| 1. |
- Elmlund, Hans, et al.
(författare)
-
A new cryo-EM single-particle Ab initio reconstruction method visualizes secondary structure elements in an ATP-Fueled AAA+ motor
- 2008
-
Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 1089-8638 .- 0022-2836. ; 375:4, s. 934-947
-
Tidskriftsartikel (refereegranskat)abstract
- The generation of ab initio three-dimensional (3D) models is a bottleneck in the studies of large macromolecular assemblies by single-particle cryo-electron microscopy. We describe here a novel method, in which established methods for two-dimensional image processing are combined with newly developed programs for joint rotational 3D alignment of a large number of class averages (RAD) and calculation of 3D volumes from aligned projections (VolRec). We demonstrate the power of the method by reconstructing an 660-kDa ATP-fueled AAA+ motor to 7.5 Å resolution, with secondary structure elements identified throughout the structure. We propose the method as a generally applicable automated strategy to obtain 3D reconstructions from unstained single particles imaged in vitreous ice.
|
|
| 2. |
- Fodje, Michel, et al.
(författare)
-
Interplay Between an AAA Module and an Integrin I Domain May Regulate the Function of Magnesium Chelatase
- 2001
-
Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 1089-8638 .- 0022-2836. ; 311:1, s. 111-122
-
Tidskriftsartikel (refereegranskat)abstract
- In chlorophyll biosynthesis, insertion of Mg2+ into protoporphyrin IX is catalysed in an ATP-dependent reaction by a three-subunit (BchI, BchD and BchH) enzyme magnesium chelatase. In this work we present the three-dimensional structure of the ATP-binding subunit BchI. The structure has been solved by the multiple wavelength anomalous dispersion method and refined at 2.1 A resolution to the crystallographic R-factor of 22.2 % (Rfree = 24.5 %). It belongs to the chaperone-like ''ATPase associated with a variety of cellular activities'' (AAA) family of ATPases, with a novel arrangement of domains: the C-terminal helical domain is located behind the nucleotide-binding site, while in other known AAA module structures it is located on the top. Examination by electron microscopy of BchI solutions in the presence of ATP demonstrated that BchI, like other AAA proteins, forms oligomeric ring structures. Analysis of the amino acid sequence of subunit BchD revealed an AAA module at the N-terminal portion of the sequence and an integrin I domain at the C terminus. An acidic, proline-rich region linking these two domains is suggested to contribute to the association of BchI and BchD by binding to a positively charged cleft at the surface of the nucleotide-binding domain of BchI. Analysis of the amino acid sequences of BchI and BchH revealed integrin I domain-binding sequence motifs. These are proposed to bind the integrin I domain of BchD during the functional cycle of magnesium chelatase, linking porphyrin metallation by BchH to ATP hydrolysis by BchI. An integrin I domain and an acidic and proline-rich region have been identified in subunit CobT of cobalt chelatase, clearly demonstrating its homology to BchD. These findings, for the first time, provide an insight into the subunit organisation of magnesium chelatase and the homologous colbalt chelatase.
|
|
| 3. |
- Karlberg, Tobias, et al.
(författare)
-
Porphyrin Binding and Distortion and Substrate Specificity in the Ferrochelatase Reaction: The Role of Active Site Residues.
- 2008
-
Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 1089-8638 .- 0022-2836. ; 378:5, s. 1074-1083
-
Tidskriftsartikel (refereegranskat)abstract
- The specific insertion of a divalent metal ion into tetrapyrrole macrocycles is catalyzed by a group of enzymes called chelatases. Distortion of the tetrapyrrole has been proposed to be an important component of the mechanism of metallation. We present the structures of two different inhibitor complexes: (1) N-methylmesoporphyrin (N-MeMP) with the His183Ala variant of Bacillus subtilis ferrochelatase; (2) the wild-type form of the same enzyme with deuteroporphyrin IX 2,4-disulfonic acid dihydrochloride (dSDP). Analysis of the structures showed that only one N-MeMP isomer out of the eight possible was bound to the protein and it was different from the isomer that was earlier found to bind to the wild-type enzyme. A comparison of the distortion of this porphyrin with other porphyrin complexes of ferrochelatase and a catalytic antibody with ferrochelatase activity using normal-coordinate structural decomposition reveals that certain types of distortion are predominant in all these complexes. On the other hand, dSDP, which binds closer to the protein surface compared to N-MeMP, does not undergo any distortion upon binding to the protein, underscoring that the position of the porphyrin within the active site pocket is crucial for generating the distortion required for metal insertion. In addition, in contrast to the wild-type enzyme, Cu(2+)-soaking of the His183Ala variant complex did not show any traces of porphyrin metallation. Collectively, these results provide new insights into the role of the active site residues of ferrochelatase in controlling stereospecificity, distortion and metallation.
|
|
| 4. |
- Lecerof, David, et al.
(författare)
-
Structural and Mechanistic Basis of Porphyrin Metallation by Ferrochelatase
- 2000
-
Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 1089-8638 .- 0022-2836. ; 297:1, s. 221-232
-
Tidskriftsartikel (refereegranskat)abstract
- Ferrochelatase, the enzyme catalyzing metallation of protoporphyrin IX at the terminal step of heme biosynthesis, was co-crystallized with an isomer mixture of the potent inhibitor N-methylmesoporphyrin (N-MeMP). The X-ray structure revealed the active site of the enzyme, to which only one of the isomers was bound, and for the first time allowed characterization of the mode of porphyrin macrocycle distortion by ferrochelatase. Crystallization of ferrochelatase and N-MeMP in the presence of Cu2+ leads to metallation and demethylation of N-MeMP. A mechanism of porphyrin distortion is proposed, which assumes that the enzyme holds pyrrole rings B, C and D in a vice-like grip and forces a 36 o tilt on ring A.
|
|
| 5. |
- Shipovskov, Stepan, et al.
(författare)
-
Metallation of the transition-state inhibitor N-methyl mesoporphyrin by ferrochelatase: Implications for the catalytic reaction mechanism
- 2005
-
Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 1089-8638 .- 0022-2836. ; 352:5, s. 1081-1090
-
Tidskriftsartikel (refereegranskat)abstract
- Insertion of metals into various tetrapyrroles is catalysed by a group of enzymes called chelatases, e.g. nickel, cobalt, magnesium and ferrochelatase. It has been proposed that catalytic metallation includes distorting the porphyrin substrate by the enzyme towards a transition state-like geometry in which at least one of the pyrrole rings will be available for metal chelation. Here, we present a study of metal insertion into the transition-state inhibitor of protoporphyrin IX ferrochelatase, N-methyl mesoporphyrin (N-MeMP), by time-resolved crystallography and mass spectrometry with and without the presence of ferrochelatase. The results show that metallation of N-MeMP has a very limited effect on the conformation of the residues that participate in porphyrin and metal binding. These findings support theoretical data, which indicate that product release is controlled largely by the strain created by metal insertion into the distorted porphyrin. The results suggest that, similar to noncatalytic metallation of N-MeMP, the ferrochelatase-assisted metallation depends on the ligand exchange rate for the respective metal. Moreover, ferrochelatase catalyses insertion of Cu(II) and Zn(II) into N-MeMP with a rate that is about 20 times faster than non-enzymatic metallation in solution, suggesting that the catalytic strategy of ferrochelatase includes a stage of acceleration of the rate of ligand exchange for the metal substrate. The greater efficiency of N-MeMP metallation by Cu(II), as compared to Zn(II), contrasts with the Km values for Zn(II), (17 mu M) and Cu(II) (170 mu M) obtained for metallation of protoporphyrin IX. We suggest that this difference in metal specificity depends on the type of distortion imposed by the enzyme on protoporphyrin IX, which is different from the intrinsic non-planar distortion of N-MeMP. A mechanism of control of metal specificity by porphyrin distortion may be general for different chelatases, and may have common features with the mechanism of metal specificity in crown ethers.(c) 2005 Elsevier Ltd. All rights reserved.
|
|
| 6. |
- Söderberg, Christopher, et al.
(författare)
-
Oligomerization Propensity and Flexibility of Yeast Frataxin Studied by X-ray Crystallography and Small-Angle X-ray Scattering.
- 2011
-
Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 1089-8638 .- 0022-2836. ; 414:5, s. 783-797
-
Tidskriftsartikel (refereegranskat)abstract
- Frataxin is a mitochondrial protein with a central role in iron homeostasis. Defects in frataxin function lead to Friedreich's ataxia, a progressive neurodegenerative disease with childhood onset. The function of frataxin has been shown to be closely associated with its ability to form oligomeric species; however, the factors controlling oligomerization and the types of oligomers present in solution are a matter of debate. Using small-angle X-ray scattering, we found that Co(2+), glycerol, and a single amino acid substitution at the N-terminus, Y73A, facilitate oligomerization of yeast frataxin, resulting in a dynamic equilibrium between monomers, dimers, trimers, hexamers, and higher-order oligomers. Using X-ray crystallography, we found that Co(2+) binds inside the channel at the 3-fold axis of the trimer, which suggests that the metal has an oligomer-stabilizing role. The results reveal the types of oligomers present in solution and support our earlier suggestions that the trimer is the main building block of yeast frataxin oligomers. They also indicate that different mechanisms may control oligomer stability and oligomerization in vivo.
|
|
| 7. |
- Tamu Dufe, Veronica, et al.
(författare)
-
Crystal structure of Plasmodium falciparum spermidine synthase in complex with the substrate decarboxylated S-adenosylmethionine and the potent inhibitors 4MCHA and AdoDATO
- 2007
-
Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 1089-8638 .- 0022-2836. ; 373:1, s. 167-177
-
Tidskriftsartikel (refereegranskat)abstract
- Plasmodium falciparum is the causative agent of the most severe type of malaria, a life-threatening disease affecting the lives of over three billion people. Factors like widespread resistance against available drugs and absence of an effective vaccine are seriously compounding control of the malaria parasite. Thus, there is an urgent need for the identification and validation of new drug targets. The enzymes of the polyamine biosynthesis pathway have been suggested as possible targets for the treatment of malaria. One of these enzymes is spermidine synthase (SPDS, putrescine aminopropyltransferase), which catalyzes the transfer of an aminopropyl moiety from decarboxylated S-adenosylmethionine (dcAdoMet) to putrescine, leading to the formation of spermidine and 5 '-methylthioadenosine. Here we present the three-dimensional structure of P falciparum spermidine synthase (pfSPDS) in apo form, in complex with dcAdoMet and two inhibitors, S-adenosyl-1,8-diamino-3-thio-octane (AdoDATO) and trans-4-methylcyclohexylamine (4MCHA). The results show that binding of dcAdoMet to pfSPDS stabilizes the conformation of the flexible gatekeeper loop of the enzyme and affects the conformation of the active-site amino acid residues, preparing the protein for binding of the second substrate. The complexes of AdoDATO and 4MCHA with pfSPDS reveal the mode of interactions of these compounds with the enzyme. While AdoDATO essentially fills the entire active-site pocket, 4MCHA only occupies part of it, which suggests that simple modifications of this compound may yield more potent inhibitors of pfSPDS. (C) 2007 Elsevier Ltd. All rights reserved.
|
|
| 8. |
- Ursby, Thomas, et al.
(författare)
-
Iron superoxide dismutase from the archaeon Sulfolobus sulfataricus : Analysis of structure and thermostability.
- 1999
-
Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 1089-8638 .- 0022-2836. ; 286:1, s. 189-205
-
Tidskriftsartikel (refereegranskat)abstract
- The crystal structure of superoxide dismutase (SOD) from the hyper thermophile Sulfolobus solfataricus has been determined at 2.3 Å resolution by molecular replacement and refined to a crystallographic R-factor of 16.8 % (Rfree 19.8 %). The crystals belong to the space group C2 (a = 76.3 Å, b = 124.3 Å,c = 60.3 Å, β = 128.8°) with two identical monomers in the asymmetric unit. The monomer has a molecular weight of 24 kDa and consists of 210 amino acid residues of which 205 are visible in the electron density map. The overall fold of the monomer of S. solfataricus SOD is similar to that of the other known Fe or Mn-SODs. S. solfataricus SOD forms a very compact tetramer of a type similar to that of SOD from the hyperthermophile Aquifex pyrophilus. Both structures show an elevated number of inter-subunit ion-pairs compared with the mesophilic SOD from Mycobacterium tuberculosis and the thermophilic SOD from Thermus thermophilus. However, in contrast to the A. pyrophilus SOD structure, the number of intra-subunit ion-pairs as well as inter-subunit hydrogen bonds is not higher than in the compared mesophilic and thermophilic SOD structures. The electron density also revealed an unexpected and unusual covalent modification of a conserved tyrosine in the active site. Its involvement in the specific activity of the enzyme is discussed.
|
|
