Molecular mechanism and evolution of guanylate kinase regulation by (p)ppGpp.

The nucleotide (p)ppGpp mediates bacterial stress responses, but its targets and underlying mechanisms of action vary among bacterial species and remain incompletely understood. Here, we characterize the molecular interaction between (p)ppGpp and guanylate kinase (GMK), revealing the importance of this interaction in adaptation to starvation. Combining structural and kinetic analyses, we show that (p)ppGpp binds the GMK active site and competitively inhibits the enzyme. The (p)ppGpp-GMK interaction prevents the conversion of GMP to GDP, resulting in GMP accumulation upon amino acid downshift. Abolishing this interaction leads to excess (p)ppGpp and defective adaptation to amino acid starvation. A survey of GMKs from phylogenetically diverse bacteria shows that the (p)ppGpp-GMK interaction is conserved in members of Firmicutes, Actinobacteria, and Deinococcus-Thermus, but not in Proteobacteria, where (p)ppGpp regulates RNA polymerase (RNAP). We propose that GMK is an ancestral (p)ppGpp target and RNAP evolved more recently as a direct target in Proteobacteria.

Sphingoid Bases Regulate the Sigma-1 Receptor-Sphingosine and N,N'-Dimethylsphingosine Are Endogenous Agonists.

Both bioactive sphingolipids and Sigma-1 receptor (S1R) chaperones occur ubiquitously in mammalian cell membranes. Endogenous compounds that regulate the S1R are important for controlling S1R responses to cellular stress. Herein, we interrogated the S1R in intact Retinal Pigment Epithelial cells (ARPE-19) with the bioactive sphingoid base, sphingosine (SPH), or the pain-provoking dimethylated SPH derivative, N,N'-dimethylsphingosine (DMS). As informed by a modified native gel approach, the basal and antagonist (BD-1047)-stabilized S1R oligomers dissociated to protomeric forms in the presence of SPH or DMS (PRE-084 as control). We, thus, posited that SPH and DMS are endogenous S1R agonists. Consistently, in silico docking of SPH and DMS to the S1R protomer showed strong associations with Asp126 and Glu172 in the cupin beta barrel and extensive van der Waals interactions of the C18 alkyl chains with the binding site including residues in helices 4 and 5. Mean docking free energies were 8.73-8.93 kcal/mol for SPH and 8.56-8.15 kcal/mol for DMS, and calculated binding constants were ~40 nM for SPH and ~120 nM for DMS. We hypothesize that SPH, DMS, and similar sphingoid bases access the S1R beta barrel via a membrane bilayer pathway. We further propose that the enzymatic control of ceramide concentrations in intracellular membranes as the primary sources of SPH dictates availability of endogenous SPH and DMS to the S1R and the subsequent control of S1R activity within the same cell and/or in cellular environments.

Structure-specific DNA replication-fork recognition directs helicase and replication restart activities of the PriA helicase.

DNA replication restart, the essential process that reinitiates prematurely terminated genome replication reactions, relies on exquisitely specific recognition of abandoned DNA replication-fork structures. The PriA DNA helicase mediates this process in bacteria through mechanisms that remain poorly defined. We report the crystal structure of a PriA/replication-fork complex, which resolves leading-strand duplex DNA bound to the protein. Interaction with PriA unpairs one end of the DNA and sequesters the 3'-most nucleotide from the nascent leading strand into a conserved protein pocket. Cross-linking studies reveal a surface on the winged-helix domain of PriA that binds to parental duplex DNA. Deleting the winged-helix domain alters PriA's structure-specific DNA unwinding properties and impairs its activity in vivo. Our observations lead to a model in which coordinated parental-, leading-, and lagging-strand DNA binding provide PriA with the structural specificity needed to act on abandoned DNA replication forks.

Trace derivatives of kynurenine potently activate the aryl hydrocarbon receptor (AHR).

Cellular metabolites act as important signaling cues, but are subject to complex unknown chemistry. Kynurenine is a tryptophan metabolite that plays a crucial role in cancer and the immune system. Despite its atypical, non-ligand-like, highly polar structure, kynurenine activates the aryl hydrocarbon receptor (AHR), a PER, ARNT, SIM (PAS) family transcription factor that responds to diverse environmental and cellular ligands. The activity of kynurenine is increased 100-1000-fold by incubation or long-term storage and relies on the hydrophobic ligand-binding pocket of AHR, with identical structural signatures for AHR induction before and after activation. We purified trace-active derivatives of kynurenine and identified two novel, closely related condensation products, named trace-extended aromatic condensation products (TEACOPs), which are active at low picomolar levels. The synthesized compound for one of the predicted structures matched the purified compound in both chemical structure and AHR pharmacology. Our study provides evidence that kynurenine acts as an AHR pro-ligand, which requires novel chemical conversions to act as a receptor agonist.

Use of a Stereochemical Strategy To Probe the Mechanism of Phenol-Soluble Modulin α3 Toxicity.

Phenol-soluble modulin α3 (PSMα3) is a cytotoxic peptide secreted by virulent strains of Staphylococcus aureus. We used a stereochemical strategy to examine the mechanism of PSMα3-mediated toxicity. One hypothesis is that PSMα3 toxicity requires fibril formation; an alternative is that toxicity is caused by soluble forms of PSMα3, possibly oligomeric. We find that the unnatural enantiomer (D residues) displays cytotoxicity comparable to that of L-PSMα3. Racemic PSMα3 is similarly toxic to enantiopure PSMα3 (L or D) under some conditions, but the toxicity is lost under conditions that cause racemic PSMα3 to aggregate. A crystal structure of racemic PSMα3-NH2 displays an α-helical secondary structure and a packing pattern that is reminiscent of the cross-α arrangement recently discovered in crystals of L-PSMα3. Our data suggest that the cytotoxicity of PSMα3 does not depend on stereospecific engagement of a target protein or other chiral macromolecule, an observation that supports a mechanism based on membrane disruption. In addition, our data support the hypothesis that toxicity is exerted by a soluble form rather than an insoluble fibrillar form.

The structural basis for tight control of PP2A methylation and function by LCMT-1.

Proper formation of protein phosphatase 2A (PP2A) holoenzymes is essential for the fitness of all eukaryotic cells. Carboxyl methylation of the PP2A catalytic subunit plays a critical role in regulating holoenzyme assembly; methylation is catalyzed by PP2A-specific methyltransferase LCMT-1, an enzyme required for cell survival. We determined crystal structures of human LCMT-1 in isolation and in complex with PP2A stabilized by a cofactor mimic. The structures show that the LCMT-1 active-site pocket recognizes the carboxyl terminus of PP2A, and, interestingly, the PP2A active site makes extensive contacts to LCMT-1. We demonstrated that activation of the PP2A active site stimulates methylation, suggesting a mechanism for efficient conversion of activated PP2A into substrate-specific holoenzymes, thus minimizing unregulated phosphatase activity or formation of inactive holoenzymes. A dominant-negative LCMT-1 mutant attenuates the cell cycle without causing cell death, likely by inhibiting uncontrolled phosphatase activity. Our studies suggested mechanisms of LCMT-1 in tight control of PP2A function, important for the cell cycle and cell survival.

Arm-in-Arm Response Regulator Dimers Promote Intermolecular Signal Transduction.

UNLABELLED: Bacteriophytochrome photoreceptors (BphPs) and their cognate response regulators make up two-component signal transduction systems which direct bacteria to mount phenotypic responses to changes in environmental light quality. Most of these systems utilize single-domain response regulators to transduce signals through unknown pathways and mechanisms. Here we describe the photocycle and autophosphorylation kinetics of RtBphP1, a red light-regulated histidine kinase from the desert bacterium Ramlibacter tataouinensis RtBphP1 undergoes red to far-red photoconversion with rapid thermal reversion to the dark state. RtBphP1 is autophosphorylated in the dark; this activity is inhibited under red light. The RtBphP1 cognate response regulator, the R. tataouinensis bacteriophytochrome response regulator (RtBRR), and a homolog, AtBRR from Agrobacterium tumefaciens, crystallize unexpectedly as arm-in-arm dimers, reliant on a conserved hydrophobic motif, hFWAhL (where h is a hydrophobic M, V, L, or I residue). RtBRR and AtBRR dimerize distinctly from four structurally characterized phytochrome response regulators found in photosynthetic organisms and from all other receiver domain homodimers in the Protein Data Bank. A unique cacodylate-zinc-histidine tag metal organic framework yielded single-wavelength anomalous diffraction phases and may be of general interest. Examination of the effect of the BRR stoichiometry on signal transduction showed that phosphorylated RtBRR is accumulated more efficiently than the engineered monomeric RtBRR (RtBRRmon) in phosphotransfer reactions. Thus, we conclude that arm-in-arm dimers are a relevant signaling intermediate in this class of two-component regulatory systems. IMPORTANCE: BphP histidine kinases and their cognate response regulators comprise widespread red light-sensing two-component systems. Much work on BphPs has focused on structural understanding of light sensing and on enhancing the natural infrared fluorescence of these proteins, rather than on signal transduction or the resultant phenotypes. To begin to address this knowledge gap, we solved the crystal structures of two single-domain response regulators encoded by a region immediately downstream of that encoding BphPs. We observed a previously unknown arm-in-arm dimer linkage. Monomerization via deletion of the C-terminal dimerization motif had an inhibitory effect on net response regulator phosphorylation, underlining the importance of these unusual dimers for signal transduction.

Quasiracemate Crystal Structures of Magainin 2 Derivatives Support the Functional Significance of the Phenylalanine Zipper Motif.

Quasiracemic crystallography has been used to explore the significance of homochiral and heterochiral associations in a set of host-defense peptide derivatives. The previously reported racemic crystal structure of a magainin 2 derivative displayed a homochiral antiparallel dimer association featuring a "phenylalanine zipper" notable for the dual roles of phenylalanines in mediating dimerization and formation of an exposed hydrophobic swath. This motif is seen as well in two new quasiracemate crystals that contain the d form of the magainin 2 derivative along with an l-peptide in which one Ala has been replaced by a ß-amino acid residue. This structural trend supports the hypothesis that the Phe zipper motif has functional significance.

The recent evolution of a symbiotic ion channel in the legume family altered ion conductance and improved functionality in calcium signaling.

Arbuscular mycorrhiza and the rhizobia-legume symbiosis are two major root endosymbioses that facilitate plant nutrition. In Lotus japonicus, two symbiotic cation channels, CASTOR and POLLUX, are indispensable for the induction of nuclear calcium spiking, one of the earliest plant responses to symbiotic partner recognition. During recent evolution, a single amino acid substitution in DOES NOT MAKE INFECTIONS1 (DMI1), the POLLUX putative ortholog in the closely related Medicago truncatula, rendered the channel solo sufficient for symbiosis; castor, pollux, and castor pollux double mutants of L. japonicus were rescued by DMI1 alone, while both Lj-CASTOR and Lj-POLLUX were required for rescuing a dmi1 mutant of M. truncatula. Experimental replacement of the critical serine by an alanine in the selectivity filter of Lj-POLLUX conferred a symbiotic performance indistinguishable from DMI1. Electrophysiological characterization of DMI1 and Lj-CASTOR (wild-type and mutants) by planar lipid bilayer experiments combined with calcium imaging in Human Embryonic Kidney-293 cells expressing DMI1 (the wild type and mutants) suggest that the serine-to-alanine substitution conferred reduced conductance with a long open state to DMI1 and improved its efficiency in mediating calcium oscillations. We propose that this single amino acid replacement in the selectivity filter made DMI1 solo sufficient for symbiosis, thus explaining the selective advantage of this allele at the mechanistic level.

Propofol binding to the resting state of the gloeobacter violaceus ligand-gated ion channel (GLIC) induces structural changes in the inter- and intrasubunit transmembrane domain (TMD) cavities.

General anesthetics exert many of their CNS actions by binding to and modulating membrane-embedded pentameric ligand-gated ion channels (pLGICs). The structural mechanisms underlying how anesthetics modulate pLGIC function remain largely unknown. GLIC, a prokaryotic pLGIC homologue, is inhibited by general anesthetics, suggesting anesthetics stabilize a closed channel state, but in anesthetic-bound GLIC crystal structures the channel appears open. Here, using functional GLIC channels expressed in oocytes, we examined whether propofol induces structural rearrangements in the GLIC transmembrane domain (TMD). Residues in the GLIC TMD that frame intrasubunit and intersubunit water-accessible cavities were individually mutated to cysteine. We measured and compared the rates of modification of the introduced cysteines by sulfhydryl-reactive reagents in the absence and presence of propofol. Propofol slowed the rate of modification of L240C (intersubunit) and increased the rate of modification of T254C (intrasubunit), indicating that propofol binding induces structural rearrangements in these cavities that alter the local environment near these residues. Propofol acceleration of T254C modification suggests that in the resting state propofol does not bind in the TMD intrasubunit cavity as observed in the crystal structure of GLIC with bound propofol (Nury, H., Van Renterghem, C., Weng, Y., Tran, A., Baaden, M., Dufresne, V., Changeux, J. P., Sonner, J. M., Delarue, M., and Corringer, P. J. (2011) Nature 469, 428-431). In silico docking using a GLIC closed channel homology model suggests propofol binds to intersubunit sites in the TMD in the resting state. Propofol-induced motions in the intersubunit cavity were distinct from motions associated with channel activation, indicating propofol stabilizes a novel closed state.

Structure-guided engineering enhances a phytochrome-based infrared fluorescent protein.

Phytochrome is a multidomain dimeric red light photoreceptor that utilizes a chromophore-binding domain (CBD), a PHY domain, and an output module to induce cellular changes in response to light. A promising biotechnology tool emerged when a structure-based substitution at Asp-207 was shown to be an infrared fluorophore that uses a biologically available tetrapyrrole chromophore. We report multiple crystal structures of this D207H variant of the Deinococcus radiodurans CBD, in which His-207 is observed to form a hydrogen bond with either the tetrapyrrole A-ring oxygen or the Tyr-263 hydroxyl. Based on the implications of this duality for fluorescence properties, Y263F was introduced and shown to have stronger fluorescence than the original D207H template. Our structures are consistent with the model that the Y263F change prevents a red light-induced far-red light absorbing phytochrome chromophore configuration. With the goal of decreasing size and thereby facilitating use as a fluorescent tag in vivo, we also engineered a monomeric form of the CBD. Unexpectedly, photoconversion was observed in the monomer despite the lack of a PHY domain. This observation underscores an interplay between dimerization and the photochemical properties of phytochrome and suggests that the monomeric CBD could be used for further studies of the photocycle. The D207H substitution on its own in the monomer did not result in fluorescence, whereas Y263F did. Combined, the D207H and Y263F substitutions in the monomeric CBD lead to the brightest of our variants, designated Wisconsin infrared phytofluor (Wi-Phy).

Differential impact of ß and γ residue preorganization on α/ß/γ-peptide helix stability in water.

Cyclic constraints have proven to be very effective for preorganizing ß-amino acid residues and thereby stabilizing ß- and α/ß-peptide helices, but little is known about possible preorganization effects among γ residues. Here we assess and compare the impact of cyclic preorganization of ß and γ residues in the context of a specific α/ß/γ-peptide helix. The results show that ß residue preorganization is critical for helix stability but that γ residue preorganization is less important.

Small-molecule tools for dissecting the roles of SSB/protein interactions in genome maintenance.

Bacterial single-stranded DNA-binding proteins (SSBs) help to recruit a diverse array of genome maintenance enzymes to their sites of action through direct protein interactions. For all cases examined to date, these interactions are mediated by the evolutionarily conserved C terminus of SSB (SSB-Ct). The essential nature of SSB protein interactions makes inhibitors that block SSB complex formation valuable biochemical tools and attractive potential antibacterial agents. Here, we identify four small molecules that disrupt complexes formed between Escherichia coli SSB and Exonuclease I (ExoI), a well-studied SSB-interacting enzyme. Each compound disrupts ExoI/SSB-Ct peptide complexes and abrogates SSB stimulation of ExoI nuclease activity. Structural and biochemical studies support a model for three of the compounds in which they compete with SSB for binding to ExoI. The fourth appears to rely on an allosteric mechanism to disrupt ExoI/SSB complexes. Subsets of the inhibitors block SSB-Ct complex formation with two other SSB-interaction partners as well, which highlights their utility as reagents for investigating the roles of SSB/protein interactions in diverse DNA replication, recombination, and repair reactions.

Stereoelectronic and steric effects in side chains preorganize a protein main chain.

Preorganization is shown to endow a protein with extraordinary conformational stability. This preorganization is achieved by installing side-chain substituents that impose stereoelectronic and steric effects that restrict main-chain torsion angles. Replacing proline residues in (ProProGly)(7) collagen strands with 4-fluoroproline and 4-methylproline leads to the most stable known triple helices, having T ( m ) values that are increased by > 50 degrees C. Differential scanning calorimetry data indicate an entropic basis to the hyperstability, as expected from an origin in preorganization. Structural data at a resolution of 1.21 A reveal a prototypical triple helix with insignificant deviations to its main chain, even though 2/3 of the residues are nonnatural. Thus, preorganization of a main chain by subtle changes to side chains can confer extraordinary conformational stability upon a protein without perturbing its structure.

Catalytic activation of histone acetyltransferase Rtt109 by a histone chaperone.

Most histone acetyltransferases (HATs) function as multisubunit complexes in which accessory proteins regulate substrate specificity and catalytic efficiency. Rtt109 is a particularly interesting example of a HAT whose specificity and catalytic activity require association with either of two histone chaperones, Vps75 or Asf1. Here, we utilize biochemical, structural, and genetic analyses to provide the detailed molecular mechanism for activation of a HAT (Rtt109) by its activating subunit Vps75. The rate-determining step of the activated complex is the transfer of the acetyl group from acetyl CoA to the acceptor lysine residue. Vps75 stimulates catalysis (> 250-fold), not by contributing a catalytic base, but by stabilizing the catalytically active conformation of Rtt109. To provide structural insight into the functional complex, we produced a molecular model of Rtt109-Vps75 based on X-ray diffraction of crystals of the complex. This model reveals distinct negative electrostatic surfaces on an Rtt109 molecule that interface with complementary electropositive ends of a symmetrical Vps75 dimer. Rtt109 variants with interface point substitutions lack the ability to be fully activated by Vps75, and one such variant displayed impaired Vps75-dependent histone acetylation functions in yeast, yet these variants showed no adverse effect on Asf1-dependent Rtt109 activities in vitro and in vivo. Finally, we provide evidence for a molecular model in which a 12 complex of Rtt109-Vps75 acetylates a heterodimer of H3-H4. The activation mechanism of Rtt109-Vps75 provides a valuable framework for understanding the molecular regulation of HATs within multisubunit complexes.

Replication fork binding triggers structural changes in the PriA helicase that govern DNA replication restart in E. coli.

Bacterial replisomes often dissociate from replication forks before chromosomal replication is complete. To avoid the lethal consequences of such situations, bacteria have evolved replication restart pathways that reload replisomes onto prematurely terminated replication forks. To understand how the primary replication restart pathway in E. coli (PriA-PriB) selectively acts on replication forks, we determined the cryogenic-electron microscopy structure of a PriA/PriB/replication fork complex. Replication fork specificity arises from extensive PriA interactions with each arm of the branched DNA. These interactions reshape the PriA protein to create a pore encircling single-stranded lagging-strand DNA while also exposing a surface of PriA onto which PriB docks. Together with supporting biochemical and genetic studies, the structure reveals a switch-like mechanism for replication restart initiation in which restructuring of PriA directly couples replication fork recognition to PriA/PriB complex formation to ensure robust and high-fidelity replication re-initiation.

Quasiracemic crystallization as a tool to assess the accommodation of noncanonical residues in nativelike protein conformations.

Quasiracemic crystallization has been used to obtain high-resolution structures of two variants of the villin headpiece subdomain (VHP) that contain a pentafluorophenylalanine (F(5)Phe) residue in the hydrophobic core. In each case, the crystal contained the variant constructed from l-amino acids and the native sequence constructed from d-amino acids. We were motivated to undertake these studies by reports that racemic proteins crystallize more readily than homochiral forms and the prospect that quasiracemic crystallization would enable us to determine whether a polypeptide containing a noncanonical residue can closely mimic the tertiary structure of the native sequence. The results suggest that quasiracemic crystallization may prove to be generally useful for assessing mimicry of naturally evolved protein folding patterns by polypeptides that contain unnatural side-chain or backbone subunits.

X-ray crystal structure of the Escherichia coli RadD DNA repair protein bound to ADP reveals a novel zinc ribbon domain.

Genome maintenance is an essential process in all cells. In prokaryotes, the RadD protein is important for survival under conditions that include DNA-damaging radiation. Precisely how RadD participates in genome maintenance remains unclear. Here we present a high-resolution X-ray crystal structure of ADP-bound Escherichia coli RadD, revealing a zinc-ribbon element that was not modelled in a previous RadD crystal structure. Insights into the mode of nucleotide binding and additional structure refinement afforded by the new RadD model will help to drive investigations into the activity of RadD as a genome stability and repair factor.

X-ray crystal structure of the bacterial conjugation factor PsiB, a negative regulator of RecA.

During bacterial conjugation, genetic material from one cell is transferred to another as single-stranded DNA. The introduction of single-stranded DNA into the recipient cell would ordinarily trigger a potentially deleterious transcriptional response called SOS, which is initiated by RecA protein filaments formed on the DNA. During F plasmid conjugation, however, the SOS response is suppressed by PsiB, an F-plasmid-encoded protein that binds and sequesters free RecA to prevent filament formation. Among the many characterized RecA modulator proteins, PsiB is unique in using sequestration as an inhibitory mechanism. We describe the crystal structure of PsiB from the Escherichia coli F plasmid. The stucture of PsiB is surprisingly similar to CapZ, a eukaryotic actin filament capping protein. Structure-directed neutralization of electronegative surfaces on PsiB abrogates RecA inhibition whereas neutralization of an electropositive surface element enhances PsiB inhibition of RecA. Together, these studies provide a first molecular view of PsiB and highlight its use as a reagent in studies of RecA activity.

Extended binding site on fibronectin for the functional upstream domain of protein F1 of Streptococcus pyogenes.

The 49-residue functional upstream domain (FUD) of Streptococcus pyogenes F1 adhesin interacts with fibronectin (FN) in a heretofore unknown manner that prevents assembly of a FN matrix. Biotinylated FUD (b-FUD) bound to adsorbed FN or its recombinant N-terminal 70-kDa fibrin- and gelatin-binding fragment (70K). Binding was blocked by FN or 70K, but not by fibrin- or gelatin-binding subfragments of 70K. Isothermal titration calorimetry showed that FUD binds with K(d) values of 5.2 and 59 nM to soluble 70K and FN, respectively. We tested sets of FUD mutants and epitope-mapped monoclonal antibodies (mAbs) for ability to compete with b-FUD for binding to FN or to block FN assembly by cultured fibroblasts. Deletions or alanine substitutions throughout FUD caused loss of both activities. mAb 4D1 to the (2)FNI module had little effect, whereas mAb 7D5 to the (4)FNI module in the fibrin-binding region, 5C3 to the (9)FNI module in the gelatin-binding region, or L8 to the G-strand of (1)FNIII module adjacent to (9)FNI caused loss of binding of b-FUD to FN and decreased FN assembly. Conversely, FUD blocked binding of 7D5, 5C3, or L8, but not of 4D1, to FN. Circular dichroism indicated that FUD binds to 70K by ß-strand addition, a possibility supported by modeling based on crystal structures of peptides bound to (2)FNI-(5)FNI of the fibrin-binding domain and (8)FNI-(9)FNI of the gelatin-binding domain. Thus, the interaction likely involves an extensive anti-parallel ß-zipper in which FUD interacts with the E-strands of (2)FNI-(5)FNI and (8)FNI-(9)FNI.