Charged Pocket Research Articles

Essential Roles for GSK-3s and GSK-3-Primed Substrates in Neurotrophin-Induced and Hippocampal Axon Growth

Glycogen synthase kinase-3beta (GSK-3beta) is thought to mediate morphological responses to a variety of extracellular signals. Surprisingly, we found no gross morphological deficits in nervous system development in GSK-3beta null mice. We therefore designed an shRNA that targeted both GSK-3 isoforms. Strong knockdown of both GSK-3alpha and beta markedly reduced axon growth in dissociated cultures and slice preparations. We then assessed the role of different GSK-3 substrates in regulating axon morphology. Elimination of activity toward primed substrates only using the GSK-3 R96A mutant was associated with a defect in axon polarity (axon branching) compared to an overall reduction in axon growth induced by a kinase-dead mutant. Consistent with this finding, moderate reduction of GSK-3 activity by pharmacological inhibitors induced axon branching and was associated primarily with effects on primed substrates. Our results suggest that GSK-3 is a downstream convergent point for many axon growth regulatory pathways and that differential regulation of primed versus all GSK-3 substrates is associated with a specific morphological outcome.

First Structure of a Eukaryotic Phosphohistidine Phosphatase

Phosphatases are a diverse group of enzymes that regulate numerous cellular processes. Much of what is known relates to the tyrosine, threonine, and serine phosphatases, whereas the histidine phosphatases have not been studied as much. The structure of phosphohistidine phosphatase (PHPT1), the first identified eukaryotic-protein histidine phosphatase, has been determined to a resolution of 1.9A using multiple-wavelength anomalous dispersion methods. This enzyme can dephosphorylate a variety of proteins (e.g. ATP-citrate lyase and the beta-subunit of G proteins). A putative active site has been identified by its electrostatic character, ion binding, and conserved protein residues. Histidine 53 is proposed to play a major role in histidine dephosphorylation based on these observations and previous mutational studies. Models of peptide binding are discussed to suggest possible mechanisms for substrate recognition.

Kinetic Effect of a Downstream Strand and Its 5′-Terminal Moieties on Single Nucleotide Gap-filling Synthesis Catalyzed by Human DNA Polymerase λ

During short-patch base excision repair, the excision of a 5'-terminal 2-deoxyribose-5-phosphate moiety of the downstream strand by the 5'-2-deoxyribose-5-phosphate lyase activity of either DNA polymerase beta or lambda is believed to occur after each respective enzyme catalyzes gap-filling DNA synthesis. Yet the effects of this 5'-terminal 2-deoxyribose-5-phosphate moiety on the polymerase activities of these two enzymes have never been quantitatively determined. Moreover, x-ray crystal structures of truncated polymerase lambda have revealed that the downstream strand and its 5'-phosphate group of gapped DNA interact intensely with the dRPase domain, but the kinetic effect of these interactions is unclear. Here, we utilized pre-steady state kinetic methods to systematically investigate the effect of a downstream strand and its 5'-moieties on the polymerase activity of the full-length human polymerase lambda. The downstream strand and its 5'-phosphate were both found to increase nucleotide incorporation efficiency (kp/Kd) by 15 and 11-fold, respectively, with the increase procured by the effect on the nucleotide incorporation rate constant kp rather than the ground state nucleotide binding affinity Kd. With 4 single nucleotide-gapped DNA substrates containing a 1,2-dideoxyribose-5-phosphate moiety, a 2-deoxyribose-5-phosphate mimic, we measured the incorporation efficiencies of 16 possible nucleotides. Our results demonstrate that although this 5'-terminal 2-deoxyribose-5-phosphate mimic does not affect the fidelity of polymerase lambda, it moderately decreased the polymerase efficiency by 3.4-fold. Moreover, this decrease in polymerase efficiency is due to a drop of similar magnitude in kp rather than Kd. The implication of the downstream strand and its 5'-moieties on the kinetics of gap-filling synthesis is discussed.

A Highly Specific Mechanism of Histone H3-K4 Recognition by Histone Demethylase LSD1

Human lysine-specific demethylase (LSD1) is a chromatin-modifying enzyme that specifically removes methyl groups from mono- and dimethylated Lys4 of histone H3 (H3-K4). We used a combination of in vivo and in vitro experiments to characterize the substrate specificity and recognition by LSD1. Biochemical assays on histone peptides show that essentially all epigenetic modifications on the 21 N-terminal amino acids of histone H3 cause a significant reduction in enzymatic activity. Replacement of Lys4 with Arg greatly enhances binding affinity, and a histone peptide incorporating this mutation has a strong inhibitory power. Conversely, a peptide bearing a trimethylated Lys4 is only a weak inhibitor of the enzyme. Rapid kinetics measurements evidence that the enzyme is efficiently reoxidized by molecular oxygen with a second-order rate constant of 9.6x10(3) M-1 s-1, and that the presence of the reaction product does not greatly influence the rate of flavin reoxidation. In vivo experiments provide a correlation between the in vitro inhibitory properties of the tested peptides and their ability of affecting endogenous LSD1 activity. Our results show that epigenetic modifications on histone H3 need to be removed before Lys4 demethylation can efficiently occur. The complex formed by LSD1 with histone deacetylases 1/2 may function as a "double-blade razor" that first eliminates the acetyl groups from acetylated Lys residues and then removes the methyl group from Lys4. We suggest that after H3-K4 demethylation, LSD1 recruits the forthcoming chromatin remodelers leading to the introduction of gene repression marks.

Crystal structure of the catalytic domain of human DUSP5, a dual specificity MAP kinase protein phosphatase

Crystal structure of the catalytic domain of human DUSP5, a dual specificity MAP kinase protein phosphatase

Trapping excess electrons in molecular charge pockets on surfaces

This work considers the use of theoretical ab initio calculations to design a novel molecular trap for excess electrons. The basic principle is to develop a model by which these electrons can be stabilized by charge pockets on the surface of molecules. Calculations reveal that systems with OH groups can form stable hydrogen-bonded networks on one side of a hydrocarbon surface (i.e. cyclohexane sheets), while the hydrogen atoms on the opposite side of this surface form a pocket of positive charge that attracts the extra negative charge.

Structural Basis for CoREST-Dependent Demethylation of Nucleosomes by the Human LSD1 Histone Demethylase

Histone methylation regulates diverse chromatin-templated processes, including transcription. Many transcriptional corepressor complexes contain lysine-specific demethylase 1 (LSD1) and CoREST that collaborate to demethylate mono- and dimethylated H3-K4 of nucleosomes. Here, we report the crystal structure of the LSD1-CoREST complex. LSD1-CoREST forms an elongated structure with a long stalk connecting the catalytic domain of LSD1 and the CoREST SANT2 domain. LSD1 recognizes a large segment of the H3 tail through a deep, negatively charged pocket at the active site and possibly a shallow groove on its surface. CoREST SANT2 interacts with DNA. Disruption of the SANT2-DNA interaction diminishes CoREST-dependent demethylation of nucleosomes by LSD1. The shape and dimension of LSD1-CoREST suggest its bivalent binding to nucleosomes, allowing efficient H3-K4 demethylation. This spatially separated, multivalent nucleosome binding mode may apply to other chromatin-modifying enzymes that generally contain multiple nucleosome binding modules.

The Crystal Structure of the Transthyretin-like Protein from Salmonella dublin, a Prokaryote 5-Hydroxyisourate Hydrolase

The mechanism of binding of thyroid hormones by the transport protein transthyretin (TTR) in vertebrates is structurally well characterised. However, a homologous family of transthyretin-like proteins (TLPs) present in bacteria as well as eukaryotes do not bind thyroid hormones, instead they are postulated to perform a role in the purine degradation pathway and function as 5-hydroxyisourate hydrolases. Here we describe the 2.5 Å X-ray crystal structure of the TLP from the Gram-negative bacterium Salmonella dublin, and compare and contrast its structure with vertebrate TTRs. The overall architecture of the homotetramer is conserved and, despite low sequence homology with vertebrate TTRs, structural differences within the monomer are restricted to flexible loop regions. However, sequence variation at the dimer–dimer interface has profound consequences for the ligand binding site and provides a structural rationalisation for the absence of thyroid hormone binding affinity in bacterial TLPs: the deep, negatively charged thyroxine-binding pocket that characterises vertebrate TTR contrasts with a shallow and elongated, positively charged cleft in S. dublin TLP. We have demonstrated that Sdu_TLP is a 5-hydroxyisourate hydrolase. Furthermore, using site-directed mutagenesis, we have identified three conserved residues located in this cleft that are critical to the enzyme activity. Together our data reveal that the active site of Sdu_TLP corresponds to the thyroxine binding site in TTRs.

Structure-Function Relationships of Glucansucrase and Fructansucrase Enzymes from Lactic Acid Bacteria

Lactic acid bacteria (LAB) employ sucrase-type enzymes to convert sucrose into homopolysaccharides consisting of either glucosyl units (glucans) or fructosyl units (fructans). The enzymes involved are labeled glucansucrases (GS) and fructansucrases (FS), respectively. The available molecular, biochemical, and structural information on sucrase genes and enzymes from various LAB and their fructan and alpha-glucan products is reviewed. The GS and FS enzymes are both glycoside hydrolase enzymes that act on the same substrate (sucrose) and catalyze (retaining) transglycosylation reactions that result in polysaccharide formation, but they possess completely different protein structures. GS enzymes (family GH70) are large multidomain proteins that occur exclusively in LAB. Their catalytic domain displays clear secondary-structure similarity with alpha-amylase enzymes (family GH13), with a predicted permuted (beta/alpha)(8) barrel structure for which detailed structural and mechanistic information is available. Emphasis now is on identification of residues and regions important for GS enzyme activity and product specificity (synthesis of alpha-glucans differing in glycosidic linkage type, degree and type of branching, glucan molecular mass, and solubility). FS enzymes (family GH68) occur in both gram-negative and gram-positive bacteria and synthesize beta-fructan polymers with either beta-(2-->6) (inulin) or beta-(2-->1) (levan) glycosidic bonds. Recently, the first high-resolution three-dimensional structures have become available for FS (levansucrase) proteins, revealing a rare five-bladed beta-propeller structure with a deep, negatively charged central pocket. Although these structures have provided detailed mechanistic insights, the structural features in FS enzymes dictating the synthesis of either beta-(2-->6) or beta-(2-->1) linkages, degree and type of branching, and fructan molecular mass remain to be identified.

A structural basis for selection and cross-species reactivity of the semi-invariant NKT cell receptor in CD1d/glycolipid recognition.

Little is known regarding the basis for selection of the semi-invariant αβ T cell receptor (TCR) expressed by natural killer T (NKT) cells or how this mediates recognition of CD1d–glycolipid complexes. We have determined the structures of two human NKT TCRs that differ in their CDR3β composition and length. Both TCRs contain a conserved, positively charged pocket at the ligand interface that is lined by residues from the invariant TCR α- and semi-invariant β-chains. The cavity is centrally located and ideally suited to interact with the exposed glycosyl head group of glycolipid antigens. Sequences common to mouse and human invariant NKT TCRs reveal a contiguous conserved “hot spot” that provides a basis for the reactivity of NKT cells across species. Structural and functional data suggest that the CDR3β loop provides a plasticity mechanism that accommodates recognition of a variety of glycolipid antigens presented by CD1d. We propose a model of NKT TCR–CD1d–glycolipid interaction in which the invariant CDR3α loop is predicted to play a major role in determining the inherent bias toward CD1d. The findings define a structural basis for the selection of the semi-invariant αβ TCR and the unique antigen specificity of NKT cells.

Predictions of Charge Drift in a Concept Electrosprayed DISI Engine

Limited to nonvaporizing spray cases, this work discusses the transport of charged droplets within a cylinder of a motored axisymmetric model electrosprayed direct injection spark ignition (eDISI) engine with electrified walls. The concept engine investigated here is assumed to operate with an electrostatic atomizer previously studied for application in fuel burners [Yule et al., 1994, Fuel, 74(7), pp. 1094–1103]. A split/multiple injection strategy is employed in which three pulses of 5mg each are made at crank angles of 80, 150 and 300 deg ATDC of the intake, which fall within the intervals for stable combustion of either early or late injection modes of operation of DISI engines [Jackson et al., 1997, SAE Paper No. 970543]. The direct Simulation Monte Carlo (DSMC) approach embodied in an in-house CFD research code is used to simulate the discrete phase flow with the electrical charge distribution for different instants within the computational cells being computed by simple addition of the droplet charges residing in particular cells at particular instants of time. It is shown in the half engine cycle investigated that the use of charged sprays in eDISI engines may help to reduce the in-cylinder wall-wetting phenomenon. In addition, pockets of highest electrical charge are found to populate the region near the spark plug by 345 deg CA, which may be a path for improved combustion efficiency.

Structural Analysis of Divalent Metals Binding to the Bacillus subtilis Response Regulator Spo0F: The Possibility for In Vitro Metalloregulation in the Initiation of Sporulation

The presence of a divalent metal ion in a negatively charged aspartic acid pocket is essential for phosphorylation of response regulator proteins. Here, we present metal binding studies of the Bacillus subtilis response regulator Spo0F using NMR and microESI-MS. NMR studies show that the divalent metals Ca(2+), Mg(2+) and Mn(2+) primarily bind, as expected, in the Asp pocket phosphorylation site. However, identical studies with Cu(2+) show distinct binding effects in three specific locations: (i) the Asp pocket, (ii) a grouping of charged residues at a site opposite of the Asp pocket, and (iii) on the beta 4-alpha 4 loop and the beta 5/alpha 5 interface, particularly around and including H101. microESI-MS studies stoichiometrically confirm the NMR studies and demonstrate that most divalent metal ions bind to Spo0F primarily in a 1:1 ratio. Again, in the case of Cu(2+), multiple metal-bound species are observed. Subsequent experiments reveal that Mg(2+) supports phosphotransfer between KinA and Spo0F, while Cu(2+) fails to support KinA phosphotransfer. Additionally, the presence of Cu(2+) at non-lethal concentrations in sporulation media for B. subtilis and the related organism Pasteuria penetrans was found to inhibit spore formation while continuing to permit vegetative growth. Depending on the type of divalent metal ion present, in vitro phosphorylation of Spo0F by its cognate kinase KinA can be inhibited.

The Integrity of the Charged Pocket in the BTB/POZ Domain Is Essential for the Phenotype Induced by the Leukemia-Associated t(11;17) Fusion Protein PLZF/RARα

Acute myeloid leukemia is characterized by a differentiation block as well as by an increased self-renewal of hematopoietic precursors in the bone marrow. This phenotype is induced by specific acute myeloid leukemia-associated translocations, such as t(15;17) and t(11;17), which involve an identical portion of the retinoic acid receptor alpha (RARalpha) and either the promyelocytic leukemia (PML) or promyelocytic zinc finger (PLZF) genes, respectively. The resulting fusion proteins form high molecular weight complexes and aberrantly bind several histone deacetylase-recruiting nuclear corepressor complexes. The amino-terminal BTB/POZ domain is indispensable for the capacity of PLZF to form high molecular weight complexes. Here, we studied the role of dimerization and binding to histone deacetylase-recruiting nuclear corepressor complexes for the induction of the leukemic phenotype by PLZF/RARalpha and we show that (a) the BTB/POZ domain mediates the oligomerization of PLZF/RARalpha; (b) mutations that inhibit dimerization of PLZF do the same in PLZF/RARalpha; (c) the PLZF/RARalpha-related block of differentiation requires an intact BTB/POZ domain; (d) the mutations interfering with either folding of the BTB/POZ domain or with its charged pocket prevent the self-renewal of PLZF/RARalpha-positive hematopoietic stem cells. Taken together, these data provide evidence that the dimerization capacity and the formation of a functionally charged pocket are indispensable for the PLZF/RARalpha-induced leukemogenesis.

Mutagenesis at Two Distinct Phosphate-Binding Sites Unravels Their Differential Roles in Regulation of Rubisco Activation and Catalysis

Orthophosphate (P(i)) has two antagonistic effects on ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), stimulation of activation and inhibition of catalysis by competition with the substrate RuBP. The enzyme binds P(i) at three distinct sites, two within the catalytic site (where 1P and 5P of ribulose 1,5-bisphosphate [RuBP] bind), and the third at the latch site (a positively charged pocket involved in active-site closure during catalysis). We examined the role of the latch and 5P sites in regulation of Rubisco activation and catalysis by introducing specific mutations in the enzyme of the cyanobacterium Synechocystis sp. strain PCC 6803. Whereas mutations at both sites abolished the P(i)-stimulated Rubisco activation, substitution of residues at the 5P site, but not at the latch site, affected the P(i) inhibition of Rubisco catalysis. Although some of these mutations substantially reduced the catalytic turnover of Rubisco and increased the K(m)(RuBP), they had little to moderate effect on the rate of photosynthesis and no effect on photoautotrophic growth. These findings suggest that in cyanobacteria, Rubisco does not limit photosynthesis to the extent previously estimated. These results indicate that both the latch and 5P sites participate in regulation of Rubisco activation, whereas P(i) binding only at the 5P site inhibits catalysis in a competitive manner.

Crystal Structure of Baculovirus RNA Triphosphatase Complexed with Phosphate

Baculovirus RNA 5'-triphosphatase (BVP) exemplifies a family of RNA-specific cysteine phosphatases that includes the RNA triphosphatase domains of metazoan and plant mRNA capping enzymes. Here we report the crystal structure of BVP in a phosphate-bound state at 1.5 A resolution. BVP adopts the characteristic cysteine-phosphatase alpha/beta fold and binds two phosphate ions in the active site region, one of which is proposed to mimic the phosphate of the product complex after hydrolysis of the covalent phosphoenzyme intermediate. The crystal structure highlights the role of backbone amides and side chains of the P-loop motif (118)HCTHGXNRT(126) in binding the cleavable phosphate and stabilizing the transition state. Comparison of the BVP structure to the apoenzyme of mammalian RNA triphosphatase reveals a concerted movement of the Arg-125 side chain (to engage the phosphate directly) and closure of an associated surface loop over the phosphate in the active site. The structure highlights a direct catalytic role of Asn-124, which is the signature P-loop residue of the RNA triphosphatase family and a likely determinant of the specificity of BVP for hydrolysis of phosphoanhydride linkages.

Role of T-loop Phosphorylation in PDK1 Activation, Stability, and Substrate Binding

3-Phosphoinositide-dependent protein kinase-1 (PDK1) phosphorylates the T-loop of several AGC (cAMP-dependent, cGMP-dependent, protein kinase C) family protein kinases, resulting in their activation. Previous structural studies have revealed that the alpha C-helix, located in the small lobe of the kinase domain of PDK1, is a key regulatory element, as it links a substrate interacting site termed the hydrophobic motif (HM) pocket with the phosphorylated Ser-241 in the T-loop. In this study we have demonstrated by mutational analysis that interactions between the phosphorylated Ser-241 and the alpha C-helix are not required for PDK1 activity or substrate binding through the HM-pocket but are necessary for PDK1 to be activated or stabilized by a peptide that binds to this site. The structure of an inactive T-loop mutant of PDK1, in which Ser-241 is changed to Ala, was also determined. This structure, together with surface plasmon resonance binding studies, demonstrates that the PDK1(S241A)-inactive mutant possesses an intact HM-pocket as well as an ordered alpha C-helix. These findings reveal that the integrity of the alpha C-helix and HM-pocket in PDK1 is not regulated by T-loop phosphorylation.

Crystal Structure of the PTPL1/FAP-1 Human Tyrosine Phosphatase Mutated in Colorectal Cancer: EVIDENCE FOR A SECOND PHOSPHOTYROSINE SUBSTRATE RECOGNITION POCKET

Protein-tyrosine phosphatase-L1 (PTPL1, also known as FAP-1, PTP1E, PTP-BAS, and PTPN13) is mutated in a significant number of colorectal tumors and may play a role in down-regulating signaling responses mediated by phosphatidylinositol 3-kinase, although the precise substrates are as yet unknown. In this study, we describe a 1.8 A resolution crystal structure of a fully active fragment of PTPL1 encompassing the catalytic domain. PTPL1 adopts the standard PTP fold, albeit with an unusually positioned additional N-terminal helix, and shows an ordered phosphate in the active site. Interestingly, a positively charged pocket is located near the PTPL1 catalytic site, reminiscent of the second phosphotyrosine binding site in PTP1B, which is required to dephosphorylate peptides containing two adjacent phosphotyrosine residues (as occurs for example in the activated insulin receptor). We demonstrate that PTPL1, like PTP1B, interacts with and dephosphorylates a bis-phosphorylated insulin receptor peptide more efficiently than monophosphorylated peptides, indicating that PTPL1 may down-regulate the phosphatidylinositol 3-kinase pathway, by dephosphorylating insulin or growth factor receptors that contain tandem phosphotyrosines. The structure also reveals that four out of five PTPL1 mutations found in colorectal cancers are located on solvent-exposed regions remote from the active site, consistent with these mutants being normally active. In contrast, the fifth mutation, which changes Met-2307 to Thr, is close to the active site cysteine and decreases activity significantly. Our studies provide the first molecular description of the PTPL1 catalytic domain and give new insight into the function of PTPL1.

Affinity Chromatography of Tryptases: Design, Synthesis and Characterization of a Novel Matrix‐Bound Bivalent Inhibitor

beta-Tryptases are mast cell-derived serine proteases that are enzymatically active in the form of an oligomer consisting of four subunits each with trypsin-like activity. The active-site clefts, which are directed toward the central pore of the tetramer, form spatial arrays of four negatively charged S1 binding pockets. Therefore, dibasic inhibitors of appropriate geometry can bind in a bivalent fashion to neighboring subunits. We have recently identified a potent bivalent inhibitor (K(i)=18 nM), based on the bifunctional scaffold cyclo-(-D-Asp-L-Asp-) and the arginine mimetic dl-3-aminomethyl-phenylalanine methyl ester as a ligand for S1 pockets that takes advantage of the this unique tetrameric geometry. To generate an affinity matrix, the bivalent ligand was modified and immobilized on a Sepharose matrix by use of the PEG derivative Jeffamine ED 900 as spacer. This matrix selectively recognizes and binds beta-tryptase from crude protein mixtures and thus is useful as a geometry-driven means of isolating and purifying human mast cell tryptases.

Dual substrate recognition of aminotransferases

Pyridoxal 5'-phosphate-dependent aminotransferases reversibly catalyzes the transamination reaction in which the alpha-amino group of amino acid 1 is transferred to the 2-oxo acid of amino acid 2 (usually 2-oxoglutarate) to produce the 2-oxo acid of amino acid 1 and amino acid 2 (glutamate). An aminotransferase must thus be able to recognize and bind two kinds of amino acids (amino acids 1 and 2), the side chains of which are different in shape and properties, from among many other small molecules. The dual substrate recognition mechanism has been discovered based on three-dimensional structures of aromatic amino acids, histidinol phosphate, glutamine:phenylpyruvate, acetylornithine, and branched-chain amino acid aminotransferases. There are two representative strategies for dual substrate recognition. An aromatic amino acid aminotransferase prepares charged and neutral pockets for acidic and aromatic side chains, respectively, at the same place by a large-scale rearrangement of the hydrogen-bond network caused by the induced fit. In a branched-chain aminotransferase, the same hydrophobic cavity implanted with hydrophilic sites accommodates both hydrophobic and acidic side chains without side-chain rearrangements of the active-site residues, which is reminiscent of the lock and key mechanism. Dual substrate recognition in other aminotransferases is attained by combining the two representative methods.

Structural Features and Thermodynamics of the J4/5 Loop from the Candida albicans and Candida dubliniensis Group I Introns,

The J4/5 loop of group I introns has tertiary interactions with the P1 helix that position the P1 substrate for the self-splicing reaction. The J4/5 loop of Candida albicans and Candida dubliniensis, 5'GAAGG3'/3'UAAUU5', potentially contains two A.A pairs flanked by one G.U pair on one side and two G.U pairs on the other side. Results from optical melting, nuclear magnetic resonance spectroscopy, and functional group substitution experiments with a mimic of the C. albicans and C. dubliniensis J4/5 loop are consistent with the adenosines forming tandem sheared A.A pairs with a cross-strand stack and only the G.U pair not adjacent to an A.A pair forming a static wobble G.U pair. The two G.U pairs adjacent to the tandem A.A pairs are likely in a dynamic equilibrium between multiple conformations. Although Co(NH(3))(6)(3+) stabilizes the loop by several kilocalories per mole at 37 degrees C, addition of Mg(2+) or Co(NH(3))(6)(3+) has no effect on the structure of the loop. The tandem G.U pairs provide a pocket of negative charge for Co(NH(3))(6)(3+) to bind. The results contribute to understanding the structure and dynamics of purine-rich internal loops and potential G.U pairs adjacent to internal loops.