| 1. |
- Andriianov, Aleksandr, et al.
(author)
-
Phage T3 overcomes the BREX defense through SAM cleavage and inhibition of SAM synthesis by SAM lyase
- 2023
-
In: Cell Reports. - : Elsevier. - 2211-1247. ; 42:8
-
Journal article (peer-reviewed)abstract
- Bacteriophage T3 encodes a SAMase that, through cleavage of S-adenosyl methionine (SAM), circumvents the SAM-dependent type I restriction-modification (R-M) defense. We show that SAMase also allows T3 to evade the BREX defense. Although SAM depletion weakly affects BREX methylation, it completely inhibits the defensive function of BREX, suggesting that SAM could be a co-factor for BREX-mediated exclusion of phage DNA, similar to its anti-defense role in type I R-M. The anti-BREX activity of T3 SAMase is mediated not just by enzymatic degradation of SAM but also by direct inhibition of MetK, the host SAM synthase. We present a 2.8 A cryoelectron microscopy (cryo-EM) structure of the eight-subunit T3 SAMase-MetK complex. Structure-guided mutagenesis reveals that this interaction stabilizes T3 SAMase in vivo, further stimulating its anti-BREX activity. This work provides insights in the versatility of bacteriophage counterdefense mech-anisms and highlights the role of SAM as a co-factor of diverse bacterial immunity systems.
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| 2. |
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| 3. |
- Brewster, Jodi L., et al.
(author)
-
Structures and kinetics of Thermotoga maritima MetY reveal new insights into the predominant sulfurylation enzyme of bacterial methionine biosynthesis
- 2021
-
In: Journal of Biological Chemistry. - : Elsevier. - 0021-9258 .- 1083-351X. ; 296
-
Journal article (peer-reviewed)abstract
- Bacterial methionine biosynthesis can take place by either the trans-sulfurylation route or direct sulfurylation. The enzymes responsible for trans-sulfurylation have been characterized extensively because they occur in model organisms such as Escherichia coli. However, direct sulfurylation is actually the predominant route for methionine biosynthesis across the phylogenetic tree. In this pathway, most bacteria use an O-acetylhomoserine aminocarboxypropyltransferase (MetY) to catalyze the formation of homocysteine from O-acetylhomoserine and bisulfide. Despite the widespread distribution of MetY, this pyridoxal 5′-phosphate–dependent enzyme remains comparatively understudied. To address this knowledge gap, we have characterized the MetY from Thermotoga maritima (TmMetY). At its optimal temperature of 70 °C, TmMetY has a turnover number (apparent kcat = 900 s−1) that is 10- to 700-fold higher than the three other MetY enzymes for which data are available. We also present crystal structures of TmMetY in the internal aldimine form and, fortuitously, with a β,γ-unsaturated ketimine reaction intermediate. This intermediate is identical to that found in the catalytic cycle of cystathionine γ-synthase (MetB), which is a homologous enzyme from the trans-sulfurylation pathway. By comparing the TmMetY and MetB structures, we have identified Arg270 as a critical determinant of specificity. It helps to wall off the active site of TmMetY, disfavoring the binding of the first MetB substrate, O-succinylhomoserine. It also ensures a strict specificity for bisulfide as the second substrate of MetY by occluding the larger MetB substrate, cysteine. Overall, this work illuminates the subtle structural mechanisms by which homologous pyridoxal 5′-phosphate–dependent enzymes can effect different catalytic, and therefore metabolic, outcomes.
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| 4. |
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| 5. |
- Chen, Yang, et al.
(author)
-
Staphylococcus aureus elongation factor G - structure and analysis of a target for fusidic acid
- 2010
-
In: The FEBS Journal. - : Wiley. - 1742-464X .- 1742-4658. ; 277:18, s. 3789-3803
-
Journal article (peer-reviewed)abstract
- Fusidic acid (FA) is a bacteriostatic antibiotic that locks elongation factor G (EF-G) on the ribosome in a post-translocational state. It is used clinically against Gram-positive bacteria such as pathogenic strains of Staphylococcus aureus, but no structural information has been available for EF-G from these species. We have solved the apo crystal structure of EF-G from S. aureus to 1.9 A resolution. This structure shows a dramatically different overall conformation from previous structures of EF-G, although the individual domains are highly similar. Between the different structures of free or ribosome-bound EF-G, domains III-V move relative to domains I-II, resulting in a displacement of the tip of domain IV relative to domain G. In S. aureus EF-G, this displacement is about 25 A relative to structures of Thermus thermophilus EF-G in a direction perpendicular to that in previous observations. Part of the switch I region (residues 46-56) is ordered in a helix, and has a distinct conformation as compared with structures of EF-Tu in the GDP and GTP states. Also, the switch II region shows a new conformation, which, as in other structures of free EF-G, is incompatible with FA binding. We have analysed and discussed all known fusA-based fusidic acid resistance mutations in the light of the new structure of EF-G from S. aureus, and a recent structure of T. thermophilus EF-G in complex with the 70S ribosome with fusidic acid [Gao YG et al. (2009) Science326, 694-699]. The mutations can be classified as affecting FA binding, EF-G-ribosome interactions, EF-G conformation, and EF-G stability.
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| 6. |
- Chen, Yang
(author)
-
Structural and Biochemical Studies of Antibiotic Resistance and Ribosomal Frameshifting
- 2013
-
Doctoral thesis (other academic/artistic)abstract
- Protein synthesis, translation, performed by the ribosome, is a fundamental process of life and one of the main targets of antibacterial drugs. This thesis provides structural and biochemical understanding of three aspects of bacterial translation.Elongation factor G (EF-G) is the target for the antibiotic fusidic acid (FA). FA binds to EF-G only on the ribosome after GTP hydrolysis and prevents EF-G dissociation from the ribosome. Point mutations in EF-G can lead to FA resistance but are often accompanied by a fitness cost in terms of slower growth of the bacteria. Secondary mutations can compensate for this fitness cost while resistance is maintained. Here we present the crystal structure of the clinical FA drug target, Staphylococcus aureus EF-G, together with the mapping and analysis of all known FA-resistance mutations in EF-G. We also present crystal structures of the FA-resistant mutant F88L, the FA-hypersensitive mutant M16I and the FA-resistant but fitness-compensated double mutant F88L/M16I. Analysis of mutant structures together with biochemical data allowed us to propose that fitness loss and compensation are caused by effects on the conformational dynamics of EF-G on the ribosome.Aminoglycosides are another group of antibiotics that target the decoding region of the 30S ribosomal subunit. Resistance to aminoglycosides can be acquired by inactivation of the drugs via enzymatic modification. Here, we present the first crystal structure an aminoglycoside 3’’ adenyltransferase, AadA from Salmonella enterica. AadA displays two domains and unlike related structures most likely functions as a monomer.Frameshifts are deviations the standard three-base reading frame of translation. -1 frameshifting can be caused by normal tRNASer3 at GCA alanine codons and tRNAThr3 at CCA/CCG proline codons. This process has been proposed to involve doublet decoding using non-standard codon-anticodon interactions. In our study, we showed by equilibrium binding that these tRNAs bind with low micromolar Kd to the frameshift codons. Our results support the doublet-decoding model and show that non-standard anticodon loop structures need to be adopted for the frameshifts to happen.These findings provide new insights in antibiotic resistance and reading-frame maintenance and will contribute to a better understanding of the translation elongation process.
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| 7. |
- Chen, Yang, et al.
(author)
-
Structure of AadA from Salmonella enterica : a monomeric aminoglycoside (3'')(9) adenyltransferase
- 2015
-
In: Acta Crystallographica Section D. - 0907-4449 .- 1399-0047. ; 71, s. 2267-2277
-
Journal article (peer-reviewed)abstract
- Aminoglycoside resistance is commonly conferred by enzymatic modification of drugs by aminoglycoside-modifying enzymes such as aminoglycoside nucleo\-tidyltransferases (ANTs). Here, the first crystal structure of an ANT(3$^\prime$$^\prime$)(9) adenyltransferase, AadA from Salmonella enterica, is presented. AadA catalyses the magnesium-dependent transfer of adenosine monophosphate from ATP to the two chemically dissimilar drugs streptomycin and spectinomycin. The structure was solved using selenium SAD phasing and refined to 2.5Å resolution. AadA consists of a nucleotidyltransferase domain and an α-helical bundle domain. AadA crystallizes as a monomer and is a monomer in solution as confirmed by small-angle X-ray scattering, in contrast to structurally similar homodimeric adenylating enzymes such as kanamycin nucleotidyltransferase. Isothermal titration calorimetry experiments show that ATP binding has to occur before binding of the aminoglycoside substrate, and structure analysis suggests that ATP binding repositions the two domains for aminoglycoside binding in the interdomain cleft. Candidate residues for ligand binding and catalysis were subjected to site-directed mutagenesis. In vivo resistance and in vitro binding assays support the role of Glu87 as the catalytic base in adenylation, while Arg192 and Lys205 are shown to be critical for ATP binding.
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| 8. |
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| 9. |
- Chi, Celestine, 1978-
(author)
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Post-synaptic Density Disc Large Zo-1 (PDZ) Domains : From Folding and Binding to Drug Targeting
- 2010
-
Doctoral thesis (other academic/artistic)abstract
- Understanding how proteins fold and bind is interesting since these processes are central to most biological activity. Protein folding and protein-protein interaction are by themselves very complex but using a good and robust system to study them could ease some of the hurdles. In this thesis I have tried to answer some of the fundamental questions of protein folding and binding. I chose to work with PDZ domains, which are protein domains consisting of 90-100 amino acids. They are found in more than 400 human proteins and function mostly as protein-protein interaction units. These proteins are very stable, easy to express and purify and their folding reaction is reversible under most laboratory conditions. I have characterized the interaction of PSD-95 PDZ3 domain with its putative ligand under different experimental conditions and found out that its binding kinetics is sensitive to salt and pH. I also demonstrated that the two conserved residues R318 and H372 in PDZ3 are responsible for the salt and pH effect, respectively, on the binding reaction. Moreover, I determined that for PSD 95 PDZ3 coupling of distal residues to peptide binding was better described by a distance relationship and there was a very weak evidence of an allosteric network. Further, I showed that another PDZ domain, SAP97 PDZ2 undergoes conformational change upon ligand binding. Also, I characterized the binding mechanism of a dimeirc ligand/PDZ1-2 tandem interaction and showed that despite its apparent complexity the binding reaction is best described by a square scheme. Additionally, I determined that for the SAP 97 PDZ/HPV E6 interaction that all three PDZ domains each bind one molecule of the E6 protein and that a set of residues in the PDZ2 of SAP 97 could operate in an unexpected long-range manner during E6 interaction. Finally, I showed that perhaps all members in the PDZ family could fold via a three state folding mechanism. I characterized the folding mechanism of five different PDZ domains having similar overall fold but different primary structure and the results indicate that all five fold via an intermediate with two transition states. Transition state one is rate limiting at low denaturant concentration and vice versa for transition state two. Comparing and characterizing the structures of the transition states of two PDZ domains using phi value analysis indicated that their early transition states are less similar as compared to their late transition states.
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| 10. |
- Dunham, Christine M., et al.
(author)
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Structures of tRNAs with an expanded anticodon loop in the decoding center of the 30S ribosomal subunit
- 2007
-
In: RNA. - : Cold Spring Harbor Laboratory. - 1355-8382 .- 1469-9001. ; 13:6, s. 817-823
-
Journal article (peer-reviewed)abstract
- During translation, some +1 frameshift mRNA sites are decoded by frameshift suppressor tRNAs that contain an extra base in their anticodon loops. Similarly engineered tRNAs have been used to insert nonnatural amino acids into proteins. Here, we report crystal structures of two anticodon stem–loops (ASLs) from tRNAs known to facilitate +1 frameshifting bound to the 30S ribosomal subunit with their cognate mRNAs. ASLCCCG and ASLACCC (5'–3' nomenclature) form unpredicted anticodon–codon interactions where the anticodon base 34 at the wobble position contacts either the fourth codon base or the third and fourth codon bases. In addition, we report the structure of ASLACGA bound to the 30S ribosomal subunit with its cognate mRNA. The tRNA containing this ASL was previously shown to be unable to facilitate +1 frameshifting in competition with normal tRNAs (Hohsaka et al. 2001), and interestingly, it displays a normal anticodon–codon interaction. These structures show that the expanded anticodon loop of +1 frameshift promoting tRNAs are flexible enough to adopt conformations that allow three bases of the anticodon to span four bases of the mRNA. Therefore it appears that normal triplet pairing is not an absolute constraint of the decoding center.
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| 11. |
- Durall de la Fuente, Claudia, et al.
(author)
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Oligomerization and characteristics of phosphoenolpyruvate carboxylase in Synechococcus PCC 7002
- 2020
-
In: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 10
-
Journal article (peer-reviewed)abstract
- Phosphoenolpyruvate carboxylase (PEPc) is an essential enzyme in plants. A photosynthetic form is present both as dimer and tetramer in C4 and CAM metabolism. Additionally, non-photosynthetic PEPcs are also present. The single, non-photosynthetic PEPc of the unicellular cyanobacterium Synechococcus PCC 7002 (Synechococcus), involved in the TCA cycle, was examined. Using size exclusion chromatography (SEC) and small angle X-ray scattering (SAXS), we observed that PEPc in Synechococcus exists as both a dimer and a tetramer. This is the first demonstration of two different oligomerization states of a non-photosynthetic PEPc. High concentration of Mg2+, the substrate PEP and a combination of low concentration of Mg2+ and HCO3− induced the tetramer form of the carboxylase. Using SEC-SAXS analysis, we showed that the oligomerization state of the carboxylase is concentration dependent and that, among the available crystal structures of PEPc, the scattering profile of PEPc of Synechococcus agrees best with the structure of PEPc from Escherichia coli. In addition, the kinetics of the tetramer purified in presence of Mg2+ using SEC, and of the mixed population purified in presence of Mg2+ using a Strep-tagged column were examined. Moreover, the enzyme showed interesting allosteric regulation, being activated by succinate and inhibited by glutamine, and not affected by either malate, 2-oxoglutarate, aspartic acid or citric acid.
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| 12. |
- Fedorov, R V, et al.
(author)
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Structure of ribosomal protein TL5 complexed with RNA provides new insights into the CTC family of stress proteins
- 2001
-
In: Acta Crystallographica. Section D: Biological Crystallography. - 1399-0047. ; D57:7, s. 968-976
-
Journal article (peer-reviewed)abstract
- The crystal structure of Thermus thermophilus ribosomal protein TL5 in complex with a fragment of Escherichia coli 5S rRNA has been determined at 2.3 Å resolution. The protein consists of two domains. The structure of the N-terminal domain is close to the structure of E. coli ribosomal protein L25, but the C-terminal domain represents a new fold composed of seven -strands connected by long loops. TL5 binds to the RNA through its N-terminal domain, whereas the C-terminal domain is not included in this interaction. Cd2+ ions, the presence of which improved the crystal quality significantly, bind only to the protein component of the complex and stabilize the protein molecule itself and the interactions between the two molecules in the asymmetric unit of the crystal. The TL5 sequence reveals homology to the so-called general stress protein CTC. The hydrophobic cores which stabilize both TL5 domains are highly conserved in CTC proteins. Thus, all CTC proteins may fold with a topology close to that of TL5.
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| 13. |
- Gao, YG, et al.
(author)
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The structure of the ribosome with elongation factor G trapped in the posttranslocational state
- 2009
-
In: Science. - : American Association for the Advancement of Science (AAAS). - 0036-8075 .- 1095-9203. ; 326:5953, s. 694-699
-
Journal article (peer-reviewed)abstract
- Elongation factor G (EF-G) is a guanosine triphosphatase (GTPase) that plays a crucial role in the translocation of transfer RNAs (tRNAs) and messenger RNA (mRNA) during translation by the ribosome. We report a crystal structure refined to 3.6 angstrom resolution of the ribosome trapped with EF-G in the posttranslocational state using the antibiotic fusidic acid. Fusidic acid traps EF-G in a conformation intermediate between the guanosine triphosphate and guanosine diphosphate forms. The interaction of EF-G with ribosomal elements implicated in stimulating catalysis, such as the L10-L12 stalk and the L11 region, and of domain IV of EF-G with the tRNA at the peptidyl-tRNA binding site (P site) and with mRNA shed light on the role of these elements in EF-G function. The stabilization of the mobile stalks of the ribosome also results in a more complete description of its structure.
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| 14. |
- González-López, Adrián, et al.
(author)
-
Structures of the Staphylococcus aureus ribosome inhibited by fusidic acid and fusidic acid cyclopentane
- 2024
-
In: Scientific Reports. - : Springer Nature. - 2045-2322. ; 14:1
-
Journal article (peer-reviewed)abstract
- The antibiotic fusidic acid (FA) is used to treat Staphylococcus aureus infections. It inhibits protein synthesis by binding to elongation factor G (EF-G) and preventing its release from the ribosome after translocation. While FA, due to permeability issues, is only effective against gram-positive bacteria, the available structures of FA-inhibited complexes are from gram-negative model organisms. To fill this knowledge gap, we solved cryo-EM structures of the S. aureus ribosome in complex with mRNA, tRNA, EF-G and FA to 2.5 Å resolution and the corresponding complex structures with the recently developed FA derivative FA-cyclopentane (FA-CP) to 2.0 Å resolution. With both FA variants, the majority of the ribosomal particles are observed in chimeric state and only a minor population in post-translocational state. As expected, FA binds in a pocket between domains I, II and III of EF-G and the sarcin-ricin loop of 23S rRNA. FA-CP binds in an identical position, but its cyclopentane moiety provides additional contacts to EF-G and 23S rRNA, suggesting that its improved resistance profile towards mutations in EF-G is due to higher-affinity binding. These high-resolution structures reveal new details about the S. aureus ribosome, including confirmation of many rRNA modifications, and provide an optimal starting point for future structure-based drug discovery on an important clinical drug target.
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| 15. |
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| 16. |
- Guo, Xiaohu
(author)
-
Life will find a way : Structural and evolutionary insights into FusB and HisA
- 2015
-
Doctoral thesis (other academic/artistic)abstract
- How do microbes adapt to challenges from the environment? In this thesis, two distinct cases were examined through structural and biochemical methods. In the first, we followed a real-time protein evolution of HisA to a novel function. The second case was fusidic acid (FA) resistance mediated by the protein FusB in Staphylococcus aureus.In the first study, the aim was to understand how mutants of HisA from the histidine biosynthetic pathway could evolve a novel TrpF activity and further evolve to generalist or specialist enzymes. We solved the crystal structure of wild type Salmonella enterica HisA in its apo-state and the structures of the mutants D7N and D7N/D176A in complex with the substrate ProFAR. These two distinct complex structures showed us the coupled conformational changes of HisA and ProFAR before catalysis. We also solved crystal structures of ten mutants, some in complex with substrate or product. The structures indicate that bi-functional mutants adopt distinct loop conformations linked to the two functions and that mutations in specialist enzymes favor one of the conformations. We also observed biphasic relationships in which small changes in the activities of low-performance enzymes had large effects on fitness, until a threshold, above which large changes in enzyme performance had little effect on fitness.Fusidic acid blocks protein translation by locking elongation factor G (EF-G) to the ribosome after GTP hydrolysis in elongation and recycling of bacterial protein synthesis. To understand the rescue mechanism, we solved the crystal structure of FusB at 1.6Å resolution. The structure showed that FusB is a two-domain protein and C-terminal domain contains a treble clef zinc finger. Using hybrid constructs between S. aureus EF-G that binds to FusB, and E. coli EF-G that does not, the binding determinants were located to domain IV of EF-G. This was further supported by small-angle X-ray scattering studies of the FusB·EF-G complex. Using single-molecule methods, we observed FusB frequently binding to the ribosome and rescue of FA-inhibited elongation by effects on the non-rotated state ribosome. Ribosome binding of FusB was confirmed by isothermal titration calorimetry.
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| 17. |
- Guo, Xiaohu, et al.
(author)
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Structure and function of FusB : an elongation factor G-binding fusidic acid resistance protein active in ribosomal translocation and recycling
- 2012
-
In: Open Biology. - : The Royal Society. - 2046-2441. ; 2, s. 120016-
-
Journal article (peer-reviewed)abstract
- Fusidic acid (FA) is a bacteriostatic antibiotic that locks elongation factor G (EF-G) to the ribosome after GTP hydrolysis during elongation and ribosome recycling. The plasmid pUB101-encoded protein FusB causes FA resistance in clinical isolates of Staphylococcus aureus through an interaction with EF-G. Here, we report 1.6 and 2.3 angstrom crystal structures of FusB. We show that FusB is a two-domain protein lacking homology to known structures, where the N-terminal domain is a four-helix bundle and the C-terminal domain has an alpha/beta fold containing a C4 treble clef zinc finger motif and two loop regions with conserved basic residues. Using hybrid constructs between S. aureus EF-G that binds to FusB and Escherichia coli EF-G that does not, we show that the sequence determinants for FusB recognition reside in domain IV and involve the C-terminal helix of S. aureus EF-G. Further, using kinetic assays in a reconstituted translation system, we demonstrate that FusB can rescue FA inhibition of tRNA translocation as well as ribosome recycling. We propose that FusB rescues S. aureus from FA inhibition by preventing formation or facilitating dissociation of the FA-locked EF-G-ribosome complex.
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| 18. |
- Guo, Xiaohu, et al.
(author)
-
Structure and mechanism of a phage-encoded SAM lyase revises catalytic function of enzyme family
- 2021
-
In: eLIFE. - : eLife Sciences Publications Ltd. - 2050-084X. ; 10
-
Journal article (peer-reviewed)abstract
- The first S-adenosyl methionine (SAM) degrading enzyme (SAMase) was discovered in bacteriophage T3, as a counter-defense against the bacterial restriction-modification system, and annotated as a SAM hydrolase forming 5’-methyl-thioadenosine (MTA) and L-homoserine. From environmental phages, we recently discovered three SAMases with barely detectable sequence similarity to T3 SAMase and without homology to proteins of known structure. Here, we present the very first phage SAMase structures, in complex with a substrate analogue and the product MTA. The structure shows a trimer of alpha–beta sandwiches similar to the GlnB-like superfamily, with active sites formed at the trimer interfaces. Quantum-mechanical calculations, thin-layer chromatography, and nuclear magnetic resonance spectroscopy demonstrate that this family of enzymes are not hydrolases but lyases forming MTA and L-homoserine lactone in a unimolecular reaction mechanism. Sequence analysis and in vitro and in vivo mutagenesis support that T3 SAMase belongs to the same structural family and utilizes the same reaction mechanism.
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| 19. |
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| 20. |
- Gustafsson, Robert, et al.
(author)
-
Structure and Characterization of Phosphoglucomutase 5 from Atlantic and Baltic Herring : An Inactive Enzyme with Intact Substrate Binding
- 2020
-
In: Biomolecules. - : MDPI AG. - 2218-273X. ; 10:12
-
Journal article (peer-reviewed)abstract
- Phosphoglucomutase 5 (PGM5) in humans is known as a structural muscle protein without enzymatic activity, but detailed understanding of its function is lacking. PGM5 belongs to the alpha-D-phosphohexomutase family and is closely related to the enzymatically active metabolic enzyme PGM1. In the Atlantic herring, Clupea harengus, PGM5 is one of the genes strongly associated with ecological adaptation to the brackish Baltic Sea. We here present the first crystal structures of PGM5, from the Atlantic and Baltic herring, diering by a single substitution Ala330Val. The structure of PGM5 is overall highly similar to structures of PGM1. The structure of the Baltic herring PGM5 in complex with the substrate glucose-1-phosphate shows conserved substrate binding and active site compared to human PGM1, but both PGM5 variants lack phosphoglucomutase activity under the tested conditions. Structure comparison and sequence analysis of PGM5 and PGM1 from fish and mammals suggest that the lacking enzymatic activity of PGM5 is related to dierences in active-site loops that are important for flipping of the reaction intermediate. The Ala330Val substitution does not alter structure or biophysical properties of PGM5 but, due to its surface-exposed location, could affect interactions with protein-binding partners.
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| 21. |
- Haq, Syed Raza, et al.
(author)
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The plastic energy landscape of protein folding : a triangular folding mechanism with an equilibrium intermediate for a small protein domain
- 2010
-
In: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 285:23, s. 18051-18059
-
Journal article (peer-reviewed)abstract
- Protein domains usually fold without or with only transiently populated intermediates, possibly to avoid misfolding, which could result in amyloidogenic disease. Whether observed intermediates are productive and obligatory species on the folding reaction pathway or dispensable by-products is a matter of debate. Here, we solved the crystal structure of a small protein domain, SAP97 PDZ2 I342W C378A, and determined its folding pathway. The presence of a folding intermediate was demonstrated both by single and double-mixing kinetic experiments using urea-induced (un) folding as well as ligand-induced folding. This protein domain was found to fold via a triangular scheme, where the folding intermediate could be either on-or off-pathway, depending on the experimental conditions. Furthermore, we found that the intermediate was present at equilibrium, which is rarely seen in folding reactions of small protein domains. The folding mechanism observed here illustrates the roughness and plasticity of the protein folding energy landscape, where several routes may be employed to reach the native state. The results also reconcile the folding mechanisms of topological variants within the PDZ domain family.
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| 22. |
- Henriksson, Linda, 1973-
(author)
-
Structural and functional studies of a novel Botulinum neurotoxin and of MTH1
- 2018
-
Doctoral thesis (other academic/artistic)abstract
- X-ray crystallography visualizes the three dimensional molecular structures of proteins at atomic resolution. Seeing the molecular structure of a biomedically interesting protein enables a higher understanding of its function. The process of producing pure protein from genetic material to generate crystals and determine the molecular structure can be a long and challenging process. My thesis involves structural and functional studies of two different proteins, which are both biomedically interesting and important to learn about. X-ray crystallography is the method which has been used to determine the majority of the protein structures that we know of today and is also the method used in the results presented in my thesis. Today there are no cancer therapies defeating all types of cancers and they do not come without side effects. Battling cancer diseases often include long and painful treatments. Finding an anti-cancer drug targeting phenotypes characteristic of cancer cells is a compelling thought. MutT homolog-1 (MTH1) is an enzyme present in all proliferating cells. The enzyme seems to be crucial for cancer cell survival but not for the viability of normal cells. MTH1 cleans out oxidized and thereby damaged nucleotides from the free nucleotide pool and stops them from being used in DNA synthesis. This process is very important in fast proliferating cancer cells. The hypothesis is to inhibit MTH1 and thereby allow a limitless amount of DNA damage in the cancer cells. This action will eventually kill cancer cells while not affecting normal cells. The molecular structure of MTH1 with (PDB ID: 3ZR0) and without a product bound (PDB ID: 3ZR1) was determined and is presented in my thesis. These two structures aided in the synthesis of inhibitors. Botulinum neurotoxins (BoNTs) are the most potent toxins known. As little as one gram of pure toxin could potentially kill one million people. Due to its potency BoNT is a potential bioterrorism threat. The toxin is also a very potent drug used clinically to relieve the symptoms of an array of neuromuscular disorders. Most people know this neurotoxin by one of its commercial names: Botox™. Additionally BoNTs are the cause of botulism. BoNTs are neuro-specific enzymes that target neuromuscular signaling, inducing flaccid paralysis and potentially death. It is of importance to learn more about these toxins to enable the development of new countermeasures, vaccines or more efficient neuroparalytic drugs. BoNTs consist of three domains with different functions, all crucial for intoxication. The toxins are fragile and can easily be destroyed by harsh surroundings if not protected by non-toxic non-hemagglutinin (NTNH) proteins. The complex of some BoNT serotypes and their protective NTNH have proven to be pH-dependent. Parts of the intoxication process are not yet clear and their mechanisms are still puzzling researchers. Until recently seven BoNT serotypes were identified. We have now identified and characterized a novel serotype called BoNT/X. The molecular structure of the active domain is presented here (PDB ID: 6F47). The pH-dependent mechanism forming a complex as seen in other serotypes, is confirmed to be present in BoNT/X as well.
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| 23. |
- Hirokawa, G, et al.
(author)
-
Post-termination complex disassembly by ribosome recycling factor, a functional tRNA mimic
- 2002
-
In: EMBO Journal. - : Wiley. - 1460-2075. ; 21:9, s. 2272-2281
-
Journal article (peer-reviewed)abstract
- Ribosome recycling factor (RRF) together with elongation factor G (EF-G) disassembles the post- termination ribosomal complex. Inhibitors of translocation, thiostrepton, viomycin and aminoglycosides, inhibited the release of tRNA and mRNA from the post-termination complex. In contrast, fusidic acid and a GTP analog that fix EF-G to the ribosome, allowing one round of tRNA translocation, inhibited mRNA but not tRNA release from the complex. The release of tRNA is a prerequisite for mRNA release but partially takes place with EF-G alone. The data are consistent with the notion that RRF binds to the A-site and is translocated to the P-site, releasing deacylated tRNA from the P- and E-sites. The final step, the release of mRNA, is accompanied by the release of RRF and EF-G from the ribosome. With the model post-termination complex, 70S ribosomes were released from the post-termination complex by the RRF reaction and were then dissociated into subunits by IF3.
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| 24. |
- Hultqvist, Greta, 1980-, et al.
(author)
-
Energetic pathway sampling in a protein interaction domain
- 2013
-
In: Structure. - : Elsevier BV. - 0969-2126 .- 1878-4186. ; 21, s. 1193-1202
-
Journal article (other academic/artistic)abstract
- The affinity and specificity of protein-ligand interactions are influenced by energeticcrosstalk within the protein domain. However, the molecular details of such intradomain allostery are still unclear. Here, we have experimentally detected and computationally predicted interactionpathways in the postsynaptic density 95/discs large/zonula occludens 1 (PDZ)-peptide ligand model system using wild-type and circularly permuted PDZ proteins. The circular permutant introduced small perturbations in the tertiary structure and a concomitant rewiring of allosteric pathways, allowing us to describe how subtle changes may reshape energetic signaling. The results were analyzed in the context of other members of the PDZ family, which were found to contain distinct interaction pathways for different peptide ligands. The data reveal a fascinating scenario whereby several energetic pathways are sampled within one single domain and distinct pathways are activated by specific protein ligands.
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| 25. |
- Hultqvist, Greta, 1980-, et al.
(author)
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Tolerance of Protein Folding to a Circular Permutation in a PDZ Domain
- 2012
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In: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 7:11, s. e50055-
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Journal article (peer-reviewed)abstract
- Circular permutation is a common molecular mechanism for evolution of proteins. However, such re-arrangement of secondary structure connectivity may interfere with the folding mechanism causing accumulation of folding intermediates, which in turn can lead to misfolding. We solved the crystal structure and investigated the folding pathway of a circularly permuted variant of a PDZ domain, SAP97 PDZ2. Our data illustrate how well circular permutation may work as a mechanism for molecular evolution. The circular permutant retains the overall structure and function of the native protein domain. Further, unlike most examples in the literature, this circular permutant displays a folding mechanism that is virtually identical to that of the wild type. This observation contrasts with previous data on the circularly permuted PDZ2 domain from PTP-BL, for which the folding pathway was remarkably affected by the same mutation in sequence connectivity. The different effects of this circular permutation in two homologous proteins show the strong influence of sequence as compared to topology. Circular permutation, when peripheral to the major folding nucleus, may have little effect on folding pathways and could explain why, despite the dramatic change in primary structure, it is frequently tolerated by different protein folds.
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