A Novel Signal Transduction Protein PII Variant from Synechococcus elongatus PCC 7942 Indicates a Two-Step Process for NAGK-PII Complex Formation

P_II signal transduction proteins are highly conserved in bacteria, archaea and plants and have key functions in coordination of central metabolism by integrating signals from the carbon, nitrogen and energy status of the cell. In the cyanobacterium Synechococcus elongatus PCC 7942, P_II binds ATP and 2-oxoglutarate (2-OG) in a synergistic manner, with the ATP binding sites also accepting ADP. Depending on its effector molecule binding status, P_II (from this cyanobacterium and other oxygenic phototrophs) complexes and regulates the arginine-controlled enzyme of the cyclic ornithine pathway, N-acetyl-l-glutamate kinase (NAGK), to control arginine biosynthesis. To gain deeper insights into the process of P_II binding to NAGK, we searched for P_II variants with altered binding characteristics and found P_II variants I86N and I86T to be able to bind to an NAGK variant (R233A) that was previously shown to be unable to bind wild-type P_II protein. Analysis of interactions between these P_II variants and wild-type NAGK as well as with the NAGK R233A variant suggested that the P_II I86N variant was a superactive NAGK binder. To reveal the structural basis of this property, we solved the crystal structure of the P_II I86N variant at atomic resolution. The large T-loop, which prevails in most receptor interactions of P_II proteins, is present in a tightly bended conformation that mimics the T-loop of S. elongatus P_II after having latched onto NAGK. Moreover, both P_II I86 variants display a specific defect in 2-OG binding, implying a role of residue I86 in 2-OG binding. We propose a two-step model for the mechanism of P_II-NAGK complex formation: in an initiating step, a contact between R233 of NAGK and E85 of P_II initiates the bending of the extended T-loop of P_II, followed by a second step, where a bended T-loop deeply inserts into the NAGK clefts to form the tight complex.     

Cooperative Binding of MgATP and MgADP in the Trimeric PII Protein GlnK2 from Archaeoglobus fulgidus

P_II-like proteins, such as GlnK, found in a wide variety of organisms from prokaryotes to plants constitute a family of cytoplasmic signaling proteins that play a central regulatory role in the assimilation of nitrogen for biosyntheses. They specifically bind and are modulated by effector molecules such as adenosine triphosphate, adenosine diphosphate and 2-oxoglutarate. Their highly conserved, trimeric structure suggests that cooperativity in effector binding might be the basis for the ability to integrate and respond to a wide range of concentrations, but to date no direct quantification of this cooperative behavior has been presented. The hyperthermophilic archaeon Archaeoglobus fulgidus contains three GlnK proteins, functionally associated with ammonium transport proteins (Amt). We have characterized GlnK2 and its interaction with effectors by high-resolution X-ray crystallography and isothermal titration calorimetry. Binding of adenosine nucleotides resulted in distinct, cooperative behavior for ATP and ADP. While 2-oxoglutarate has been shown to interact with other GlnK proteins, GlnK2 was completely insensitive to this key indicator of a low level of intracellular nitrogen. These findings point to different regulation and modulation patterns and add to our understanding of the flexibility and versatility of the GlnK family of signaling proteins.     

Differential regulation of hexameric and dodecameric hemocyanin from A. leptodactylus

The oxygen binding properties of hemocyanins are regulated on a short time scale by effectors such as l-lactate, urate and protons, and on longer time scales by expression of the different types of subunits. For Astacus leptodactylus it was shown previously that acclimation to higher temperatures leads to increased levels of a 6-meric hemocyanin species, whereas at lower temperatures the 12-meric form prevails. Here we show that the temperature dependence of the two forms supports the idea, that the maintenance of high affinity towards oxygen is the driving force for the differential expression of these hemocyanins. Furthermore, the two different types of hemocyanin differ not only in the affinity to oxygen, but also with respect to their interaction with l-lactate: while the 12-meric form displays a normal shift in oxygen affinity upon the addition of l-lactate this allosteric regulation is absent in the 6-meric form. Exclusive binding of l-lactate to the 12-meric form was supported by isothermal titration calorimetry. These results indicate that l-lactate binds either at the interface between the two hexamers or at subunit α′ which is responsible for the formation of the 12-mers and is not present in the 6-meric form. Urate has a comparable effect on the oxygen affinity of 6-meric and 12-meric forms and also binds to a similar extent to the oxygenated state as determined by isothermal titration calorimetry. Thus, urate and l-lactate do not seem to share the same binding sites. Interestingly, urate binding sites with no allosteric effect seem to exist, which is unusual. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.     

Structural Basis of Substrate Binding in WsaF, a Rhamnosyltransferase from Geobacillus stearothermophilus

Carbohydrate polymers are medically and industrially important. The S-layer of many Gram-positive organisms comprises protein and carbohydrate polymers and forms an almost paracrystalline array on the cell surface. Not only is this array important for the bacteria but it has potential application in the manufacture of commercially important polysaccharides and glycoconjugates as well. The S-layer glycoprotein glycan from Geobacillus stearothermophilus NRS 2004/3a is mainly composed of repeating units of three rhamnose sugars linked by α-1,3-, α-1,2-, and β-1,2-linkages. The formation of the β-1,2-linkage is catalysed by the enzyme WsaF. The rational use of this system is hampered by the fact that WsaF and other enzymes in the pathway share very little homology to other enzymes. We report the structural and biochemical characterisation of WsaF, the first such rhamnosyltransferase to be characterised. Structural work was aided by the surface entropy reduction method. The enzyme has two domains, the N-terminal domain, which binds the acceptor (the growing rhamnan chain), and the C-terminal domain, which binds the substrate (dTDP-β-l-rhamnose). The structure of WsaF bound to dTDP and dTDP-β-l-rhamnose coupled to biochemical analysis identifies the residues that underlie catalysis and substrate recognition. We have constructed and tested by site-directed mutagenesis a model for acceptor recognition.     

Binding Kinetics of Histone Chaperone Chz1 and Variant Histone H2A.Z-H2B by Relaxation Dispersion NMR Spectroscopy

The genome of eukaryotic cells is packed into a compact structure called chromatin that consists of DNA as well as histone and non-histone proteins. Histone chaperones associate with histone proteins and play important roles in the assembly of chromatin structure and transport of histones in the cell. The recently discovered histone chaperone Chz1 associates with the variant histone H2A.Z of budding yeast and plays a critical role in the exchange of the canonical histone pair H2A-H2B for the variant H2A.Z-H2B. Here, we present an NMR approach that provides accurate estimates for the rates of association and dissociation of Chz1 and H2A.Z-H2B. The methodology exploits the fact that in a 1:1 mixture of Chz1 and H2A.Z-H2B, the small amounts of unbound proteins that are invisible in spectra produce line broadening of signals from the complex that can be quantified in terms of the thermodynamics and kinetics of the exchange process. The dissociation rate constant measured, 22 ± 2 s^− 1, provides an upper bound for the rate of transfer of H2A.Z-H2B to the chromatin remodeling complex, and the faster-than-diffusion association rate, 10⁸ ± 10⁷ M^− 1 s^− 1, establishes the importance of attractive electrostatic interactions that form the chaperone-histone complex.     

Structural and binding studies of the three-metal center in two mycobacterial PPM Ser/Thr protein phosphatases

Phospho-Ser/Thr protein phosphatases (PPs) are dinuclear metalloenzymes classed into two large families, PPP and PPM, on the basis of sequence similarity and metal ion dependence. The archetype of the PPM family is the α isoform of human PP2C (PP2Cα), which folds into an α/β domain similar to those of PPP enzymes. The recent structural studies of three bacterial PPM phosphatases, Mycobacterium tuberculosis MtPstP, Mycobacterium smegmatis MspP, and Streptococcus agalactiae STP, confirmed the conservation of the overall fold and dinuclear metal center in the family, but surprisingly revealed the presence of a third conserved metal-binding site in the active site. To gain insight into the roles of the three-metal center in bacterial enzymes, we report structural and metal-binding studies of MtPstP and MspP. The structure of MtPstP in a new trigonal crystal form revealed a fully active enzyme with the canonical dinuclear metal center but without the third metal ion bound to the catalytic site. The absence of metal correlates with a partially unstructured flap segment, indicating that the third manganese ion contributes to reposition the flap, but is dispensable for catalysis. Studies of metal binding to MspP using isothermal titration calorimetry revealed that the three Mn²⁺-binding sites display distinct affinities, with dissociation constants in the nano- and micromolar range for the two catalytic metal ions and a significantly lower affinity for the third metal-binding site. In agreement, the structure of inactive MspP at acidic pH was determined at atomic resolution and shown to lack the third metal ion in the active site. Structural comparisons of all bacterial phosphatases revealed positional variations in the third metal-binding site that are correlated with the presence of bound substrate and the conformation of the flap segment, supporting a role of this metal ion in assisting enzyme-substrate interactions.     

Binding the Mammalian High Mobility Group Protein AT-hook 2 to AT-Rich Deoxyoligonucleotides: Enthalpy-Entropy Compensation

HMGA2 is a DNA minor-groove binding protein. We previously demonstrated that HMGA2 binds to AT-rich DNA with very high binding affinity where the binding of HMGA2 to poly(dA-dT)₂ is enthalpy-driven and to poly(dA)poly(dT) is entropy-driven. This is a typical example of enthalpy-entropy compensation. To further study enthalpy-entropy compensation of HMGA2, we used isothermal-titration-calorimetry to examine the interactions of HMGA2 with two AT-rich DNA hairpins: 5′-CCAAAAAAAAAAAAAAAGCCCCCGCTTTTTTTTTTTTTTTGG-3′ (FL-AT-1) and 5′-CCATATATATATATATAGCCCCCGCTATATATATATATATGG-3′ (FL-AT-2). Surprisingly, we observed an atypical isothermal-titration-calorimetry-binding curve at low-salt aqueous solutions whereby the apparent binding-enthalpy decreased dramatically as the titration approached the end. This unusual behavior can be attributed to the DNA-annealing coupled to the ligand DNA-binding and is eliminated by increasing the salt concentration to ∼200 mM. At this condition, HMGA2 binding to FL-AT-1 is entropy-driven and to FL-AT-2 is enthalpy-driven. Interestingly, the DNA-binding free energies for HMGA2 binding to both hairpins are almost temperature independent; however, the enthalpy-entropy changes are dependent on temperature, which is another aspect of enthalpy-entropy compensation. The heat capacity change for HMGA2 binding to FL-AT-1 and FL-AT-2 are almost identical, indicating that the solvent displacement and charge-charge interaction in the coupled folding/binding processes for both binding reactions are similar.     

Structure of PP4397 Reveals the Molecular Basis for Different c-di-GMP Binding Modes by Pilz Domain Proteins

Cyclic diguanylate (c-di-GMP) is a global regulator that modulates pathogen virulence and biofilm formation in bacteria. Although a bioinformatic study revealed that PilZ domain proteins are the long-sought c-di-GMP binding proteins, the mechanism by which c-di-GMP regulates them is uncertain. Pseudomonas putida PP4397 is one such protein that contains YcgR-N and PilZ domains and the apo-PP4397 structure was solved earlier by the Joint Center for Structural Genomics. We determined the crystal structure of holo-PP4397 and found that two intercalated c-di-GMPs fit into the junction of its YcgR-N and PilZ domains. Moreover, c-di-GMP binding induces PP4397 to undergo a dimer-to-monomer transition. Interestingly, another PilZ domain protein, VCA0042, binds to a single molecule of c-di-GMP, and both its apo and holo forms are dimeric. Mutational studies and the additional crystal structure of holo-VCA0042 (L135R) showed that the Arg122 residue of PP4397 is crucial for the recognition of two molecules of c-di-GMP. Thus, PilZ domain proteins exhibit different c-di-GMP binding stoichiometry and quaternary structure, and these differences are expected to play a role in generating diverse forms of c-di-GMP-mediated regulation.     

Thermodynamics of GTP and GDP Binding to Bacterial Initiation Factor 2 Suggests Two Types of Structural Transitions

During initiation of messenger RNA translation in bacteria, the GTPase initiation factor (IF) 2 plays major roles in the assembly of the preinitiation 30S complex and its docking to the 50S ribosomal subunit leading to the 70S initiation complex, ready to form the first peptide bond in a nascent protein. Rapid and accurate initiation of bacterial protein synthesis is driven by conformational changes in IF2, induced by GDP-GTP exchange and GTP hydrolysis. We have used isothermal titration calorimetry and linear extrapolation to characterize the thermodynamics of the binding of GDP and GTP to free IF2 in the temperature interval 4-37 °C. IF2 binds with about 20-fold and 2-fold higher affinity for GDP than for GTP at 4 and 37 °C, respectively. The binding of IF2 to both GTP and GDP is characterized by a large heat capacity change (− 868 ± 25 and − 577 ± 23 cal mol^− 1 K^− 1, respectively), associated with compensatory changes in binding entropy and enthalpy. From our data, we propose that GTP binding to IF2 leads to protection of hydrophobic amino acid residues from solvent by the locking of switch I and switch II loops to the γ-phosphate of GTP, as in the case of elongation factor G. From the large heat capacity change (also upon GDP binding) not seen in the case of elongation factor G, we propose the existence of yet another type of conformational change in IF2, which is induced by GDP and GTP alike. Also, this transition is likely to protect hydrophobic groups from solvent, and its functional relevance is discussed.     

Thermodynamics and folding pathway of tetraloop receptor-mediated RNA helical packing

Little is known about the thermodynamic forces that drive the folding pathways of higher-order RNA structure. In this study, we employ calorimetric [isothermal titration calorimetry (ITC) and differential scanning calorimetry (DSC)] and spectroscopic (NMR and UV) methods to characterize the thermodynamics of the GAAA tetraloop-receptor interaction, utilizing a previously described bivalent construct. ITC studies indicate that the bivalent interaction is enthalpy driven and highly stable, with a binding constant (K(obs)) of 5.5x10(6) M(-1) and enthalpy (DeltaH(obs)(o)) of -33.8 kcal/mol at 45 degrees C in 20 mM KCl and 2 mM MgCl(2). Thus, we derive the DeltaH(obs)(o) for a single tetraloop-receptor interaction to be -16.9 kcal/mol at these conditions. UV absorbance data indicate that an increase in base stacking quality contributes to the enthalpy of complex formation. These highly favorable thermodynamics are consistent with the known critical role for the tetraloop-receptor motif in the folding of large RNAs. Additionally, a significant heat capacity change (DeltaC(p,obs)(o)) of -0.24 kcal mol(-1) K(-1) was determined by ITC. DSC and UV-monitored thermal denaturation experiments indicate that the bivalent tetraloop-receptor construct follows a minimally five-state unfolding pathway and suggest the observed DeltaC(p,obs)(o) for the interaction results from a temperature-dependent unbound receptor RNA structure.     

More than a Simple Lipophilic Contact: A Detailed Thermodynamic Analysis of Nonbasic Residues in the S1 Pocket of Thrombin

The field of medicinal chemistry aims to design and optimize small molecule leads into drug candidates that may positively interfere with pathological disease situations in humans or combat the growth of infective pathogens. From the plethora of crystal structures of protein-inhibitor complexes we have learned how molecules recognize each other geometrically, but we still have rather superficial understanding of why they bind to each other. This contribution surveys a series of 26 thrombin inhibitors with small systematic structural differences to elucidate the rationale for their widely deviating binding affinity from 185 μM to 4 nM as recorded by enzyme kinetic measurements. Five well-resolved (resolution 2.30 - 1.47 Å) crystal structures of thrombin-inhibitor complexes and an apo-structure of the uncomplexed enzyme (1.50 Å) are correlated with thermodynamic data recorded by isothermal titration calorimetry with 12 selected inhibitors from the series. Taking solubility data into account, the variation in physicochemical properties allows conclusions to be reached about the relative importance of the enthalpic binding features as well as to estimate the importance of the parameters more difficult to capture, such as residual ligand entropy and desolvation properties. The collected data reveal a comprehensive picture of the thermodynamic signature that explains the so far poorly understood attractive force experienced by m-chloro-benzylamides to thrombin.     

The Outer Membrane Usher Guarantees the Formation of Functional Pili by Selectively Catalyzing Donor-Strand Exchange between Subunits That Are Adjacent in the Mature Pilus

Type 1 pili from uropathogenic Escherichia coli are a prototype of adhesive surface organelles assembled and secreted by the conserved chaperone/usher pathway. They are composed of four different homologous protein subunits that need to be assembled in a defined order. In the periplasm, the pilus chaperone FimC donates a β-strand segment to the subunits to complete their imperfect immunoglobulin-like fold. During subunit assembly, this segment of the chaperone is displaced by an amino-terminal extension of an incoming subunit in a reaction termed donor-strand exchange. To date, the molecular mechanisms underlying the coordinated subunit assembly, in particular the role of the outer membrane usher FimD, are still poorly understood. Here we show that the binding of complexes between FimC and the different pilus subunits to the amino-terminal substrate recognition domain of FimD is an extremely fast process, with association rate constants in the range of 10⁷-10⁸ M^− 1 s^− 1 at 20 °C. Furthermore, we demonstrate that the ordered assembly of pilus subunits is a consequence of the usher's ability to selectively catalyze the assembly of defined subunit-subunit pairs that are adjacent in the mature pilus. The usher therefore coordinates the assembly of pilus subunits at the stage of donor-strand exchange between pairs of subunits and not at the level of the initial binding of chaperone-subunit complexes.     

Novel Insights into the Mechanisms of CIN85 SH3 Domains Binding to Cbl Proteins: Solution-Based Investigations and In Vivo Implications

CIN85 is a multifunctional protein that plays key roles in endocytic down-regulation of receptor tyrosine kinases, apoptosis, cell adhesion, and cytoskeleton rearrangement. Its three SH3 domains (CIN85A, CIN85B, and CIN85C) allow it to recruit multiple binding partners. To understand the manifold interactions of CIN85, we present a detailed high-resolution solution structural study of CIN85A and CIN85B binding to proline-arginine peptides derived from the cognate ligands Cbl and Cbl-b. We report the structure of CIN85B and provide evidence that both CIN85A and CIN85B, in isolation or when linked, form heterodimeric complexes with the peptides. We report unusual curved chemical shift changes for several residues of CIN85A when titrated with Cbl-b peptide, indicating the existence of more than one complex form. Here we demonstrate that CIN85A and CIN85B use different mechanisms for peptide binding.     

Thermodynamic Characterization of ppGpp Binding to EF-G or IF2 and of Initiator tRNA Binding to Free IF2 in the Presence of GDP, GTP, or ppGpp

In addition to their natural substrates GDP and GTP, the bacterial translational GTPases initiation factor (IF) 2 and elongation factor G (EF-G) interact with the alarmone molecule guanosine tetraphosphate (ppGpp), which leads to GTPase inhibition. We have used isothermal titration calorimetry to determine the affinities of ppGpp for IF2 and EF-G at a temperature interval of 5-25 °C. We find that ppGpp has a higher affinity for IF2 than for EF-G (1.7-2.8 μM K_dversus 9.1-13.9 μM K_d at 10-25 °C), suggesting that during stringent response in vivo, IF2 is more responsive to ppGpp than to EF-G. We investigated the effects of ppGpp, GDP, and GTP on IF2 interactions with fMet-tRNA^fMet demonstrating that IF2 binds to initiator tRNA with submicromolar K_d and that affinity is altered by the G nucleotides only slightly. This—in conjunction with earlier reports on IF2 interactions with fMet-tRNA^fMet in the context of the 30S initiation complex, where ppGpp was suggested to strongly inhibit fMet-tRNA^fMet binding and GTP was suggested to strongly promote fMet-tRNA^fMet binding—sheds new light on the mechanisms of the G-nucleotide-regulated fMet-tRNA^fMet selection.     

Unusual Diheme Conformation of the Heme-Degrading Protein from Mycobacterium tuberculosis

Heme degradation plays a pivotal role in the availability of the essential nutrient, iron, in pathogenic bacteria. A previously unannotated protein from Mycobacterium tuberculosis, Rv3592, which shares homology to heme-degrading enzymes, has been identified. Biochemical analyses confirm that Rv3592, which we have termed MhuD (mycobacterial heme utilization, degrader), is able to bind and degrade heme. Interestingly, contrary to previously reported stoichiometry for the Staphylococcus aureus heme degraders, iron-regulated surface determinant (Isd)G and IsdI, MhuD has the ability to bind heme in a 1:2 protein-to-heme ratio, although the MhuD-diheme complex is inactive. Furthermore, the 1.75-Å crystal structure of the MhuD-diheme complex reveals two stacked hemes forming extensive contacts with residues in the active site. In particular, the solvent-exposed heme is axially liganded by His75 and is stacked planar upon the solvent-protected heme. The solvent-protected heme is coordinated by a chloride ion, which is, in turn, stabilized by Asn7. Structural comparison between MhuD-diheme and inactive IsdG and IsdI bound to only one highly distorted metalloporphyrin ring reveals that several residues located in α-helix 2 and the subsequent loop appear to be responsible for heme stoichiometric differences and suggest open and closed conformations for substrate entry and product exit.     

Regulation of Structural Dynamics within a Signal Recognition Particle Promotes Binding of Protein Targeting Substrates

Protein targeting is critical in all living organisms and involves a signal recognition particle (SRP), an SRP receptor, and a translocase. In co-translational targeting, interactions among these proteins are mediated by the ribosome. In chloroplasts, the light-harvesting chlorophyll-binding protein (LHCP) in the thylakoid membrane is targeted post-translationally without a ribosome. A multidomain chloroplast-specific subunit of the SRP, cpSRP43, is proposed to take on the role of coordinating the sequence of targeting events. Here, we demonstrate that cpSRP43 exhibits significant interdomain dynamics that are reduced upon binding its SRP binding partner, cpSRP54. We showed that the affinity of cpSRP43 for the binding motif of LHCP (L18) increases when cpSRP43 is complexed to the binding motif of cpSRP54 (cpSRP54_pep). These results support the conclusion that substrate binding to the chloroplast SRP is modulated by protein structural dynamics in which a major role of cpSRP54 is to improve substrate binding efficiency to the cpSRP.     

Binding thermodynamics of paromomycin,neomycin, neomycin‐dinucleotide and ‐diPNA conjugates to bacterial and human rRNA

Isothermal titration calorimetry (ITC) is a powerful technique able to evaluate the energetics of target‐drug binding within the context of drug discovery. In this work, the interactions of RNAs reproducing bacterial and human ribosomal A‐site, with two well‐known antibiotic aminoglycosides, Paromomycin and Neomycin, as well as several Neomycin‐dinucleotide and ‐diPNA conjugates, have been evaluated by ITC and the corresponding thermodynamic quantities determined. The comparison of the thermodynamic data of aminoglycosides and their chemical analogues allowed to select Neomycin‐diPNA conjugates as the best candidates for antimicrobial activity. Copyright © 2015 John Wiley & Sons, Ltd.