| 1. |
- Barends, Thomas R. M., et al.
(författare)
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Structure and mechanism of a bacterial light-regulated cyclic nucleotide phosphodiesterase
- 2009
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Ingår i: Nature. - : Springer Science and Business Media LLC. - 0028-0836 .- 1476-4687. ; 459, s. 1015-1018
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Tidskriftsartikel (refereegranskat)abstract
- The ability to respond to light is crucial for most organisms. BLUF is a recently identified photoreceptor protein domain that senses blue light using a FAD chromophore. BLUF domains are present in various proteins from the Bacteria, Euglenozoa and Fungi. Although structures of single-domain BLUF proteins have been determined, none are available for a BLUF protein containing a functional output domain; the mechanism of light activation in this new class of photoreceptors has thus remained poorly understood. Here we report the biochemical, structural and mechanistic characterization of a full-length, active photoreceptor, BlrP1 (also known as KPN_01598), from Klebsiella pneumoniae. BlrP1 consists of a BLUF sensor domain and a phosphodiesterase EAL output domain which hydrolyses cyclic dimeric GMP (c-di-GMP). This ubiquitous second messenger controls motility, biofilm formation, virulence and antibiotic resistance in the Bacteria. Crystal structures of BlrP1 complexed with its substrate and metal ions involved in catalysis or in enzyme inhibition provide a detailed understanding of the mechanism of the EAL-domain c-di-GMP phosphodiesterases. These structures also sketch out a path of light activation of the phosphodiesterase output activity. Photon absorption by the BLUF domain of one subunit of the antiparallel BlrP1 homodimer activates the EAL domain of the second subunit through allosteric communication transmitted through conserved domain-domain interfaces.
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| 2. |
- Carter, Megan, et al.
(författare)
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Crystal structure, biochemical and cellular activities demonstrate separate functions of MTH1 and MTH2
- 2015
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Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 6
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Tidskriftsartikel (refereegranskat)abstract
- Deregulated redox metabolism in cancer leads to oxidative damage to cellular components including deoxyribonucleoside triphosphates (dNTPs). Targeting dNTP pool sanitizing enzymes, such as MTH1, is a highly promising anticancer strategy. The MTH2 protein, known as NUDT15, is described as the second human homologue of bacterial MutT with 8-oxo-dGTPase activity. We present the first NUDT15 crystal structure and demonstrate that NUDT15 prefers other nucleotide substrates over 8-oxo-dGTP. Key structural features are identified that explain different substrate preferences for NUDT15 and MTH1. We find that depletion of NUDT15 has no effect on incorporation of 8-oxo-dGTP into DNA and does not impact cancer cell survival in cell lines tested. NUDT17 and NUDT18 were also profiled and found to have far less activity than MTH1 against oxidized nucleotides. We show that NUDT15 is not a biologically relevant 8-oxo-dGTPase, and that MTH1 is the most prominent sanitizer of the cellular dNTP pool known to date.
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| 3. |
- Diamanti, Riccardo, et al.
(författare)
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Comparative structural analysis provides new insights into the function of R2-like ligand-binding oxidase
- 2022
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Ingår i: FEBS Letters. - : John Wiley & Sons. - 0014-5793 .- 1873-3468. ; 596:12, s. 1600-1610
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Tidskriftsartikel (refereegranskat)abstract
- R2-like ligand-binding oxidase (R2lox) is a ferritin-like protein that harbours a heterodinuclear manganese–iron active site. Although R2lox function is yet to be established, the enzyme binds a fatty acid ligand coordinating the metal centre and catalyses the formation of a tyrosine–valine ether cross-link in the protein scaffold upon O2 activation. Here, we characterized the ligands copurified with R2lox by mass spectrometry-based metabolomics. Moreover, we present the crystal structures of two new homologs of R2lox, from Saccharopolyspora erythraea and Sulfolobus acidocaldarius, at 1.38 Å and 2.26 Å resolution, respectively, providing the highest resolution structure for R2lox, as well as new insights into putative mechanisms regulating the function of the enzyme.
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| 4. |
- Fenn, Sebastian, et al.
(författare)
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Crystal Structure of an Anti-Ang2 CrossFab Demonstrates Complete Structural and Functional Integrity of the Variable Domain
- 2013
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 8:4
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Tidskriftsartikel (refereegranskat)abstract
- Bispecific antibodies are considered as a promising class of future biotherapeutic molecules. They comprise binding specificities for two different antigens, which may provide additive or synergistic modes of action. There is a wide variety of design alternatives for such bispecific antibodies, including the "CrossMab" format. CrossMabs contain a domain crossover in one of the antigen-binding (Fab) parts, together with the "knobs-and-holes" approach, to enforce the correct assembly of four different polypeptide chains into an IgG-like bispecific antibody. We determined the crystal structure of a hAng-2-binding Fab in its crossed and uncrossed form and show that CH1-CL-domain crossover does not induce significant perturbations of the structure and has no detectable influence on target binding.
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| 5. |
- Grāve, Kristīne, et al.
(författare)
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Redox-induced structural changes in the di-iron and di-manganese forms of Bacillus anthracis ribonucleotide reductase subunit NrdF suggest a mechanism for gating of radical access
- 2019
-
Ingår i: Journal of Biological Inorganic Chemistry. - : Springer Science and Business Media LLC. - 0949-8257 .- 1432-1327. ; 24:6, s. 849-861
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Tidskriftsartikel (refereegranskat)abstract
- Class Ib ribonucleotide reductases (RNR) utilize a di-nuclear manganese or iron cofactor for reduction of superoxide or molecular oxygen, respectively. This generates a stable tyrosyl radical (Y center dot) in the R2 subunit (NrdF), which is further used for ribonucleotide reduction in the R1 subunit of RNR. Here, we report high-resolution crystal structures of Bacillus anthracis NrdF in the metal-free form (1.51 angstrom) and in complex with manganese (Mn-II/Mn-II, 1.30 angstrom). We also report three structures of the protein in complex with iron, either prepared anaerobically (Fe-II/Fe-II form, 1.32 angstrom), or prepared aerobically in the photo-reduced Fe-II/Fe-II form (1.63 angstrom) and with the partially oxidized metallo-cofactor (1.46 angstrom). The structures reveal significant conformational dynamics, likely to be associated with the generation, stabilization, and transfer of the radical to the R1 subunit. Based on observed redox-dependent structural changes, we propose that the passage for the superoxide, linking the FMN cofactor of NrdI and the metal site in NrdF, is closed upon metal oxidation, blocking access to the metal and radical sites. In addition, we describe the structural mechanics likely to be involved in this process.
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| 6. |
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| 7. |
- Grāve, Kristīne, et al.
(författare)
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The Bacillus anthracis class Ib ribonucleotide reductase subunit NrdF intrinsically selects manganese over iron
- 2020
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Ingår i: Journal of Biological Inorganic Chemistry. - : Springer Science and Business Media LLC. - 0949-8257 .- 1432-1327. ; 25:4, s. 571-582
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Tidskriftsartikel (refereegranskat)abstract
- Correct protein metallation in the complex mixture of the cell is a prerequisite for metalloprotein function. While some metals, such as Cu, are commonly chaperoned, specificity towards metals earlier in the Irving-Williams series is achieved through other means, the determinants of which are poorly understood. The dimetal carboxylate family of proteins provides an intriguing example, as different proteins, while sharing a common fold and the same 4-carboxylate 2-histidine coordination sphere, are known to require either a Fe/Fe, Mn/Fe or Mn/Mn cofactor for function. We previously showed that the R2lox proteins from this family spontaneously assemble the heterodinuclear Mn/Fe cofactor. Here we show that the class Ib ribonucleotide reductase R2 protein from Bacillus anthracis spontaneously assembles a Mn/Mn cofactor in vitro, under both aerobic and anoxic conditions, when the metal-free protein is subjected to incubation with Mn-II and Fe-II in equal concentrations. This observation provides an example of a protein scaffold intrinsically predisposed to defy the Irving-Williams series and supports the assumption that the Mn/Mn cofactor is the biologically relevant cofactor in vivo. Substitution of a second coordination sphere residue changes the spontaneous metallation of the protein to predominantly form a heterodinuclear Mn/Fe cofactor under aerobic conditions and a Mn/Mn metal center under anoxic conditions. Together, the results describe the intrinsic metal specificity of class Ib RNR and provide insight into control mechanisms for protein metallation.
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| 8. |
- Griese, Julia J., et al.
(författare)
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Assembly of a heterodinuclear Mn/Fe cofactor is coupled to tyrosine-valine ether cross-link formation in the R2-like ligand-binding oxidase
- 2019
-
Ingår i: Journal of Biological Inorganic Chemistry. - : Springer Science and Business Media LLC. - 0949-8257 .- 1432-1327. ; 24:2, s. 211-221
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Tidskriftsartikel (refereegranskat)abstract
- R2-like ligand-binding oxidases (R2lox) assemble a heterodinuclear Mn/Fe cofactor which performs reductive dioxygen (O-2) activation, catalyzes formation of a tyrosine-valine ether cross-link in the protein scaffold, and binds a fatty acid in a putative substrate channel. We have previously shown that the N-terminal metal binding site 1 is unspecific for manganese or iron in the absence of O-2, but prefers manganese in the presence of O-2, whereas the C-terminal site 2 is specific for iron. Here, we analyze the effects of amino acid exchanges in the cofactor environment on cofactor assembly and metalation specificity using X-ray crystallography, X-ray absorption spectroscopy, and metal quantification. We find that exchange of either the cross-linking tyrosine or the valine, regardless of whether the mutation still allows cross-link formation or not, results in unspecific manganese or iron binding at site 1 both in the absence or presence of O-2, while site 2 still prefers iron as in the wild-type. In contrast, a mutation that blocks binding of the fatty acid does not affect the metal specificity of either site under anoxic or aerobic conditions, and cross-link formation is still observed. All variants assemble a dinuclear trivalent metal cofactor in the aerobic resting state, independently of cross-link formation. These findings imply that the cross-link residues are required to achieve the preference for manganese in site 1 in the presence of O-2. The metalation specificity, therefore, appears to be established during the redox reactions leading to cross-link formation.
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| 9. |
- Griese, Julia J., et al.
(författare)
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Assembly of nonheme Mn/Fe active sites in heterodinuclear metalloproteins
- 2014
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Ingår i: Journal of Biological Inorganic Chemistry. - : Springer Science and Business Media LLC. - 0949-8257 .- 1432-1327. ; 19:6, s. 759-774
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Forskningsöversikt (refereegranskat)abstract
- The ferritin superfamily contains several protein groups that share a common fold and metal coordinating ligands. The different groups utilize different dinuclear cofactors to perform a diverse set of reactions. Several groups use an oxygen-activating di-iron cluster, while others use di-manganese or heterodinuclear Mn/Fe cofactors. Given the similar primary ligand preferences of Mn and Fe as well as the similarities between the binding sites, the basis for metal specificity in these systems remains enigmatic. Recent data for the heterodinuclear cluster show that the protein scaffold per se is capable of discriminating between Mn and Fe and can assemble the Mn/Fe center in the absence of any potential assembly machineries or metal chaperones. Here we review the current understanding of the assembly of the heterodinuclear cofactor in the two different protein groups in which it has been identified, ribonucleotide reductase R2c proteins and R2-like ligand-binding oxidases. Interestingly, although the two groups form the same metal cluster they appear to employ partly different mechanisms to assemble it. In addition, it seems that both the thermodynamics of metal binding and the kinetics of oxygen activation play a role in achieving metal specificity.
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| 10. |
- Griese, Julia J., et al.
(författare)
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Direct observation of structurally encoded metal discrimination and ether bond formation in a heterodinuclear metalloprotein
- 2013
-
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. ; 110:43, s. 17189-17194
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Tidskriftsartikel (refereegranskat)abstract
- Although metallocofactors are ubiquitous in enzyme catalysis, how metal binding specificity arises remains poorly understood, especially in the case of metals with similar primary ligand preferences such as manganese and iron. The biochemical selection of manganese over iron presents a particularly intricate problem because manganese is generally present in cells at a lower concentration than iron, while also having a lower predicted complex stability according to the Irving-Williams series (Mn-II < Fe-II < Ni-II < Co-II < Cu-II > Zn-II). Here we show that a heterodinuclear Mn/Fe cofactor with the same primary protein ligands in both metal sites self-assembles from MnII and FeII in vitro, thus diverging from the Irving-Williams series without requiring auxiliary factors such as metallochaperones. Crystallographic, spectroscopic, and computational data demonstrate that one of the two metal sites preferentially binds FeII over MnII as expected, whereas the other site is nonspecific, binding equal amounts of both metals in the absence of oxygen. Oxygen exposure results in further accumulation of the Mn/Fe cofactor, indicating that cofactor assembly is at least a two-step process governed by both the intrinsic metal specificity of the protein scaffold and additional effects exerted during oxygen binding or activation. We further show that the mixed-metal cofactor catalyzes a two-electron oxidation of the protein scaffold, yielding a tyrosine-valine ether cross-link. Theoretical modeling of the reaction by density functional theory suggests a multistep mechanism including a valyl radical intermediate.
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| 11. |
- Griese, Julia J., et al.
(författare)
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Ether cross-link formation in the R2-like ligand-binding oxidase
- 2018
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Ingår i: Journal of Biological Inorganic Chemistry. - : Springer Science and Business Media LLC. - 0949-8257 .- 1432-1327. ; 23:6, s. 879-886
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Tidskriftsartikel (refereegranskat)abstract
- R2-like ligand-binding oxidases contain a dinuclear metal cofactor which can consist either of two iron ions or one manganese and one iron ion, but the heterodinuclear Mn/Fe cofactor is the preferred assembly in the presence of Mn-II and Fe-II in vitro. We have previously shown that both types of cofactor are capable of catalyzing formation of a tyrosine-valine ether cross-link in the protein scaffold. Here we demonstrate that Mn/Fe centers catalyze cross-link formation more efficiently than Fe/Fe centers, indicating that the heterodinuclear cofactor is the biologically relevant one. We further explore the chemical potential of the Mn/Fe cofactor by introducing mutations at the cross-linking valine residue. We find that cross-link formation is possible also to the tertiary beta-carbon in an isoleucine, but not to the secondary beta-carbon or tertiary gamma-carbon in a leucine, nor to the primary beta-carbon of an alanine. These results illustrate that the reactivity of the cofactor is highly specific and directed.
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| 12. |
- Griese, Julia J., et al.
(författare)
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Location-specific quantification of protein-bound metal ions by X-ray anomalous dispersion : Q-XAD
- 2019
-
Ingår i: Acta Crystallographica Section D. - 2059-7983. ; D75, s. 764-771
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Tidskriftsartikel (refereegranskat)abstract
- Here, a method is described which exploits X-ray anomalous dispersion (XAD) to quantify mixtures of metal ions in the binding sites of proteins and can be applied to metalloprotein crystals of average quality. This method has successfully been used to study site-specific metal binding in a protein from the R2-like ligand-binding oxidase family which assembles a heterodinuclear Mn/Fe cofactor. While previously only the relative contents of Fe and Mn in each metal-binding site have been assessed, here it is shown that the method can be extended to quantify the relative occupancies of at least three different transition metals, enabling complex competition experiments. The number of different metal ions that can be quantified is only limited by the number of high-quality anomalous data sets that can be obtained from one crystal, as one data set has to be collected for each transition-metal ion that is present (or is suspected to be present) in the protein, ideally at the absorption edge of each metal. A detailed description of the method, Q-XAD, is provided.
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| 13. |
- Griese, Julia J., et al.
(författare)
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Structural Basis for Oxygen Activation at a Heterodinuclear Manganese/Iron Cofactor
- 2015
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Ingår i: Journal of Biological Chemistry. - : American Society for Biochemistry and Molecular Biology. - 0021-9258 .- 1083-351X. ; 290:42, s. 25254-25272
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Tidskriftsartikel (refereegranskat)abstract
- Two recently discovered groups of prokaryotic di-metal carboxylate proteins harbor a heterodinuclear Mn/Fe cofactor. These are the class Ic ribonucleotide reductase R2 proteins and a group of oxidases that are found predominantly in pathogens and extremophiles, called R2-like ligand-binding oxidases (R2lox). We have recently shown that the Mn/Fe cofactor of R2lox self-assembles from Mn-II and Fe-II in vitro and catalyzes formation of a tyrosine-valine ether cross-link in the protein scaffold (Griese, J. J., Roos, K., Cox, N., Shafaat, H. S., Branca, R.M., Lehtio , J., Graslund, A., Lubitz, W., Siegbahn, P. E., and Hogbom, M. (2013) Proc. Natl. Acad. Sci. U.S.A. 110, 1718917194). Here, we present a detailed structural analysis of R2lox in the nonactivated, reduced, and oxidized resting Mn/Fe- and Fe/Fe-bound states, as well as the nonactivated Mn/Mn-bound state. X-ray crystallography and x-ray absorption spectroscopy demonstrate that the active site ligand configuration of R2lox is essentially the same regardless of cofactor composition. Both the Mn/Fe and the diiron cofactor activate oxygen and catalyze formation of the ether cross-link, whereas the dimanganese cluster does not. The structures delineate likely routes for gated oxygen and substrate access to the active site that are controlled by the redox state of the cofactor. These results suggest that oxygen activation proceeds via similar mechanisms at the Mn/Fe and Fe/Fe center and that R2lox proteins might utilize either cofactor in vivo based on metal availability.
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| 14. |
- Griese, Julia J., et al.
(författare)
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Structure and DNA binding activity of the mouse condensin hinge domain highlight common and diverse features of SMC proteins
- 2010
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Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 38:10, s. 3454-3465
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Tidskriftsartikel (refereegranskat)abstract
- Structural Maintenance of Chromosomes (SMC) proteins are vital for a wide range of processes including chromosome structure and dynamics, gene regulation and DNA repair. Eukaryotes have three SMC complexes, consisting of heterodimeric pairs of six different SMC proteins along with several specific regulatory subunits. In addition to their other functions, all three SMC complexes play distinct roles in DNA repair. Cohesin (SMC1-SMC3) is involved in DNA double-strand break repair, condensin (SMC2-SMC4) participates in single-strand break (SSB) repair, and the SMC5-SMC6 complex functions in various DNA repair pathways. SMC proteins consist of N- and C-terminal domains that fold back onto each other to create an ATPase ’head’ domain, connected to a central ’hinge’ domain via long coiled-coils. The hinge domain mediates dimerization of SMC proteins and binds DNA, but it is not clear to what purpose this activity serves. We studied the structure and function of the condensin hinge domain from mouse. While the SMC hinge domain structure is largely conserved from prokaryotes to eukaryotes, its function seems to have diversified throughout the course of evolution. The condensin hinge domain preferentially binds single-stranded DNA. We propose that this activity plays a role in the SSB repair function of the condensin complex.
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| 15. |
- Griese, Julia J., et al.
(författare)
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Structure and DNA-binding activity of the Pyrococcus furiosus SMC protein hinge domain
- 2011
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Ingår i: Proteins. - : Wiley. - 0887-3585 .- 1097-0134. ; 79:2, s. 558-568
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Tidskriftsartikel (refereegranskat)abstract
- Structural Maintenance of Chromosomes (SMC) proteins are essential for a wide range of processes including chromosome structure and dynamics, gene regulation, and DNA repair. While bacteria and archaea have one SMC protein that forms a homodimer, eukaryotes possess three distinct SMC complexes, consisting of heterodimeric pairs of six different SMC proteins. SMC holocomplexes additionally contain several specific regulatory subunits. The bacterial SMC complex is required for chromosome condensation and segregation. In eukaryotes, this function is carried out by the condensin (SMC2-SMC4) complex. SMC proteins consist of N-terminal and C-terminal domains that fold back onto each other to create an ATPase "head" domain, connected to a central "hinge" domain via a long coiled-coil region. The hinge domain mediates dimerization of SMC proteins and binds DNA. This activity implicates a direct involvement of the hinge domain in the action of SMC proteins on DNA. We studied the SMC hinge domain from the thermophilic archaeon Pyrococcus furiosus. Its crystal structure shows that the SMC hinge domain fold is largely conserved between archaea and bacteria as well as eukarya. Like the eukaryotic condensin hinge domain, the P. furiosus SMC hinge domain preferentially binds single-stranded DNA (ssDNA), but its affinity for DNA is weaker than that of its eukaryotic counterpart, and point mutations reveal that its DNA-binding surface is more confined. The ssDNA-binding activity of its hinge domain might play a role in the DNA-loading process of the prokaryotic SMC complex during replication.
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| 16. |
- Griese, Julia J., et al.
(författare)
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X ray reduction correlates with soaking accessibility as judged from four non crystallographically related diiron sites
- 2012
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Ingår i: METALLOMICS. - : Oxford University Press (OUP). - 1756-5901 .- 1756-591X. ; 4:9, s. 894-898
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Tidskriftsartikel (refereegranskat)abstract
- X-ray crystallography is extensively used to determine the atomic structure of proteins and their cofactors. Though a commonly overlooked problem, it has been shown that structural damage to a redox active metal site may precede loss of diffractivity by more than an order of magnitude in X-ray dose. Therefore the risk of misassigning redox states is great. Adequate treatment and consideration of this issue is of paramount importance in metalloprotein science, from experimental design to interpretation of the data and results. Some metal sites appear to be much more amenable to reduction than others, but the underlying processes are poorly understood. Here, we have analyzed the four non-crystallographically related diiron sites in a crystal of the ribonucleotide reductase R2F protein from Corynebacterium ammoniagenes. We conclude that the amount of X-ray reduction a metal site suffers correlates with its soaking accessibility. This direct observation supports the hypothesis that a diffusion component is involved in the X-ray reduction process.
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| 17. |
- Griese, Julia J., et al.
(författare)
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Xenobiotic Reductase A in the Degradation of Quinoline by Pseudomonas putida 86 : Physiological Function, Structure and Mechanism of 8-Hydroxycoumarin Reduction
- 2006
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Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 361:1, s. 140-152
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Tidskriftsartikel (refereegranskat)abstract
- A continuous evolutionary pressure of the biotic and abiotic world has led to the development of a diversity of microbial pathways to degrade and biomineralize aromatic and heteroaromatic compounds. The heterogeneity of compounds metabolized by bacteria like Pseudomonas putida indicates the large variety of enzymes necessary to catalyse the required reactions. Quinoline, a N-heterocyclic aromatic compound, can be degraded by microbes along different pathways. For P. putida 86 quinoline degradation by the 8-hydroxycoumarin pathway has been described and several intermediates were identified. To select enzymes catalysing the later stages of the 8-hydroxycoumarin pathway P. putida 86 was grown with quinoline. The FMN-containing enzyme xenobiotic reductase A (XenA) was isolated and analysed for its reactivity with intermediates of the 8-hydroxycoumarin pathway. XenA catalyses the NADPH-dependent reduction of 8-hydroxycoumarin and coumarin to produce 8-hydroxy-3,4-dihydrocoumarin and 3,4-dihydrocoumarin, respectively. Crystallographic analysis of XenA alone and in complex with the two substrates revealed insights into the mechanism. XenA shows a dimeric arrangement of two (β/α)8 barrel domains each binding one FMN cofactor. High resolution crystal structures of complexes with 8-hydroxycoumarin and coumarin show different modes of binding for these molecules in the active site. While coumarin is ideally positioned for hydride transfer from N-5 of the isoalloxazine ring to C-4 of coumarin, 8-hydroxycoumarin forms a non-productive complex with oxidised XenA. Orientation of 8-hydroxycoumarin in the active site appears to be dependent on the electronic state of the flavin. We postulate that XenA catalyses the last step of the 8-hydroxycoumarin pathway before the heterocyclic ring is hydrolysed to yield 3-(2,3-dihydroxyphenyl)-propionic acid. As XenA is also found in P. putida strains unable to degrade quinoline, it appears to have more than one physiological function and is an example of how enzymes with low substrate specificity can help to explain the variety of degradation pathways possible.
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| 18. |
- Kisgeropoulos, Effie C., et al.
(författare)
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Key Structural Motifs Balance Metal Binding and Oxidative Reactivity in a Heterobimetallic Mn/Fe Protein
- 2020
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Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 0002-7863 .- 1520-5126. ; 142:11, s. 5338-5354
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Tidskriftsartikel (refereegranskat)abstract
- Heterobimetallic Mn/Fe proteins represent a new cofactor paradigm in bioinorganic chemistry and pose countless outstanding questions. The assembly of the active site defies common chemical convention by contradicting the Irving-Williams series, while the scope of reactivity remains unexplored. In this work, the assembly and C-H bond activation process in the Mn/Fe R2-like ligand-binding oxidase (R2lox) protein is investigated using a suite of biophysical techniques, including time-resolved optical spectroscopy, global kinetic modeling, X-ray crystallography, electron paramagnetic resonance spectroscopy, protein electrochemistry, and mass spectrometry. Selective metal binding is found to be under thermodynamic control, with the binding sites within the apoprotein exhibiting greater Mn-II affinity than Fe-II affinity. The comprehensive analysis of structure and reactivity of wild-type R2lox and targeted primary and secondary sphere mutants indicate that the efficiency of C-H bond activation directly correlates with the Mn/Fe cofactor reduction potentials and is inversely related to divalent metal binding affinity. These findings suggest the R2lox active site is precisely tuned for achieving both selective heterobimetallic binding and high levels of reactivity and offer a mechanism to examine the means by which proteins achieve appropriate metal incorporation.
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| 19. |
- Kositzki, Ramona, et al.
(författare)
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y Protonation State of MnFe and FeFe Cofactors in a Ligand-Binding Oxidase Revealed by X-ray Absorption, Emission, and Vibrational Spectroscopy and QM/MM Calculations
- 2016
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Ingår i: Inorganic Chemistry. - : American Chemical Society (ACS). - 0020-1669 .- 1520-510X. ; 55:19, s. 9869-9885
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Tidskriftsartikel (refereegranskat)abstract
- Enzymes with a dimetalcarboxylate cofactor catalyze reactions among the top challenges in chemistry such as methane and dioxygen (O-2) activation. Recently described proteins bind a manganeseiron cofactor (MnFe) instead of the classical diiron cofactor (FeFe). Determination of atomic-level differences of homo- versus hetero-bimetallic cofactors is crucial to understand their diverse redox reactions. We studied a ligand-binding oxidase from the bacterium Geobacillus kaustophilus (R2lox) loaded with a FeFe or MnFe cofactor, which catalyzes O-2 reduction and an unusual tyrosinevaline ether cross-link formation, as revealed by X-ray crystallography. Advanced X-ray absorption, emission, and vibrational spectroscopy methods and quantum chemical and molecular mechanics calculations provided relative Mn/Fe contents, X-ray photoreduction kinetics, metalligand bond lengths, metalmetal distances, metal oxidation states, spin configurations, valence-level degeneracy, molecular orbital composition, nuclear quadrupole splitting energies, and vibrational normal modes for both cofactors. A protonation state with an axial water (H2O) ligand at Mn or Fe in binding site 1 and a metal-bridging hydroxo group (OH) in a hydrogen-bonded network is assigned. Our comprehensive picture of the molecular, electronic, and dynamic properties of the cofactors highlights reorientation of the unique axis along the MnOH2 bond for the Mn1(III) JahnTeller ion but along the Fe-mu OH bond for the octahedral Fe1(III). This likely corresponds to a more positive redox potential of the Mn(III)Fe(III) cofactor and higher proton affinity of its mu OH group. Refined model structures for the Mn(III)Fe(III) and Fe(III)Fe(III) cofactors are presented. Implications of our findings for the site-specific metalation of R2lox and performance of the O-2 reduction and cross-link formation reactions are discussed.
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| 20. |
- Kutin, Yury, et al.
(författare)
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Chemical flexibility of heterobimetallic Mn/Fe cofactors : R2lox and R2c proteins
- 2019
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 294:48, s. 18372-18386
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Tidskriftsartikel (refereegranskat)abstract
- A heterobimetallic Mn/Fe cofactor is present in the R2 subunit of class Ic ribonucleotide reductases (R2c) and in R2-like ligand-binding oxidases (R2lox). Although the protein-derived metal ligands are the same in both groups of proteins, the connectivity of the two metal ions and the chemistry each cofactor performs are different: in R2c, a one-electron oxidant, the Mn/Fe dimer is linked by two oxygen bridges (?-oxo/?-hydroxo), whereas in R2lox, a two-electron oxidant, it is linked by a single oxygen bridge (?-hydroxo) and a fatty acid ligand. Here, we identified a second coordination sphere residue that directs the divergent reactivity of the protein scaffold. We found that the residue that directly precedes the N-terminal carboxylate metal ligand is conserved as a glycine within the R2lox group but not in R2c. Substitution of the glycine with leucine converted the resting-state R2lox cofactor to an R2c-like cofactor, a ?-oxo/?-hydroxo?bridged Mn-III/Fe-III dimer. This species has recently been observed as an intermediate of the oxygen activation reaction in WT R2lox, indicating that it is physiologically relevant. Cofactor maturation in R2c and R2lox therefore follows the same pathway, with structural and functional divergence of the two cofactor forms following oxygen activation. We also show that the leucine-substituted variant no longer functions as a two-electron oxidant. Our results reveal that the residue preceding the N-terminal metal ligand directs the cofactor's reactivity toward one- or two-electron redox chemistry, presumably by setting the protonation state of the bridging oxygens and thereby perturbing the redox potential of the Mn ion.
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| 21. |
- Kutin, Yuri, et al.
(författare)
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Divergent assembly mechanisms of the manganese/iron cofactors in R2lox and R2c proteins
- 2016
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Ingår i: Journal of Inorganic Biochemistry. - : Elsevier BV. - 0162-0134 .- 1873-3344. ; 162, s. 164-177
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Tidskriftsartikel (refereegranskat)abstract
- A manganese/iron cofactor which performs multi-electron oxidative chemistry is found in two classes of ferritin-like proteins, the small subunit (R2) of dass Ic ribonucleotide reductase (R2c) and the R2-like ligand-binding oxidase (R2lox). It is undear how a heterodimeric Mn/Fe metallocofactor is assembled in these two related proteins as opposed to a homodimeric Fe/Fe cofactor, especially considering the structural similarity and proximity of the two metal-binding sites in both protein scaffolds and the similar first coordination sphere ligand preferences of Mn-II and Fe-II. Using EPR and Mfissbauer spectroscopies as well as X-ray anomalous dispersion, we examined metal loading and cofactor activation of both proteins in vitro (in solution). We find divergent cofactor assembly mechanisms for the two systems. In both cases, excess Mn-II promotes heterobimetallic cofactor assembly. In the absence of Fe-II, R2c cooperatively binds Mn-II at both metal sites, whereas R2lox does not readily bind Mn-II at either site. Heterometallic cofactor assembly is favored at substoichiometric Feu concentrations in R2lox. Fe-II and Mn-II likely bind to the protein in a stepwise fashion, with Feu binding to site 2 initiating cofactor assembly. In R2c, however, heterometallic assembly is presumably achieved by the displacement of Mn-II by Fe-II at site 2. The divergent metal loading mechanisms are correlated with the putative in vivo functions of R2c and R2lox, and most likely with the intracellular Mn-II/Fe-II concentrations in the host organisms from which they were isolated.
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| 22. |
- Marcos-Torres, Francisco Javier, et al.
(författare)
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The bacterial iron sensor IdeR recognizes its DNA targets by indirect readout
- 2021
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Ingår i: Nucleic Acids Research. - : Oxford University Press. - 0305-1048 .- 1362-4962.
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Tidskriftsartikel (refereegranskat)abstract
- The iron-dependent regulator IdeR is the main transcriptional regulator controlling iron homeostasis genes in Actinobacteria, including species from the Corynebacterium, Mycobacterium and Streptomyces genera, as well as the erythromycin-producing bacterium Saccharopolyspora erythraea. Despite being a well-studied transcription factor since the identification of the Diphtheria toxin repressor DtxR three decades ago, the details of how IdeR proteins recognize their highly conserved 19-bp DNA target remain to be elucidated. IdeR makes few direct contacts with DNA bases in its target sequence, and we show here that these contacts are not required for target recognition. The results of our structural and mutational studies support a model wherein IdeR mainly uses an indirect readout mechanism, identifying its targets via the sequence-dependent DNA backbone structure rather than through specific contacts with the DNA bases. Furthermore, we show that IdeR efficiently recognizes a shorter palindromic sequence corresponding to a half binding site as compared to the full 19-bp target previously reported, expanding the number of potential target genes controlled by IdeR proteins.
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| 23. |
- Marcos-Torres, Francisco Javier, et al.
(författare)
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The molecular mechanisms of the bacterial iron sensor IdeR
- 2023
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Ingår i: Biochemical Society Transactions. - : Portland Press. - 0300-5127 .- 1470-8752.
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Forskningsöversikt (refereegranskat)abstract
- Life came to depend on iron as a cofactor for many essential enzymatic reactions. However, once the atmosphere was oxygenated, iron became both scarce and toxic. Therefore, complex mechanisms have evolved to scavenge iron from an environment in which it is poorly bioavailable, and to tightly regulate intracellular iron contents. In bacteria, this is typically accomplished with the help of one key regulator, an iron-sensing transcription factor. While Gram-negative bacteria and Gram-positive species with low guanine-cytosine (GC) content generally use Fur (ferric uptake regulator) proteins to regulate iron homeostasis, Gram-positive species with high GC content use the functional homolog IdeR (iron-dependent regulator). IdeR controls the expression of iron acquisition and storage genes, repressing the former, and activating the latter in an iron-dependent manner. In bacterial pathogens such as Corynebacterium diphtheriae and Mycobacterium tuberculosis, IdeR is also involved in virulence, whereas in non-pathogenic species such as Streptomyces, it regulates secondary metabolism as well. Although in recent years the focus of research on IdeR has shifted towards drug development, there is much left to learn about the molecular mechanisms of IdeR. Here, we summarize our current understanding of how this important bacterial transcriptional regulator represses and activates transcription, how it is allosterically activated by iron binding, and how it recognizes its DNA target sites, highlighting the open questions that remain to be addressed.
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| 24. |
- Maugeri, Pearson T., et al.
(författare)
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Driving Protein Conformational Changes with Light : Photoinduced Structural Rearrangement in a Heterobimetallic Oxidase
- 2018
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Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 0002-7863 .- 1520-5126. ; 140:4, s. 1471-1480
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Tidskriftsartikel (refereegranskat)abstract
- The heterobimetallic R2lox protein binds both manganese and iron ions in a site-selective fashion and activates oxygen, ultimately performing C-H bond oxidation to generate a tyrosine-valine crosslink near the active site. In this work, we demonstrate that, following assembly, R2lox undergoes photoinduced changes to the active site geometry and metal coordination motif. Through spectroscopic, structural, and mass spectrometric characterization, the photoconverted species is found to consist of a tyrosinate-bound iron center following light-induced decarboxylation of a coordinating glutamate residue and cleavage of the tyrosine-valine cross-link. This process occurs with high quantum efficiencies (Phi = 3%) using violet and near-ultraviolet light, suggesting that the photodecarboxylation is initiated via ligandto-metal charge transfer excitation. Site-directed mutagenesis and structural analysis suggest that the cross-linked tyrosine-162 is the coordinating residue. One primary product is observed following irradiation, indicating potential use of this class of proteins, which contains a putative substrate channel, for controlled photoinduced decarboxylation processes, with relevance for in vivo functionality of R2lox as well as application in environmental remediation.
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| 25. |
- Mebs, Stefan, et al.
(författare)
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Fate of oxygen species from O-2 activation at dimetal cofactors in an oxidase enzyme revealed by Fe-57 nuclear resonance X-ray scattering and quantum chemistry
- 2019
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Ingår i: Biochimica et Biophysica Acta - Bioenergetics. - : Elsevier BV. - 0005-2728 .- 1879-2650. ; 1860:12
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Tidskriftsartikel (refereegranskat)abstract
- Oxygen (O-2) activation is a central challenge in chemistry and catalyzed at prototypic dimetal cofactors in biological enzymes with diverse functions. Analysis of intermediates is required to elucidate the reaction paths of reductive O-2 cleavage. An oxidase protein from the bacterium Geobacillus kaustophilus, R2lox, was used for aerobic in-vitro reconstitution with only Fe-57(II) or Mn(II) plus Fe-57(II) ions to yield [FeFe] or [MnFe] cofactors under various oxygen and solvent isotopic conditions including O-16/18 and H/D exchange. Fe-57-specific X-ray scattering techniques were employed to collect nuclear forward scattering (NFS) and nuclear resonance vibrational spectroscopy (NRVS) data of the R2lox proteins. NFS revealed Fe/Mn(III)Fe(III) cofactor states and Mossbauer quadrupole splitting energies. Quantum chemical calculations of NRVS spectra assigned molecular structures, vibrational modes, and protonation patterns of the cofactors, featuring a terminal water (H2O) bound at iron or manganese in site 1 and a metal-bridging hydroxide (mu OH-) ligand. A procedure for quantitation and correlation of experimental and computational NRVS difference signals due to isotope labeling was developed. This approach revealed that the protons of the ligands as well as the terminal water at the R2lox cofactors exchange with the bulk solvent whereas O-18 from O-18(2) cleavage is incorporated in the hydroxide bridge. In R2lox, the two water molecules from four-electron O-2 reduction are released in a two-step reaction to the solvent. These results establish combined NRVS and QM/MM for tracking of iron-based oxygen activation in biological and chemical catalysts and clarify the reductive O-2 cleavage route in an enzyme.
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| 26. |
- Rapatskiy, Leonid, et al.
(författare)
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Characterization of Oxygen Bridged Manganese Model Complexes Using Multifrequency (17)O-Hyperfine EPR Spectroscopies and Density Functional Theory
- 2015
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Ingår i: Journal of Physical Chemistry B. - : American Chemical Society (ACS). - 1520-6106 .- 1520-5207. ; 119:43, s. 13904-13921
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Tidskriftsartikel (refereegranskat)abstract
- Multifrequency pulsed EPR data are reported for a series of oxygen bridged (μ-oxo/μ-hydroxo) bimetallic manganese complexes where the oxygen is labeled with the magnetically active isotope (17)O (I = 5/2). Two synthetic complexes and two biological metallocofactors are examined: a planar bis-μ-oxo bridged complex and a bent, bis-μ-oxo-μ-carboxylato bridge complex; the dimanganese catalase, which catalyzes the dismutation of H2O2 to H2O and O2, and the recently identified manganese/iron cofactor of the R2lox protein, a homologue of the small subunit of the ribonuclotide reductase enzyme (class 1c). High field (W-band) hyperfine EPR spectroscopies are demonstrated to be ideal methods to characterize the (17)O magnetic interactions, allowing a magnetic fingerprint for the bridging oxygen ligand to be developed. It is shown that the μ-oxo bridge motif displays a small positive isotropic hyperfine coupling constant of about +5 to +7 MHz and an anisotropic/dipolar coupling of -9 MHz. In addition, protonation of the bridge is correlated with an increase of the hyperfine coupling constant. Broken symmetry density functional theory is evaluated as a predictive tool for estimating hyperfine coupling of bridging species. Experimental and theoretical results provide a framework for the characterization of the oxygen bridge in Mn metallocofactor systems, including the water oxidizing cofactor of photosystem II, allowing the substrate/solvent interface to be examined throughout its catalytic cycle.
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| 27. |
- Shafaat, Hannah S., et al.
(författare)
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Electronic Structural Flexibility of Heterobimetallic Mn/Fe Cofactors : R2lox and R2c Proteins
- 2014
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Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 0002-7863 .- 1520-5126. ; 136:38, s. 13399-13409
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Tidskriftsartikel (refereegranskat)abstract
- The electronic structure of the Mn/Fe cofactor identified in a new class of oxidases (R2lox) described by Andersson and Hogbom [Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 5633] is reported. The R2lox protein is homologous to the small subunit of class Ic ribonucleotide reductase (R2c) but has a completely different in vivo function. Using multifrequency EPR and related pulse techniques, it is shown that the cofactor of R2lox represents an antiferromagnetically coupled Mn-III/Fe-III dimer linked by a mu-hydroxo/bis-mu-carboxylato bridging network. The Mn-III ion is coordinated by a single water ligand. The R2lox cofactor is photoactive, converting into a second form (R2lox(photo)) upon visible illumination at cryogenic temperatures (77 K) that completely decays upon warming. This second, unstable form of the cofactor more closely resembles the Mn-III/Fe-III cofactor seen in R2c. It is shown that the two forms of the R2lox cofactor differ primarily in terms of the local site geometry and electronic state of the Mn-III ion, as best evidenced by a reorientation of its unique Mn-55 hyperfine axis. Analysis of the metal hyperfine tensors in combination with density functional theory (DFT) calculations suggests that this change is triggered by deprotonation of the mu-hydroxo bridge. These results have important consequences for the mixed-metal R2c cofactor and the divergent chemistry R2lox and R2c perform.
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| 28. |
- Sustmann, Claudio, et al.
(författare)
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DuoMab : a novel CrossMab-based IgG-derived antibody format for enhanced antibody-dependent cell-mediated cytotoxicity
- 2019
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Ingår i: mAbs. - : Informa UK Limited. - 1942-0862 .- 1942-0870. ; 11:8, s. 1402-1414
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Tidskriftsartikel (refereegranskat)abstract
- High specificity accompanied with the ability to recruit immune cells has made recombinant therapeutic antibodies an integral part of drug development. Here we present a generic approach to generate two novel IgG-derived antibody formats that are based on a modification of the CrossMab technology. MoAbs harbor two heavy chains (HCs) resulting in one binding entity and one fragment crystallizable region (Fc), whereas DuoMabs are composed of four HCs harboring two binding entities and two Fc regions linked at a disulfide-bridged hinge. The latter bivalent format is characterized by avidity-enhanced target cell binding while simultaneously increasing the ‘Fc-load’ on the surface. DuoMabs were shown to be producible in high yield and purity and bind to surface cells with affinities comparable to IgGs. The increased Fc load directed at the surface of target cells by DuoMabs modulates their antibody-dependent cell-mediated cytotoxicity competency toward target cells, making them attractive for applications that require or are modulated by FcR interactions.
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| 29. |
- Tyagi, Amit, et al.
(författare)
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Photodynamics of blue-light-regulated phosphodiesterase BlrP1 protein from Klebsiella pneumoniae and its photoreceptor BLUF domain
- 2008
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Ingår i: Chemical Physics. - : Elsevier BV. - 0301-0104 .- 1873-4421. ; 354:1-3, s. 130-141
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Tidskriftsartikel (refereegranskat)abstract
- The BlrP1 protein from the enteric bacterium Klebsiella pneumoniae consists of a BLUF and an EAL domain and may activate c-di-GMP phosphodiesterase by blue-light. The full-length protein, BlrP1, and its BLUF domain, BlrP1_BLUF, are characterized by optical absorption and emission spectroscopy. The cofactor FAD in its oxidized redox state (FADox) is brought from the dark-adapted receptor state to the 10-nm red-shifted putative signalling state by violet light exposure. The recovery to the receptor state occurs with a time constant of about 1 min. The quantum yield of signalling state formation is about 0.17 for BlrP1_BLUF and about 0.08 for BlrP1. The fluorescence efficiency of the FADox cofactor is small due to photo-induced reductive electron transfer. Prolonged light exposure converts FADox in the signalling state to the fully reduced hydroquinone form FADredH- and causes low-efficient chromophore release with subsequent photo-degradation. The photo-cycle and photo-reduction dynamics in the receptor state and in the signalling state are discussed.
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| 30. |
- van der Ent, Florian, et al.
(författare)
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Computational design of the temperature optimum of an enzyme reaction
- 2023
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Ingår i: Science Advances. - : American Association for the Advancement of Science (AAAS). - 2375-2548. ; 9:26
-
Tidskriftsartikel (refereegranskat)abstract
- Cold-adapted enzymes are characterized both by a higher catalytic activity at low temperatures and by having their temperature optimum down-shifted, compared to mesophilic orthologs. In several cases, the optimum does not coincide with the onset of protein melting but reflects some other type of inactivation. In the psychrophilic a-amylase from an Antarctic bacterium, the inactivation is thought to originate from a specific enzyme-substrate interaction that breaks around room temperature. Here, we report a computational redesign of this enzyme aimed at shifting its temperature optimum upward. A set of mutations designed to stabilize the enzyme-substrate interaction were predicted by computer simulations of the catalytic reaction at different temperatures. The predictions were verified by kinetic experiments and crystal structures of the redesigned a-amylase, showing that the temperature optimum is indeed markedly shifted upward and that the critical surface loop controlling the temperature dependence approaches the target conformation observed in a mesophilic ortholog.
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| 31. |
- Xu, Hongyi, et al.
(författare)
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Solving a new R2lox protein structure by microcrystal electron diffraction
- 2019
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Ingår i: Science Advances. - : American Association for the Advancement of Science (AAAS). - 2375-2548. ; 5:8
-
Tidskriftsartikel (refereegranskat)abstract
- Microcrystal electron diffraction (MicroED) has recently shown potential for structural biology. It enables the study of biomolecules from micrometer-sized 3D crystals that are too small to be studied by conventional x-ray crystallography. However, to date, MicroED has only been applied to redetermine protein structures that had already been solved previously by x-ray diffraction. Here, we present the first new protein structure-an R2lox enzyme-solved using MicroED. The structure was phased by molecular replacement using a search model of 35% sequence identity. The resulting electrostatic scattering potential map at 3.0-angstrom resolution was of sufficient quality to allow accurate model building and refinement. The dinuclear metal cofactor could be located in the map and was modeled as a heterodinuclear Mn/Fe center based on previous studies. Our results demonstrate that MicroED has the potential to become a widely applicable tool for revealing novel insights into protein structure and function.
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