Publications (28)173.81 Total impact
-
Article: Structure and conservation of the periplasmic targeting factor Tic22 protein from plants and cyanobacteria.
[show abstract] [hide abstract]
ABSTRACT: Mitochondria and chloroplasts are of endosymbiotic origin. Their integration into cells entailed the development of protein translocons, partially by recycling bacterial proteins. We demonstrate the evolutionary conservation of the translocon component Tic22 between cyanobacteria and chloroplasts. Tic22 in Anabaena sp. PCC 7120 is essential. The protein is localized in the thylakoids and in the periplasm and can be functionally replaced by a plant orthologue. Tic22 physically interacts with the outer envelope biogenesis factor Omp85 in vitro and in vivo, the latter exemplified by immunoprecipitation after chemical cross-linking. The physical interaction together with the phenotype of a tic22 mutant comparable with the one of the omp85 mutant indicates a concerted function of both proteins. The three-dimensional structure allows the definition of conserved hydrophobic pockets comparable with those of ClpS or BamB. The results presented suggest a function of Tic22 in outer membrane biogenesis.Journal of Biological Chemistry 05/2012; 287(29):24164-73. · 4.77 Impact Factor -
Article: Assembly of the eukaryotic PLP-synthase complex from Plasmodium and activation of the Pdx1 enzyme.
[show abstract] [hide abstract]
ABSTRACT: Biosynthesis of vitamins is fundamental to malaria parasites. Plasmodia synthesize the active form of vitamin B(6) (pyridoxal 5'-phosphate, PLP) using a PLP synthase complex. The EM analysis shown here reveals a random association pattern of up to 12 Pdx2 glutaminase subunits to the dodecameric Pdx1 core complex. Interestingly, Plasmodium falciparum PLP synthase organizes in fibers. The crystal structure shows differences in complex formation to bacterial orthologs as interface variations. Alternative positioning of an α helix distinguishes an open conformation from a closed state when the enzyme binds substrate. The pentose substrate is covalently attached through its C1 and forms a Schiff base with Lys84. Ammonia transfer between Pdx2 glutaminase and Pdx1 active sites is regulated by a transient tunnel. The mutagenesis analysis allows defining the requirement for conservation of critical methionines, whereas there is also plasticity in ammonia tunnel construction as seen from comparison across different species.Structure 01/2012; 20(1):172-84. · 6.35 Impact Factor -
Article: PLP-dependent enzymes as potential drug targets for protozoan diseases.
[show abstract] [hide abstract]
ABSTRACT: The chemical properties of the B(6) vitamers are uniquely suited for wide use as cofactors in essential reactions, such as decarboxylations and transaminations. This review addresses current efforts to explore vitamin B(6) dependent enzymatic reactions as drug targets. Several current targets are described that are found amongst these enzymes. The focus is set on diseases caused by protozoan parasites. Comparison across a range of these organisms allows insight into the distribution of potential targets, many of which may be of interest in the development of broad range anti-protozoan drugs. This article is part of a Special Issue entitled: Pyridoxal Phosphate Enzymology.Biochimica et Biophysica Acta 07/2011; 1814(11):1567-76. · 4.66 Impact Factor -
Article: Mdm38 is a 14-3-3-like receptor and associates with the protein synthesis machinery at the inner mitochondrial membrane.
[show abstract] [hide abstract]
ABSTRACT: Mitochondrial ribosomes synthesize core subunits of the inner membrane respiratory chain complexes. In mitochondria, translation is regulated by mRNA-specific activator proteins and occurs on membrane-associated ribosomes. Mdm38/Letm1 is a conserved membrane receptor for mitochondrial ribosomes and specifically involved in respiratory chain biogenesis. In addition, Mdm38 and its higher eukaryotic homolog Letm1, function as K(+)/H(+) or Ca(2+)/H(+) antiporters in the inner membrane. Here, we identify the conserved ribosome-binding domain (RBD) of Mdm38 and determine the crystal structure at 2.1 Å resolution. Surprisingly, Mdm38(RBD) displays a 14-3-3-like fold despite any similarity to 14-3-3-proteins at the primary sequence level and thus represents the first 14-3-3-like protein in mitochondria. The 14-3-3-like domain is critical for respiratory chain assembly through regulation of Cox1 and Cytb translation. We show that this function can be spatially separated from the ion transport activity of the membrane integrated portion of Mdm38. On the basis of the phenotypes observed for mdm38Δ as compared to Mdm38 lacking the RBD, we suggest a model that combining ion transport and translational regulation into one molecule allows for direct coupling of ion flux across the inner membrane, and serves as a signal for the translation of mitochondrial membrane proteins via its direct association with the protein synthesis machinery.Traffic 07/2011; 12(10):1457-66. · 4.92 Impact Factor -
Article: Substrate binding disrupts dimerization and induces nucleotide exchange of the chloroplast GTPase Toc33.
[show abstract] [hide abstract]
ABSTRACT: GTPases act as molecular switches to control many cellular processes, including signalling, protein translation and targeting. Switch activity can be regulated by external effector proteins or intrinsic properties, such as dimerization. The recognition and translocation of pre-proteins into chloroplasts [via the TOC/TIC (translocator at the outer envelope membrane of chloroplasts/inner envelope membrane of chloroplasts)] is controlled by two homologous receptor GTPases, Toc33 and Toc159, whose reversible dimerization is proposed to regulate translocation of incoming proteins in a GTP-dependent manner. Toc33 is a homodimerizing GTPase. Functional analysis suggests that homodimerization is a key step in the translocation process, the molecular functions of which, as well as the elements regulating this event, are largely unknown. In the present study, we show that homodimerization reduces the rate of nucleotide exchange, which is consistent with the observed orientation of the monomers in the crystal structure. Pre-protein binding induces a dissociation of the Toc33 homodimer and results in the exchange of GDP for GTP. Thus homodimerization does not serve to activate the GTPase activity as discussed many times previously, but to control the nucleotide-loading state. We discuss this novel regulatory mode and its impact on the current models of protein import into the chloroplast.Biochemical Journal 03/2011; 436(2):313-9. · 4.90 Impact Factor -
Chapter: Vitamin Biosynthetic Pathways, the PLP Synthase Complex, and the Potential for Targeting Protein–Protein Interaction
[show abstract] [hide abstract]
ABSTRACT: Vitamin biosynthetic pathways may provide targets for the development of anti-parasitic drugs when absent from mammals or parasitic host organisms. The targeting of a specific biosynthetic route requires that the vitamin in question is essential for the parasite's survival. In this chapter, several vitamin biosynthetic pathways are discussed as malarial drug targets. Recent advances have been made in the area of vitamin B6 biosynthesis by studying a key multienzyme complex, pyridoxal 5-phosphate (PLP) synthase. Today, complex assembly and ensuing enzyme activation have been very well described, through a combination of X-ray crystallography, calorimetry, homology modeling, and kinetic analyses. The study of protein complexes using these techniques may be valuable for drug design applications, specifically for systems where protein–protein interaction can be targeted.02/2011: pages 251 - 270; , ISBN: 9783527633883 -
Article: Defining the structural requirements for ribose 5-phosphate-binding and intersubunit cross-talk of the malarial pyridoxal 5-phosphate synthase.
[show abstract] [hide abstract]
ABSTRACT: Most organisms synthesise the B(6) vitamer pyridoxal 5-phosphate (PLP) via the glutamine amidotransferase PLP synthase, a large enzyme complex of 12 Pdx1 synthase subunits with up to 12 Pdx2 glutaminase subunits attached. Deletion analysis revealed that the C-terminus has four distinct functionalities: assembly of the Pdx1 monomers, binding of the pentose substrate (ribose 5-phosphate), formation of the reaction intermediate I(320), and finally PLP synthesis. Deletions of distinct C-terminal regions distinguish between these individual functions. PLP formation is the only function that is conferred to the enzyme by the C-terminus acting in trans, explaining the cooperative nature of the complex.FEBS letters 10/2010; 584(19):4169-74. · 3.54 Impact Factor -
Article: Conserved properties of polypeptide transport-associated (POTRA) domains derived from cyanobacterial Omp85.
[show abstract] [hide abstract]
ABSTRACT: Proteins of the Omp85 family are conserved in all kingdoms of life. They mediate protein transport across or protein insertion into membranes and reside in the outer membranes of Gram-negative bacteria, mitochondria, and chloroplasts. Omp85 proteins contain a C-terminal transmembrane beta-barrel and a soluble N terminus with a varying number of polypeptide-transport-associated or POTRA domains. Here we investigate Omp85 from the cyanobacterium Anabaena sp. PCC 7120. The crystallographic three-dimensional structure of the N-terminal region shows three POTRA domains, here named P1 to P3 from the N terminus. Molecular dynamics simulations revealed a hinge between P1 and P2 but in contrast show that P2 and P3 are fixed in orientation. The P2-P3 arrangement is identical as seen for the POTRA domains from proteobacterial FhaC, suggesting this orientation is a conserved feature. Furthermore, we define interfaces for protein-protein interaction in P1 and P2. P3 possesses an extended loop unique to cyanobacteria and plantae, which influences pore properties as shown by deletion. It now becomes clear how variations in structure of individual POTRA domains, as well as the different number of POTRA domains with both rigid and flexible connections make the N termini of Omp85 proteins versatile adaptors for a plentitude of functions.Journal of Biological Chemistry 03/2010; 285(23):18016-24. · 4.77 Impact Factor -
Article: X-ray crystal structure of Saccharomyces cerevisiae Pdx1 provides insights into the oligomeric nature of PLP synthases.
[show abstract] [hide abstract]
ABSTRACT: The universal enzymatic cofactor vitamin B6 can be synthesized as pyridoxal 5-phosphate (PLP) by the glutamine amidotransferase Pdx1. We show that Saccharomyces cerevisiae Pdx1 is hexameric by analytical ultracentrifugation and by crystallographic 3D structure determination. Bacterial homologues were previously reported to exist in hexamer:dodecamer equilibrium. A small sequence insertion found in yeast Pdx1 elevates the dodecamer dissociation constant when introduced into Bacillus subtilis Pdx1. Further, we demonstrate that the yeast Pdx1 C-terminus contacts an adjacent subunit, and deletion of this segment decreases enzymatic activity 3.5-fold, suggesting a role in catalysis.FEBS letters 07/2009; 583(13):2179-86. · 3.54 Impact Factor -
Article: Dissection of contributions from invariant amino acids to complex formation and catalysis in the heteromeric pyridoxal 5-phosphate synthase complex from Bacillus subtilis.
[show abstract] [hide abstract]
ABSTRACT: Pyridoxal 5-phosphate (PLP), an active form of vitamin B(6), is one of the most versatile cofactors and is involved in numerous biochemical reactions. The main pathway for de novo PLP biosynthesis leads to direct formation of PLP from a pentose and triose. This reaction is catalyzed by the heteromeric PLP synthase, consisting of the synthase subunit Pdx1 and the glutaminase subunit Pdx2. l-Glutamine hydrolysis by Pdx2 supplies ammonia to Pdx1 for incorporation into PLP. Autonomous glutaminase Pdx2 is inactive; however, interaction with Pdx1 leads to enzymatic activity. Oxyanion hole formation in the active site of Pdx2 is required for substrate binding and was suggested as the prime event of enzyme activation. Here, we dissect interactions required for complex formation from interactions required for catalytic activation of the glutaminase. The three-dimensional structural analysis suggested a number of invariant residues that regulate complex formation and enzyme activation. We have replaced several of these invariant residues by site-directed mutagenesis in an effort to understand their function. In addition to the biochemical characterization of enzyme activity, the generated protein variants were studied by isothermal calorimetry to investigate their role in complex formation. The assembled data describe a multistep activation mechanism. Residues of helix alphaN of Pdx1 are essential for formation of the Pdx1-Pdx2 complex and also stabilize the oxyanion hole. Thus, these interactions describe the encounter complex. On the other hand, residues at the N-terminal face of the (betaalpha)(8) barrel of Pdx1 contribute to interface formation and are required for the organization of the catalytic center; thus, these interactions describe the Michaelis complex. However, the main players for formation of the Michaelis complex reside on Pdx2, as replacement of residues at the N-terminal face of the (betaalpha)(8) barrel of Pdx1 leads to reduction but not complete inactivation of the glutaminase.Biochemistry 01/2009; 48(9):1928-35. · 3.42 Impact Factor -
Article: pH sensitivity of the GTPase Toc33 as a regulatory circuit for protein translocation into chloroplasts.
[show abstract] [hide abstract]
ABSTRACT: The properties of membrane-embedded GTPases are investigated to understand translocation of preprotein across the outer envelope of chloroplasts. The homo- and heterodimerization events of the GTPases had been established previously. We show that the hydrolytic activity of the GTPase Toc33 is pH insensitive in the homodimeric conformation but has a bell-shaped pH optimum in the monomeric conformation. Further, Toc33 GTPase homodimerization and protein translocation into chloroplasts are pH sensitive as well. pH sensitivity might serve to regulate translocation; alternatively, the documented pH sensitivity might reflect a mechanistic requirement for GTPase silencing during translocation as the GTPase switches between homo- and heterodimeric conformations.Plant and Cell Physiology 12/2008; 49(12):1917-21. · 4.70 Impact Factor -
Article: Structural basis for a distinct catalytic mechanism in Trypanosoma brucei tryparedoxin peroxidase.
[show abstract] [hide abstract]
ABSTRACT: Trypanosoma brucei, the causative agent of African sleeping sickness, encodes three cysteine homologues (Px I-III) of classical selenocysteine-containing glutathione peroxidases. The enzymes obtain their reducing equivalents from the unique trypanothione (bis(glutathionyl)spermidine)/tryparedoxin system. During catalysis, these tryparedoxin peroxidases cycle between an oxidized form with an intramolecular disulfide bond between Cys(47) and Cys(95) and the reduced peroxidase with both residues in the thiol state. Here we report on the three-dimensional structures of oxidized T. brucei Px III at 1.4A resolution obtained by x-ray crystallography and of both the oxidized and the reduced protein determined by NMR spectroscopy. Px III is a monomeric protein unlike the homologous poplar thioredoxin peroxidase (TxP). The structures of oxidized and reduced Px III are essentially identical in contrast to what was recently found for TxP. In Px III, Cys(47), Gln(82), and Trp(137) do not form the catalytic triad observed in the selenoenzymes, and related proteins and the latter two residues are unaffected by the redox state of the protein. The mutational analysis of three conserved lysine residues in the vicinity of the catalytic cysteines revealed that exchange of Lys(107) against glutamate abrogates the reduction of hydrogen peroxide, whereas Lys(97) and Lys(99) play a crucial role in the interaction with tryparedoxin.Journal of Biological Chemistry 09/2008; 283(44):30401-11. · 4.77 Impact Factor -
Article: A direct role for phosphatidylinositol-4,5-bisphosphate in unconventional secretion of fibroblast growth factor 2.
[show abstract] [hide abstract]
ABSTRACT: Fibroblast growth factor 2 (FGF-2) is a mitogen that is exported from cells by an endoplasmic reticulum/Golgi-independent secretory pathway. Recent findings have shown that FGF-2 export occurs by direct translocation from the cytoplasm across the plasma membrane into the extracellular space. Here, we report that FGF-2 contains a binding site for phosphatidylinositol-4,5-bisphosphate [PI(4,5)P(2)], the principal phosphoinositide species associated with plasma membranes. Intriguingly, in the context of a lipid bilayer, the interaction between FGF-2 and PI(4,5)P(2) is shown to depend on a lipid background that resembles plasma membranes. We show that the interaction with PI(4,5)P(2) is critically important for FGF-2 secretion as experimental conditions reducing cellular levels of PI(4,5)P(2) resulted in a substantial drop in FGF-2 export efficiency. Likewise, we have identified FGF-2 variant forms deficient for binding to PI(4,5)P(2) that were found to be severely impaired with regard to export efficiency. These data show that a transient interaction with PI(4,5)P(2) associated with the inner leaflet of plasma membranes represents the initial step of the unconventional secretory pathway of FGF-2.Traffic 08/2008; 9(7):1204-17. · 4.92 Impact Factor -
Article: On the significance of Toc-GTPase homodimers.
[show abstract] [hide abstract]
ABSTRACT: Precursor protein translocation across the outer chloroplast membrane depends on the action of the Toc complex, containing GTPases as recognizing receptor components. The G domains of the GTPases are known to dimerize. In the dimeric conformation an arginine contacts the phosphate moieties of bound nucleotide in trans. Kinetic studies suggested that the arginine in itself does not act as an arginine finger of a reciprocal GTPase-activating protein (GAP). Here we investigate the specific function of the residue in two GTPase homologues. Arginine to alanine replacement variants have significantly reduced affinities for dimerization compared with wild-type GTPases. The amino acid exchange does not impact on the overall fold and nucleotide binding, as seen in the monomeric x-ray crystallographic structure of the Arabidopsis Toc33 arginine-alanine replacement variant at 2.0A. We probed the catalytic center with the transition state analogue GDP/AlF(x) using NMR and analytical ultracentrifugation. AlF(x) binding depends on the arginine, suggesting the residue can play a role in catalysis despite the non-GAP nature of the homodimer. Two non-exclusive functional models are discussed: 1) the coGAP hypothesis, in which an additional factor activates the GTPase in homodimeric form; and 2) the switch hypothesis, in which a protein, presumably the large Toc159 GTPase, exchanges with one of the homodimeric subunits, leading to activation.Journal of Biological Chemistry 07/2008; 283(34):23104-12. · 4.77 Impact Factor -
Article: The GTPase cycle of the chloroplast import receptors Toc33/Toc34: implications from monomeric and dimeric structures.
[show abstract] [hide abstract]
ABSTRACT: Transport of precursor proteins across chloroplast membranes involves the GTPases Toc33/34 and Toc159 at the outer chloroplast envelope. The small GTPase Toc33/34 can homodimerize, but the regulation of this interaction has remained elusive. We show that dimerization is independent of nucleotide loading state, based on crystal structures of dimeric Pisum sativum Toc34 and monomeric Arabidopsis thaliana Toc33. An arginine residue is--in the dimer--positioned to resemble a GAP arginine finger. However, GTPase activation by dimerization is sparse and active site features do not explain catalysis, suggesting that the homodimer requires an additional factor as coGAP. Access to the catalytic center and an unusual switch I movement in the dimeric structure support this finding. Potential binding sites for interactions within the Toc translocon or with precursor proteins can be derived from the structures.Structure 05/2008; 16(4):585-96. · 6.35 Impact Factor -
Article: Policing Tic 'n' Toc, the doorway to chloroplasts.
[show abstract] [hide abstract]
ABSTRACT: The organization of eukaryotic cells into different membrane-enclosed compartments requires an ordered and regulated system for targeting and translocating proteins synthesized in the cytosol across organellar membranes. Protein translocation through integral membrane proteinaceous complexes shares common principles in different organelles, whereas molecular mechanisms and energy requirements are diverse. Translocation into mitochondria and plastids requires most proteins to cross two membranes, and translocation must be regulated to accommodate environmental or metabolic changes. In the last decade, the first ideas were formulated about the regulation of protein translocation into chloroplasts, thereby laying the foundation for this field. Here, we describe recent models for the regulation of translocation by precursor protein phosphorylation, receptor dimerization, redox sensing and calcium signaling. We suggest how these mechanisms might fit within the regulatory framework for the entry of proteins into chloroplasts.Trends in cell biology 02/2008; 18(1):19-27. · 12.12 Impact Factor -
Article: Structural and thermodynamic insights into the assembly of the heteromeric pyridoxal phosphate synthase from Plasmodium falciparum.
[show abstract] [hide abstract]
ABSTRACT: Pyridoxal 5'-phosphate (PLP) is required as a cofactor by many enzymes. The predominant de novo biosynthetic route is catalyzed by a heteromeric glutamine amidotransferase consisting of the synthase subunit Pdx1 and the glutaminase subunit Pdx2. Previously, Bacillus subtilis PLP synthase was studied by X-ray crystallography and complex assembly had been characterized by isothermal titration calorimetry. The fully assembled PLP synthase complex contains 12 individual Pdx1/Pdx2 glutamine amidotransferase heterodimers. These studies revealed the occurrence of an encounter complex that is tightened in the Michaelis complex when the substrate l-glutamine binds. In this study, we have characterized complex formation of PLP synthase from the malaria-causing human pathogen Plasmodium falciparum using isothermal titration calorimetry. The presence of l-glutamine increases the tightness of the interaction about 30-fold and alters the thermodynamic signature of complex formation. The thermodynamic data are integrated in a 3D homology model of P. falciparum PLP synthase. The negative experimental heat capacity (C(p)) describes a protein interface that is dominated by hydrophobic interactions. In the absence of l-glutamine, the experimental C(p) is less negative than in its presence, contrasting to the previously characterised bacterial PLP synthase. Thus, while the encounter complexes differ, the Michaelis complexes of plasmodial and bacterial systems have similar characteristics concerning the relative contribution of apolar/polar surface area. In addition, we have verified the role of the N-terminal region of PfPdx1 for complex formation. A "swap mutant" in which the complete alphaN-helix of plasmodial Pdx1 was exchanged with the corresponding segment from B. subtilis shows cross-binding to B. subtilis Pdx2. The swap mutant also partially elicits glutaminase activity in BsPdx2, demonstrating that formation of the protein complex interface via alphaN and catalytic activation of the glutaminase are linked processes.Journal of Molecular Biology 12/2007; 374(3):732-48. · 4.00 Impact Factor -
Article: Two independent routes of de novo vitamin B6 biosynthesis: not that different after all.
[show abstract] [hide abstract]
ABSTRACT: Vitamin B6 is well known in its biochemically active form as pyridoxal 5'-phosphate, an essential cofactor of numerous metabolic enzymes. The vitamin is also implicated in numerous human body functions ranging from modulation of hormone function to its recent discovery as a potent antioxidant. Its de novo biosynthesis occurs only in bacteria, fungi and plants, making it an essential nutrient in the human diet. Despite its paramount importance, its biosynthesis was predominantly investigated in Escherichia coli, where it is synthesized from the condensation of deoxyxylulose 5-phosphate and 4-phosphohydroxy-L-threonine catalysed by the concerted action of PdxA and PdxJ. However, it has now become clear that the majority of organisms capable of producing this vitamin do so via a different route, involving precursors from glycolysis and the pentose phosphate pathway. This alternative pathway is characterized by the presence of two genes, Pdx1 and Pdx2. Their discovery has sparked renewed interest in vitamin B6, and numerous studies have been conducted over the last few years to characterize the new biosynthesis pathway. Indeed, enormous progress has been made in defining the nature of the enzymes involved in both pathways, and important insights have been provided into their mechanisms of action. In the present review, we summarize the recent advances in our knowledge of the biosynthesis of this versatile molecule and compare the two independent routes to the biosynthesis of vitamin B6. Surprisingly, this comparison reveals that the key biosynthetic enzymes of both pathways are, in fact, very similar both structurally and mechanistically.Biochemical Journal 11/2007; 407(1):1-13. · 4.90 Impact Factor -
Article: Functional analysis of PDX2 from Arabidopsis, a glutaminase involved in vitamin B6 biosynthesis.
[show abstract] [hide abstract]
ABSTRACT: Vitamin B6 is an essential metabolite in all organisms, being required as a cofactor for a wide variety of biochemical reactions. De novo biosynthesis of the vitamin occurs in microorganisms and plants, but animals must obtain it from their diet. Two distinct and mutually exclusive de novo pathways have been identified to date, namely deoxyxylulose 5-phosphate dependent, which is restricted to a subset of eubacteria, and deoxyxylulose 5-phosphate independent, present in archaea, fungi, plants, protista, and most eubacteria. In these organisms, pyridoxal 5'-phosphate (PLP) formation is catalyzed by a single glutamine amidotransferase (PLP synthase) composed of a glutaminase domain, PDX2, and a synthase domain, PDX1. Despite plants being an important source of vitamin B6, very little is known about its biosynthesis. Here, we provide information for Arabidopsis thaliana. The functionality of PDX2 is demonstrated, using both in vitro and in vivo analyses. The expression pattern of PDX2 is assessed at both the RNA and protein level, providing insight into the spatial and temporal pattern of vitamin B6 biosynthesis. We then provide a detailed biochemical analysis of the plant PLP synthase complex. While the active sites of PDX1 and PDX2 are remote from each other, coordination of catalysis is much more pronounced with the plant proteins than its bacterial counterpart, Bacillus subtilis. Based on a model of the PDX1/PDX2 complex, mutation of a single residue uncouples enzyme coordination and in turn provides tangible evidence for the existence of the recently proposed ammonia tunnel through the core of PDX1.Plant physiology 07/2007; 144(2):915-25. · 6.53 Impact Factor -
Article: The structure of the regulatory domain of the adenylyl cyclase Rv1264 from Mycobacterium tuberculosis with bound oleic acid.
[show abstract] [hide abstract]
ABSTRACT: The universal secondary messenger cAMP is produced by adenylyl cyclases (ACs). Most bacterial and all eukaryotic ACs belong to class III of six divergent classes. A class III characteristic is formation of the catalytic pocket at a dimer interface and the presence of additional regulatory domains. Mycobacterium tuberculosis possesses 15 class III ACs, including Rv1264, which is activated at acidic pH due to pH-dependent structural transitions of the Rv1264 dimer. It has been shown by X-ray crystallography that the N-terminal regulatory and C-terminal catalytic domains of Rv1264 interact in completely different ways in the active and inhibited states. Here, we report an in-depth structural and functional analysis of the regulatory domain of Rv1264. The 1.6 A resolution crystal structure shows the protein in a tight, disk-shaped dimer, formed around a helical bundle, and involving a protein chain crossover. To understand pH regulation, we determined structures at acidic and basic pH values and employed structure-based mutagenesis in the holoenzyme to elucidate regulation using an AC activity assay. It has been shown that regulatory and catalytic domains must be linked in a single protein chain. The new studies demonstrate that the length of the linker segment is decisive for regulation. Several amino acids on the surface of the regulatory domain, when exchanged, altered the pH-dependence of AC activity. However, these residues are not conserved amongst a number of related ACs. The closely related mycobacterial Rv2212, but not Rv1264, is strongly activated by the addition of fatty acids. The structure resolved the presence of a deeply embedded fatty acid, characterised as oleic acid by mass spectrometry, which may serve as a hinge. From these data, we conclude that the regulatory domain is a structural scaffold used for distinct regulatory purposes.Journal of Molecular Biology 07/2007; 369(5):1282-95. · 4.00 Impact Factor
Top co-authors
Top Journals
Institutions
-
2011–2012
-
Goethe-Universität Frankfurt am Main
Frankfurt am Main, Hesse, Germany -
Justus-Liebig-Universität Gießen
Gießen, Hesse, Germany -
University of Southampton
- Biological Sciences
Southampton, ENG, United Kingdom
-
-
2005–2011
-
Universität Heidelberg
- Institute of Hygiene
Heidelberg, Baden-Wuerttemberg, Germany
-
-
2007–2009
-
Technische Universität Graz
- Institut für Biochemie
Graz, Styria, Austria
-
