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Biomedical subjects

Mair E A Churchill

Publications and source records attributed to Mair E A Churchill.

16 recordsLinked to original sources
PubMed2006

Structural basis for the histone chaperone activity of Asf1.

Anti-silencing function 1 (Asf1) is a highly conserved chaperone of histones H3/H4 that assembles or disassembles chromatin during transcription, replication, and repair. The structure of the globular domain of Asf1 bound to H3/H4 determined by X-ray crystallography to a resolution of 1.7 Angstroms shows how Asf1 binds the H3/H4 heterodimer, enveloping the C terminus of histone H3 and physically blocking formation of the H3/H4 heterotetramer. Unexpectedly, the C terminus of histone H4 that forms a mini-beta sheet with histone H2A in the nucleosome undergoes a major conformational change upon binding to Asf1 and adds a beta strand to the Asf1 beta sheet sandwich. Interactions with both H3 and H4 were required for Asf1 histone chaperone function in vivo and in vitro. The Asf1-H3/H4 structure suggests a "strand-capture" mechanism whereby the H4 tail acts as a lever to facilitate chromatin disassembly/assembly that may be used ubiquitously by histone chaperones.

Amino Acid Substitution↗
PubMed2006

Structure of the progesterone receptor-deoxyribonucleic acid complex: novel interactions required for binding to half-site response elements.

The DNA binding domain (DBD) of nuclear hormone receptors contains a highly conserved globular domain and a less conserved carboxyl-terminal extension (CTE). Despite previous observations that the CTEs of some classes of nuclear receptors are structured and interact with DNA outside of the hexanucleotide hormone response element (HRE), there has been no evidence for such a CTE among the steroid receptors. We have determined the structure of the progesterone receptor (PR)-DBD-CTE DNA complex at a resolution of 2.5 A, which revealed binding of the CTE to the minor groove flanking the HREs. Alanine substitutions of the interacting CTE residues reduced affinity for inverted repeat HREs separated by three nucleotides, and essentially abrogated binding to a single HRE. A highly compressed minor groove of the trinucleotide spacer and a novel dimerization interface were also observed. A PR binding site selection experiment revealed sequence preferences in the trinucleotide spacer and flanking DNA. These results, taken together, support the notion that sequences outside of the HREs influence the DNA binding affinity and specificity of steroid receptors.

Alanine↗
PubMed2006

A new GNAT in bacterial signaling?

Acyl-amino acids were discovered in the search for novel therapeutic agents produced by uncultured microorganisms. In this issue of Structure, Van Wagoner and Clardy (2006) find that the N-acyl-amino acid synthase FeeM bound to acyl-tyrosine is the latest member of the GCN5-related N-acyltransferase superfamily.

Bacteria↗
PubMed2006

Specificity of acyl-homoserine lactone synthases examined by mass spectrometry.

Many gram-negative bacteria produce a specific set of N-acyl-L-homoserine-lactone (AHL) signaling molecules for the purpose of quorum sensing, which is a means of regulating coordinated gene expression in a cell-density-dependent manner. AHLs are produced from acylated acyl-carrier protein (acyl-ACP) and S-adenosyl-L-methionine by the AHL synthase enzyme. The appearance of specific AHLs is due in large part to the intrinsic specificity of the enzyme for subsets of acyl-ACP substrates. Structural studies of the Pantoea stewartii enzyme EsaI and AHL-sensitive bioassays revealed that threonine 140 in the acyl chain binding pocket directs the enzyme toward production of 3-oxo-homoserine lactones. Mass spectrometry was used to examine the range of AHL molecular species produced by AHL synthases under a variety of conditions. An AHL selective normal-phase chromatographic purification with addition of a deuterated AHL internal standard was followed by reverse-phase liquid chromatography-tandem mass spectrometry in order to obtain estimates of the relative amounts of different AHLs from biological samples. The AHLs produced by wild-type and engineered EsaI and LasI AHL synthases show that intrinsic specificity and different cellular conditions influence the production of AHLs. The threonine at position 140 in EsaI is important for the preference for 3-oxo-acyl-ACPs, but the role of the equivalent threonine in LasI is less clear. In addition, LasI expressed in Escherichia coli produces a high proportion of unusual AHLs with acyl chains consisting of an odd number of carbons. Furthermore, these studies offer additional methods that will be useful for surveying and quantitating AHLs from different sources.

4-Butyrolactone↗
PubMed2006

Quorum-sensing signal synthesis by the Yersinia pestis acyl-homoserine lactone synthase YspI.

The acyl-homoserine lactone molecular species (AHLs) produced by the Yersinia pestis AHL synthase YspI were identified by biochemical and physical/chemical techniques. Bioassays of extracts from culture supernatants of the recombinant YspI and wild-type Yersinia pestis showed similar profiles of AHLs. Analysis by liquid chromatography-mass spectrometry revealed that the predominant AHLs were N-3-oxooctanoyl-L-homoserine lactone and N-3-oxo-hexanoyl-L-homoserine lactone.

4-Butyrolactone↗
PubMed2005

ASF1 binds to a heterodimer of histones H3 and H4: a two-step mechanism for the assembly of the H3-H4 heterotetramer on DNA.

The first step in the formation of the nucleosome is commonly assumed to be the deposition of a histone H3-H4 heterotetramer onto DNA. Antisilencing function 1 (ASF1) is a major histone H3-H4 chaperone that deposits histones H3 and H4 onto DNA. With a goal of understanding the mechanism of deposition of histones H3 and H4 onto DNA, we have determined the stoichiometry of the Asf1-H3-H4 complex. We have established that a single molecule of Asf1 binds to an H3-H4 heterodimer using gel filtration, amino acid, reversed-phase chromatography, and analytical ultracentrifugation analyses. We demonstrate that Asf1 blocks formation of the H3-H4 heterotetramer by a mechanism that likely involves occlusion of the H3-H3 dimerization interface.

Cell Cycle Proteins↗
PubMed2005

The Aurora A and Aurora B protein kinases: a single amino acid difference controls intrinsic activity and activation by TPX2.

The Aurora A and B protein kinases are key players in mitotic control and the etiology of human cancer. Despite the near identity of amino acid sequence in the catalytic domain, monomeric Aurora B is 50 fold lower in activity than monomeric Aurora A, and previous studies have shown that TPX2 binding to the catalytic domain activates Aurora A but not Aurora B. Here we identify G205 in Xenopus Aurora A as a key determinant of both intrinsic activity and regulation by TPX2. Mutation of G205 in Aurora A to N, the equivalent residue in Aurora B, had no effect on autophosphorylation of the T-loop but led to a 10-fold loss of specific activity, whereas mutation of N158 in Aurora B to G caused a 350-fold increase in specific activity. G205 N Aurora A was still activated by TPX2, but protection of pT295 from dephosphorylation by protein phosphatase 1 was abolished. Structural analysis of these effects suggests that the G205 forms a pivot point in the enzyme that results in movement of the N-terminal domain glycine-rich loop closer to the ATP binding site of the enzyme and also moves the C-helix slightly closer to the activation loop. Changes in these positions are comparable to those reported for other protein kinases and demonstrate that phosphorylation of the activation loop alone is not sufficient for enzyme activation. The generation of an activated mutant of Aurora B will be important for studying its role in cell cycle control and tumorigenesis.

Amino Acid Sequence↗
PubMed2004

DNA binding and bending by HMG boxes: energetic determinants of specificity.

To clarify the physical basis of DNA binding specificity, the thermodynamic properties and DNA binding and bending abilities of the DNA binding domains (DBDs) of sequence-specific (SS) and non-sequence-specific (NSS) HMG box proteins were studied with various DNA recognition sequences using micro-calorimetric and optical methods. Temperature-induced unfolding of the free DBDs showed that their structure does not represent a single cooperative unit but is subdivided into two (in the case of NSS DBDs) or three (in the case of SS DBDs) sub-domains, which differ in stability. Both types of HMG box, most particularly SS, are partially unfolded even at room temperature but association with DNA results in stabilization and cooperation of all the sub-domains. Binding and bending measurements using fluorescence spectroscopy over a range of ionic strengths, combined with calorimetric data, allowed separation of the electrostatic and non-electrostatic components of the Gibbs energies of DNA binding, yielding their enthalpic and entropic terms and an estimate of their contributions to DNA binding and bending. In all cases electrostatic interactions dominate non-electrostatic in the association of a DBD with DNA. The main difference between SS and NSS complexes is that SS are formed with an enthalpy close to zero and a negative heat capacity effect, while NSS are formed with a very positive enthalpy and a positive heat capacity effect. This indicates that formation of SS HMG box-DNA complexes is specified by extensive van der Waals contacts between apolar groups, i.e. a more tightly packed interface forms than in NSS complexes. The other principal difference is that DNA bending by the NSS DBDs is driven almost entirely by the electrostatic component of the binding energy, while DNA bending by SS DBDs is driven mainly by the non-electrostatic component. The basic extensions of both categories of HMG box play a similar role in DNA binding and bending, making solely electrostatic interactions with the DNA.

Amino Acid Sequence↗
PubMed2004

Crystallization of Pseudomonas aeruginosa AHL synthase LasI using beta-turn crystal engineering.

In Gram-negative bacteria, intercellular communication and virulence regulation is mediated by the diffusible chemical signal acyl-homoserine-L-lactone (AHL). The AHL synthase enzymes produce a variety of AHLs from the substrates S-adenosyl-L-methionine and acyl-acyl carrier protein. LasI, the AHL synthase from Pseudomonas aeruginosa, has low solubility and has failed to crystallize despite extensive crystallization trials. Based on the previously determined structure of the AHL synthase EsaI, active soluble LasI was produced by re-engineering residues in a tight turn to produce a type I' beta-turn. The resulting protein is active, more stable than the wild-type LasI and has been crystallized in the cubic space group F23, with unit-cell parameters a = b = c = 154.90 A.

4-Butyrolactone↗
PubMed2004

Structure of the Pseudomonas aeruginosa acyl-homoserinelactone synthase LasI.

The LasI/LasR quorum-sensing system plays a pivotal role in virulence gene regulation of the opportunistic human pathogen, Pseudomonas aeruginosa. Here we report the crystal structure of the acyl-homoserine lactone (AHL) synthase LasI that produces 3-oxo-C12-AHL from the substrates 3-oxo-C12-acyl-carrier protein (acyl-ACP) and S-adenosyl-L-methionine. The LasI six-stranded beta sheet platform, buttressed by three alpha helices, forms a V-shaped substrate-binding cleft that leads to a tunnel passing through the enzyme that can accommodate the acyl-chain of acyl-ACP. This tunnel places no apparent restriction on acyl-chain length, in contrast to a restrictive hydrophobic pocket seen in the AHL-synthase EsaI. Interactions of essential conserved N-terminal residues, Arg23, Phe27 and Trp33, suggest that the N-terminus forms an enclosed substrate-binding pocket for S-adenosyl-L-methionine. Analysis of AHL-synthase surface residues identified a binding site for acyl-ACP, a role that was supported by in vivo reporter assay analysis of the mutated residues, including Arg154 and Lys150. This structure and the novel explanation of AHL-synthase acyl-chain-length selectivity promise to guide the design of Pseudomonas aeruginosa-specific quorum-sensing inhibitors as antibacterial agents.

Acyl Carrier Protein↗
PubMed2003

DNA binding of a non-sequence-specific HMG-D protein is entropy driven with a substantial non-electrostatic contribution.

The thermal properties of two forms of the Drosophila melanogaster HMG-D protein, with and without its highly basic 26 residue C-terminal tail (D100 and D74) and the thermodynamics of their non-sequence-specific interaction with linear DNA duplexes were studied using scanning and titration microcalorimetry, spectropolarimetry, fluorescence anisotropy and FRET techniques at different temperatures and salt concentrations. It was shown that the C-terminal tail of D100 is unfolded at all temperatures, whilst the state of the globular part depends on temperature in a rather complex way, being completely folded only at temperatures close to 0 degrees C and unfolding with significant heat absorption at temperatures below those of the gross denaturational changes. The association constant and thus Gibbs energy of binding for D100 is much greater than for D74 but the enthalpies of their association are similar and are large and positive, i.e. DNA binding is a completely entropy-driven process. The positive entropy of association is due to release of counterions and dehydration upon forming the protein/DNA complex. Ionic strength variation showed that electrostatic interactions play an important but not exclusive role in the DNA binding of the globular part of this non-sequence-specific protein, whilst binding of the positively charged C-terminal tail of D100 is almost completely electrostatic in origin. This interaction with the negative charges of the DNA phosphate groups significantly enhances the DNA bending. An important feature of the non-sequence-specific association of these HMG boxes with DNA is that the binding enthalpy is significantly more positive than for the sequence-specific association of the HMG box from Sox-5, despite the fact that these proteins bend the DNA duplex to a similar extent. This difference shows that the enthalpy of dehydration of apolar groups at the HMG-D/DNA interface is not fully compensated by the energy of van der Waals interactions between these groups, i.e. the packing density at the interface must be lower than for the sequence-specific Sox-5 HMG box.

Amino Acid Sequence↗
PubMed2003

The role of intercalating residues in chromosomal high-mobility-group protein DNA binding, bending and specificity.

Ubiquitous high-mobility-group (HMGB) chromosomal proteins bind DNA in a non-sequence- specific fashion to promote chromatin function and gene regulation. Minor groove DNA binding of the HMG domain induces substantial DNA bending toward the major groove, and several interfacial residues contribute by DNA intercalation. The role of the intercalating residues in DNA binding, bending and specificity was systematically examined for a series of mutant Drosophila HMGB (HMG-D) proteins. The primary intercalating residue of HMG-D, Met13, is required both for high-affinity DNA binding and normal DNA bending. Leu9 and Tyr12 directly interact with Met13 and are required for HMG domain stability in addition to linear DNA binding and bending, which is an important function for these residues. In contrast, DNA binding and bending is retained in truncations of intercalating residues Val32 and Thr33 to alanine, but DNA bending is decreased for the glycine substitutions. Furthermore, substitution of the intercalating residues with those predicted to be involved in the specificity of the HMG domain transcription factors results in increased DNA affinity and decreased DNA bending without increased specificity. These studies reveal the importance of residues that buttress intercalating residues and suggest that features of the HMG domain other than a few base-specific hydrogen bonds distinguish the sequence-specific and non-sequence-specific HMG domain functions.

Amino Acid Sequence↗
PubMed2003

Structures of liganded and unliganded RsrI N6-adenine DNA methyltransferase: a distinct orientation for active cofactor binding.

The structures of RsrI DNA methyltransferase (M.RsrI) bound to the substrate S-adenosyl-l-methionine (AdoMet), the product S-adenosyl-l-homocysteine (AdoHcy), the inhibitor sinefungin, as well as a mutant apo-enzyme have been determined by x-ray crystallography. Two distinct binding configurations were observed for the three ligands. The substrate AdoMet adopts a bent shape that directs the activated methyl group toward the active site near the catalytic DPPY motif. The product AdoHcy and the competitive inhibitor sinefungin bind with a straight conformation in which the amino acid moiety occupies a position near the activated methyl group in the AdoMet complex. Analysis of ligand binding in comparison with other DNA methyltransferases reveals a small, common subset of available conformations for the ligand. The structures of M.RsrI with the non-substrate ligands contained a bound chloride ion in the AdoMet carboxylate-binding pocket, explaining its inhibition by chloride salts. The L72P mutant of M.RsrI is the first DNA methyltransferase structure without bound ligand. With respect to the wild-type protein, it had a larger ligand-binding pocket and displayed movement of a loop (223-227) that is responsible for binding the ligand, which may account for the weaker affinity of the L72P mutant for AdoMet. These studies show the subtle changes in the tight specific interactions of substrate, product, and an inhibitor with M.RsrI and help explain how each displays its unique effect on the activity of the enzyme.

Adenosine↗
PubMed2002

The C-terminal extension (CTE) of the nuclear hormone receptor DNA binding domain determines interactions and functional response to the HMGB-1/-2 co-regulatory proteins.

Previously, we and others reported that the high mobility group proteins, HMGB-1/-2, enhance DNA binding in vitro and transactivation in situ by the steroid hormone subgroup of nuclear receptors but did not influence these functions of class II receptors. We show here that the DNA binding domain (DBD) is sufficient to account for the selective influence of HMGB-1/-2 on the steroid class of receptors. Furthermore, the use of chimeric DBDs reveals that this selectivity is dependent on the C-terminal extension (CTE), amino acid sequences adjacent to the zinc finger core DBD. HMGB-1/-2 interact directly with the DBDs of steroid but not class II receptors, and this interaction requires the CTE. This in vitro interaction correlates with a requirement of the CTE for maximal HMGB-1/-2 enhancement of DNA binding in vitro and transcriptional activation in cells. Finally, class II receptor DBDs have a much higher intrinsic affinity for DNA than steroid receptor DBDs, and this affinity difference is also dependent on the CTE. These results reveal the importance of the steroid receptor CTE for DNA binding affinity and functional response to HMGB-1/-2.

Amino Acid Sequence↗
PubMed2002

Structural basis and specificity of acyl-homoserine lactone signal production in bacterial quorum sensing.

Synthesis and detection of acyl-homoserine lactones (AHLs) enables many gram-negative bacteria to engage in quorum sensing, an intercellular signaling mechanism that activates differentiation to virulent and biofilm lifestyles. The AHL synthases catalyze acylation of S-adenosyl-L-methionine by acyl-acyl carrier protein and lactonization of the methionine moiety to give AHLs. The crystal structure of the AHL synthase, EsaI, determined at 1.8 A resolution, reveals a remarkable structural similarity to the N-acetyltransferases and defines a common phosphopantetheine binding fold as the catalytic core. Critical residues responsible for catalysis and acyl chain specificity have been identified from a modeled substrate complex and verified through functional analysis in vivo. A mechanism for the N-acylation of S-adenosyl-L-methionine by 3-oxo-hexanoyl-acyl carrier protein is proposed.

4-Butyrolactone↗