Elena Blagova

The structure of CodY, a GTP- and isoleucine-responsive regulator of stationary phase and virulence in gram-positive bacteria

CodY is a global regulator of transcription in Gram-positive bacteria. It represses during growth genes required for adaptation to nutrient limitation, including virulence genes in some human pathogens. CodY activity is regulated by GTP and branched chain amino acids, metabolites whose intracellular concentrations drop as cells enter stationary phase. Although CodY has a highly conserved sequence, it has no significant similarity to proteins of known structure. Here we report crystal structures of two fragments of CodY from Bacillus subtilis that clearly constitute its cofactor and DNA binding domains and reveal that CodY is a chimera of previously observed folding units. The N-terminal cofactor-binding fragment adopts a fold reminiscent of the GAF domains found in cyclic nucleotide phosphodiesterases and adenylate cyclases. It is a dimer stabilized by an intermolecular six α-helical bundle that buries an extensive apolar surface rich in residues invariant in CodY orthologues. The branched chain amino acid ligands reside in hydrophobic pockets of each monomer distal to the dimer-forming surface. The structure of the C-terminal DNA binding domain belongs to the winged helix-turn-helix family. The implications of the structure for DNA binding by CodY and its control by cofactor binding are discussed.

The Crystal Structures of Human S100A12 in Apo Form and in Complex with Zinc: New Insights Into S100A12 Oligomerisation.

The functions of the members of the S100 family of EF-hand proteins are modulated by calcium and, in a number of cases, by zinc or copper. One such protein is S100A12, which is implicated in inflammation and host-parasite responses. Previously, we reported the structures of human S100A12 in both low (dimeric) and high (hexameric) calcium forms and, in addition, that of a complex with copper and calcium. Here we report the crystal structures of the metal-free apo form of human S100A12 at 1.77 A resolution and of the zinc complex in two crystal forms (P2(1)2(1)2(1) and F222) to 1.88 A and 1.73 A resolution, respectively. These are the first structures of a zinc-only complex of an S100 protein to be determined. The zinc complex structure shows significant differences from those of both calcium-loaded and apo-S100A12 structures, and comparisons suggest an explanation for the zinc-induced 1500-fold increase in calcium affinity. In addition, the new structures provide insight into the role of zinc-calcium interplay in the transition of S100A12 from a dimer through a tetramer to a hexamer. The role of both zinc and calcium in target binding by S100A12 during host-parasite responses is confirmed by experiments with paramyosin from the tropical parasites Onchocerca volvulus and Brugia malayi.

Implementation of semi-automated cloning and prokaryotic expression screening: the impact of SPINE

The implementation of high-throughput (HTP) cloning and expression screening in Escherichia coli by 14 laboratories in the Structural Proteomics In Europe (SPINE) consortium is described. Cloning efficiencies of greater than 80% have been achieved for the three non-ligation-based cloning techniques used, namely Gateway, ligation-indendent cloning of PCR products (LIC-PCR) and In-Fusion, with LIC-PCR emerging as the most cost-effective. On average, two constructs have been made for each of the approximately 1700 protein targets selected by SPINE for protein production. Overall, HTP expression screening in E. coli has yielded 32% soluble constructs, with at least one for 70% of the targets. In addition to the implementation of HTP cloning and expression screening, the development of two novel technologies is described, namely library-based screening for soluble constructs and parallel small-scale high-density fermentation.

An ATP-binding cassette-type cysteine transporter in Campylobacter jejuni inferred from the structure of an extracytoplasmic solute receptor protein.

Campylobacter jejuni is a Gram‐negative food‐borne pathogen associated with gastroenteritis in humans as well as cases of the autoimmune disease Guillain–Barré syndrome. C. jejuni is asaccharolytic because it lacks an active glycolytic pathway for the use of sugars as a carbon source. This suggests an increased reliance on amino acids as nutrients and indeed the genome sequence of this organism indicates the presence of a number of amino acid uptake systems. Cj0982, also known as CjaA, is a putative extracytoplasmic solute receptor for one such uptake system as well as a major surface antigen and vaccine candidate. The crystal structure of Cj0982 reveals a two‐domain protein with density in the enclosed cavity between the domains that clearly defines the presence of a bound cysteine ligand. Fluorescence titration experiments were used to demonstrate that Cj0982 binds cysteine tightly and specifically with a Kd of ∼10−7 M consistent with a role as a receptor for a high‐affinity transporter. These data imply that Cj0982 is the binding protein component of an ABC‐type cysteine transporter system and that cysteine uptake is important in the physiology of C. jejuni.

Crystal structure of dihydrodipicolinate synthase (BA3935) from Bacillus anthracis at 1.94 Å resolution

Introduction. As part of a structural genomics program, we are determining structures of proteins from the causative agent of anthrax, Bacillus anthracis, a Grampositive spore-forming bacterium. Among our initial candidates for crystallographic analysis are the products of essential genes based on knock-out studies in Bacillus subtilis. The BA3935 gene of B. anthracis (www.tigr.org), annotated as DapA2, encodes a putative protein consisting of 292 amino acid residues with a subunit molecular weight of 31,233 Da. The predicted protein has 60% identity with dihydrodipicolinate synthase (DHDPS or DapA) from B. subtilis and 40% amino acid sequence identity to its orthologue in Escherichia coli. dapA is one of only 271 out of the total of 4118 genes in B. subtilis that are indispensable for growth of the organism on standard laboratory media. 1 DHDPS catalyses the condensation of aspartate semialdehyde and pyruvate and is the first committed step on the pathway to diaminopimelate and L-lysine in prokaryotes, some fungi, and higher plants (Scheme 1). The product released from the E. coli enzyme has been shown to be 4-hydroxy-2,3,4,5-tetrahydro-(2S)-dipicolinic acid (HTDPA) rather than L-2,3-dihydrodipicolinic acid (DHDPA); as the name of the enzyme suggests, 2 DHDPA can be formed spontaneously from HTDPA by elimination of water. The steps of aspartate semialdehyde synthesis from aspartate are shared with the biosynthetic pathways leading methionine and threonine. The diaminopimelate/ lysine pathway is thought to be of particular importance in Gram-positive bacteria because diaminopimelate makes up a higher proportion of the dry cell weight than it does in Gram-negative bacteria, a consequence of the thicker cell wall in the former. In Bacilli, the product of the DHDPS reaction is further reduced by dipicolinate synthase to dipicolinate, which makes up 10% of the dry weight of spores. Crystal structures have been determined of DHDPS from E. coli, 3 Nicotiana sylvestris, 4 and most recently from Thermotoga maritima. 5 In this article, we report the crystal structure of DHDPS from B. anthracis (Ba DHDPS), the first structure of a dihydrodipicolinate synthase from a Gram-positive bacterium. The structure of Ba DHDPS was determined by molecular replacement using the coordinate set for the E. coli orthologue (PDB code 1DHP) as the search model. 3 Two structures have been determined to 1.9 and 2.2 A resolution in different orthorhombic crystal forms. Data collection, refinement, and model-building statistics are summarized in Table I. The structures in the two crystal forms are very similar and unless otherwise stated, the commentary here will refer to the structure refined to higher resolution

Structure of components of an intercellular channel complex in sporulating Bacillus subtilis

Following asymmetric cell division during spore formation in Bacillus subtilis, a forespore expressed membrane protein SpoIIQ, interacts across an intercellular space with a mother cell-expressed membrane protein, SpoIIIAH. Their interaction can serve as a molecular “ratchet” contributing to the migration of the mother cell membrane around that of the forespore in a phagocytosis-like process termed engulfment. Upon completion of engulfment, SpoIIQ and SpoIIIAH are integral components of a recently proposed intercellular channel allowing passage from the mother cell into the forespore of factors required for late gene expression in this compartment. Here we show that the extracellular domains of SpoIIQ and SpoIIIAH form a heterodimeric complex in solution. The crystal structure of this complex reveals that SpoIIQ has a LytM-like zinc-metalloprotease fold but with an incomplete zinc coordination sphere and no metal. SpoIIIAH has an α-helical subdomain and a protruding β-sheet subdomain, which mediates interactions with SpoIIQ. SpoIIIAH has sequence and structural homology to EscJ, a type III secretion system protein that forms a 24-fold symmetric ring. Superposition of the structures of SpoIIIAH and EscJ reveals that the SpoIIIAH protomer overlaps with two adjacent protomers of EscJ, allowing us to generate a dodecameric SpoIIIAH ring by using structural homology. Following this superposition, the SpoIIQ chains also form a closed dodecameric ring abutting the SpoIIIAH ring, producing an assembly surrounding a 60 Å channel. The dimensions and organization of the proposed complex suggest it is a plausible model for the extracellular component of a gap junction-like intercellular channel.

Structure of the catalytic domain of the human mitochondrial Lon protease: proposed relation of oligomer formation and activity.

ATP‐dependent proteases are crucial for cellular homeostasis. By degrading short‐lived regulatory proteins, they play an important role in the control of many cellular pathways and, through the degradation of abnormally misfolded proteins, protect the cell from a buildup of aggregates. Disruption or disregulation of mammalian mitochondrial Lon protease leads to severe changes in the cell, linked with carcinogenesis, apoptosis, and necrosis. Here we present the structure of the proteolytic domain of human mitochondrial Lon at 2 Å resolution. The fold resembles those of the three previously determined Lon proteolytic domains from Escherichia coli, Methanococcus jannaschii, and Archaeoglobus fulgidus. There are six protomers in the asymmetric unit, four arranged as two dimers. The intersubunit interactions within the two dimers are similar to those between adjacent subunits of the hexameric ring of E. coli Lon, suggesting that the human Lon proteolytic domain also forms hexamers. The active site contains a 310 helix attached to the N‐terminal end of α‐helix 2, which leads to the insertion of Asp852 into the active site, as seen in M. jannaschii. Structural considerations make it likely that this conformation is proteolytically inactive. When comparing the intersubunit interactions of human with those of E. coli Lon taken with biochemical data leads us to propose a mechanism relating the formation of Lon oligomers with a conformational shift in the active site region coupled to a movement of a loop in the oligomer interface, converting the proteolytically inactive form seen here to the active one in the E. coli hexamer.

Structural rearrangement accompanying ligand binding in the GAF domain of CodY from Bacillus subtilis.

The GAF domain is a simple module widespread in proteins of diverse function, including cell signalling proteins and transcription factors. Its structure, typically spanning 150 residues, has three tiers: a basal layer of two or more alpha-helices, a middle layer of beta-pleated sheet and a top layer formed by segments of the polypeptide that connect strands of the beta-sheet. In structures of GAF domains in complex with their effectors, these polypeptide segments envelop the ligand, enclosing it in a cavity whose base is formed by the beta-sheet, such that ligand binding and release must be accompanied by conformational rearrangements of the distal portion of the structure. Descriptions of binding are presently limited by the absence of a GAF domain for which both liganded and unliganded structures are known. Earlier, we solved the crystal structure of the GAF domain of CodY, a branched-chain amino acid and GTP-responsive regulator of the transcription of stationary-phase and virulence genes in Bacillus, in complexes with isoleucine and valine. Here, we report the structure of this domain in its unliganded form, allowing definition of the structural changes accompanying ligand binding. The core of the protein and its dimerisation interface are essentially unchanged, in agreement with circular dichroism spectroscopy experiments that show that the secondary structure composition is unperturbed by ligand binding. There is however extensive refolding of the binding site loops, with up to 15-A movements of the coiled segment linking beta3 and beta4, such that the binding pocket is not formed in the absence of the ligand. The implications of these structural rearrangements for ligand affinity and specificity are discussed. Finally, saturation-transfer-difference NMR spectroscopy showed binding of isoleucine but not that of GTP to the GAF domain, suggesting that the two cofactors do not have a common binding site.

Three-dimensional structure of Serratia marcescens nuclease at 1.7 A resolution and mechanism of its action.

The three‐dimensional crystal structure of Serratia marcescens (Sm) nuclease has been refined at 1.7 Å resolution to the R‐factor of 17.3% and R‐free of 22.2%. The final model consists of 3678 non‐hydrogen atoms and 443 water molecules. The analysis of the secondary and the tertiary structures of the Sm nuclease suggests a topology which reveals essential inner symmetry in all the three layers forming the monomer. We propose the plausible mechanism of its action based on a concerted participation of the catalytically important amino acid residues of the enzyme active site.

Crystallization of the GTP‐dependent transcriptional regulator CodY from Bacillus subtilis

CodY is a GTP sensor that represses transcription of early stationary phase and sporulation genes in Bacillus subtilis. As nutrients become limiting, GTP levels fall and CodY-mediated repression is relieved. Crystals of CodY have been grown in the presence and absence of GTP from sodium citrate buffered solutions containing lithium sulfate and diffraction data have been collected extending to 3.5 A spacing.