro predominantly exists in a "closed" conformation.
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