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1.
Antimicrob Agents Chemother ; 60(2): 717-28, 2016 Feb.
Article in English | MEDLINE | ID: mdl-26525784

ABSTRACT

Class C ß-lactamases poorly hydrolyze cephamycins (e.g., cefoxitin, cefotetan, and moxalactam). In the past 2 decades, a new family of plasmid-based AmpC ß-lactamases conferring resistance to cefoxitin, the FOX family, has grown to include nine unique members descended from the Aeromonas caviae chromosomal AmpC. To understand the basis for the unique cephamycinase activity in the FOX family, we determined the first X-ray crystal structures of FOX-4, apo enzyme and the acyl-enzyme with its namesake compound, cefoxitin, using the Y150F deacylation-deficient variant. Notably, recombinant expression of N-terminally tagged FOX-4 also yielded an inactive adenylylated enzyme form not previously observed in ß-lactamases. The posttranslational modification (PTM), which occurs on the active site Ser64, would not seem to provide a selective advantage, yet might present an opportunity for the design of novel antibacterial drugs. Substantial ligand-induced changes in the enzyme are seen in the acyl-enzyme complex, particularly the R2 loop and helix H10 (P289 to N297), with movement of F293 by 10.3 Å. Taken together, this study provides the first picture of this highly proficient class C cephamycinase, uncovers a novel PTM, and suggests a possible cephamycin resistance mechanism involving repositioning of the substrate due to the presence of S153P, N289P, and N346I substitutions in the ligand binding pocket.


Subject(s)
Anti-Bacterial Agents/pharmacology , Bacterial Proteins/ultrastructure , Cefoxitin/pharmacology , Drug Resistance, Multiple, Bacterial/genetics , Escherichia coli Proteins/ultrastructure , beta-Lactamases/ultrastructure , Aeromonas caviae/drug effects , Amino Acid Sequence , Anti-Bacterial Agents/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Cefoxitin/metabolism , Crystallography, X-Ray , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Microbial Sensitivity Tests , Models, Molecular , Molecular Sequence Data , Protein Isoforms/genetics , Protein Isoforms/metabolism , Protein Isoforms/ultrastructure , Protein Processing, Post-Translational , Sequence Alignment , Tandem Mass Spectrometry , beta-Lactamases/genetics , beta-Lactamases/metabolism
2.
J Mol Biol ; 394(1): 143-59, 2009 Nov 20.
Article in English | MEDLINE | ID: mdl-19857646

ABSTRACT

Type IV pili (T4P) are bacterial virulence factors responsible for attachment to surfaces and for twitching motility, a motion that involves a succession of pilus extension and retraction cycles. In the opportunistic pathogen Pseudomonas aeruginosa, the PilM/N/O/P proteins are essential for T4P biogenesis, and genetic and biochemical analyses strongly suggest that they form an inner-membrane complex. Here, we show through co-expression and biochemical analysis that the periplasmic domains of PilN and PilO interact to form a heterodimer. The structure of residues 69-201 of the periplasmic domain of PilO was determined to 2.2 A resolution and reveals the presence of a homodimer in the asymmetric unit. Each monomer consists of two N-terminal coiled coils and a C-terminal ferredoxin-like domain. This structure was used to generate homology models of PilN and the PilN/O heterodimer. Our structural analysis suggests that in vivo PilN/O heterodimerization would require changes in the orientation of the first N-terminal coiled coil, which leads to two alternative models for the role of the transmembrane domains in the PilN/O interaction. Analysis of PilN/O orthologues in the type II secretion system EpsL/M revealed significant similarities in their secondary structures and the tertiary structures of PilO and EpsM, although the way these proteins interact to form inner-membrane complexes appears to be different in T4P and type II secretion. Our analysis suggests that PilN interacts directly, via its N-terminal tail, with the cytoplasmic protein PilM. This work shows a direct interaction between the periplasmic domains of PilN and PilO, with PilO playing a key role in the proper folding of PilN. Our results suggest that PilN/O heterodimers form the foundation of the inner-membrane PilM/N/O/P complex, which is critical for the assembly of a functional T4P complex.


Subject(s)
Bacterial Proteins/chemistry , Periplasm/chemistry , Protein Multimerization , Pseudomonas aeruginosa/chemistry , Amino Acid Sequence , Conserved Sequence , Models, Molecular , Molecular Sequence Data , Protein Binding , Protein Stability , Protein Structure, Secondary , Protein Structure, Tertiary , Protein Subunits/chemistry , Structural Homology, Protein
3.
Allergy ; 64(4): 569-80, 2009 Apr.
Article in English | MEDLINE | ID: mdl-19243361

ABSTRACT

BACKGROUND: The major timothy grass pollen allergen, Phl p 1, resembles the allergenic epitopes of natural group I grass pollen allergens and is recognized by more than 95% of grass-pollen-allergic patients. Our objective was the construction, purification and immunologic characterization of a genetically modified derivative of the major timothy grass pollen allergen, Phl p 1 for immunotherapy of grass pollen allergy. METHODS: A mosaic protein was generated by PCR-based re-assembly and expression of four cDNAs coding for Phl p 1 fragments and compared to the Phl p 1 wild-type by circular dichroism analysis, immunoglobulin E (IgE)-binding capacity, basophil activation assays and enzyme-linked immunosorbent assay competition assays. Immune responses to the derivative were studied in BALB/c mice. RESULTS: Grass-pollen-allergic patients exhibited greater than an 85% reduction in IgE reactivity to the mosaic as compared with the Phl p 1 allergen and basophil activation experiments confirmed the reduced allergenic activity of the mosaic. It also induced less Phl p 1-specific IgE antibodies than Phl p 1 upon immunization of mice. However, immunization of mice and rabbits with the mosaic induced IgG antibodies that inhibited patients' IgE-binding to the wild-type allergen and Phl p 1-induced degranulation of basophils. CONCLUSION: We have developed a strategy based on rational molecular reassembly to convert one of the clinically most relevant allergens into a hypoallergenic derivative for allergy vaccination.


Subject(s)
Allergens/biosynthesis , Allergens/immunology , Desensitization, Immunologic/methods , Plant Proteins/biosynthesis , Plant Proteins/immunology , Recombinant Proteins/biosynthesis , Recombinant Proteins/immunology , Adult , Aged , Allergens/chemistry , Amino Acid Sequence , Animals , Basophils/immunology , Basophils/metabolism , Epitopes, T-Lymphocyte/chemistry , Epitopes, T-Lymphocyte/immunology , Female , Histamine/biosynthesis , Histamine/immunology , Humans , Immunoglobulin E/blood , Immunoglobulin E/immunology , Immunoglobulin G/blood , Immunoglobulin G/immunology , Lymphocyte Activation/immunology , Male , Mice , Mice, Inbred BALB C , Middle Aged , Molecular Sequence Data , Plant Proteins/chemistry , Polymerase Chain Reaction , Protein Structure, Quaternary , Rabbits , Rats , Recombinant Proteins/chemical synthesis , Rhinitis, Allergic, Seasonal/immunology , Rhinitis, Allergic, Seasonal/prevention & control , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , T-Lymphocytes/immunology
4.
Microbiology (Reading) ; 153(Pt 4): 995-1005, 2007 Apr.
Article in English | MEDLINE | ID: mdl-17379709

ABSTRACT

The sequencing of prfA, encoding the transcriptional regulator of virulence genes, in 26 low-virulence field Listeria monocytogenes strains showed that eight strains exhibited the same single amino-acid substitution: PrfAK220T. These strains exhibited no expression of PrfA-regulated proteins and thus no virulence. This substitution inactivated PrfA, since expression of the PrfAK220T mutant gene in an EGDDeltaprfA strain did not restore the haemolytic and phosphatidylcholine phospholipase C activities, in contrast to the wild-type prfA gene. The substitution of the lysine at position 220 occurred in the helix alphaH. However, the data showed that the PrfAK220T protein is dimerized just as well as its wild-type counterpart, but does not bind to PrfA-boxes. PrfAK220T did not form a PrfA-DNA complex in electrophoretic mobility shift assays, but low concentrations of CI complexes (PrfAK220T-RNA polymerase-DNA complex) were formed by adding RNA polymerase, suggesting that PrfA interacted with RNA polymerase in solution in the absence of DNA. Formation of some transcriptionally active complexes was confirmed by in vitro runoff transcription assays and quantitative RT-PCR. Crystallographic analyses described the structure of native PrfA and highlighted the key role of allosteric changes in the activity of PrfA and especially the role of the Lys220 in the conformation of the helix-turn-helix (HTH) motif.


Subject(s)
DNA, Bacterial/metabolism , Listeria monocytogenes/pathogenicity , Peptide Termination Factors/genetics , Peptide Termination Factors/metabolism , Amino Acid Substitution , Animals , Crystallography, X-Ray , Dimerization , Helix-Turn-Helix Motifs , Hemolysin Factors/genetics , Humans , Listeria monocytogenes/chemistry , Listeria monocytogenes/metabolism , Models, Molecular , Mutation , Peptide Termination Factors/chemistry , Protein Conformation , Trans-Activators/chemistry , Trans-Activators/genetics , Trans-Activators/metabolism , Transcription, Genetic , Virulence
5.
Cell Motil Cytoskeleton ; 59(2): 141-52, 2004 Oct.
Article in English | MEDLINE | ID: mdl-15362118

ABSTRACT

The main regulators of Arp2/3 activity appear to be N-WASP and the other members of the Scar/WAVE family of proteins. We show here that after EGF stimulation, N-WASP is recruited to the nucleation zone of the dynamic leading edge compartment of carcinoma cells, with maximal recruitment of N-WASP within 1 min after EGF stimulation. The timing of N-WASP recruitment mirrors the timing of barbed-end formation at the leading edge. To determine the cellular activation of N-WASP after EGF stimulation, we made a conformation-sensitive antibody (CSA) against the CRIB domain of N-WASP that is predicted to recognize N-WASP in its open, active conformation, but not in its closed, inactive conformation. The ability of CSA to detect only active N-WASP was demonstrated by in vitro experiments using immunoprecipitation of active N-WASP from EGF-stimulated cells and Cdc42 activation of N-WASP activity. In cell staining experiments, N-WASP is maximally accessible to CSA 40 sec after EGF stimulation and this activated N-WASP is in the nucleation zone. These results indicate that active N-WASP is present at the leading edge of lamellipods, an unexpected finding given its reported involvement in filopod formation. This work establishes the feasibility of using antibodies directed against specific conformations or epitopes with changing accessibilities as a window on the status and localization of activity.


Subject(s)
Actin Cytoskeleton/metabolism , Cell Shape/physiology , Cytoskeleton/metabolism , Nerve Tissue Proteins/metabolism , Pseudopodia/metabolism , Animals , Antibodies/immunology , Cell Shape/drug effects , Epidermal Growth Factor/pharmacology , Immunoprecipitation , Molecular Conformation , Rats , Tumor Cells, Cultured , Wiskott-Aldrich Syndrome Protein, Neuronal , cdc42 GTP-Binding Protein/metabolism
6.
Proc Natl Acad Sci U S A ; 100(10): 5760-5, 2003 May 13.
Article in English | MEDLINE | ID: mdl-12732734

ABSTRACT

The structures of Saccharomyces cerevisiae, Dictyostelium, and Caenorhabditis elegans actin bound to gelsolin segment-1 have been solved and refined at resolutions between 1.9 and 1.75 A. These structures reveal several features relevant to the ATP hydrolytic mechanism, including identification of the nucleophilic water and the roles of Gln-137 and His-161 in positioning and activating the catalytic water, respectively. The involvement of these residues in the catalytic mechanism is consistent with yeast genetics studies. This work highlights both structural and mechanistic similarities with the small and trimeric G proteins and restricts the types of mechanisms responsible for the considerable enhancement of ATP hydrolysis associated with actin polymerization. The conservation of functionalities involved in nucleotide binding and catalysis also provide insights into the mechanistic features of members of the family of actin-related proteins.


Subject(s)
Actins/chemistry , Adenosine Triphosphate/metabolism , Gelsolin/chemistry , Actins/metabolism , Amino Acid Sequence , Animals , Binding Sites , Caenorhabditis elegans , Dictyostelium , Gelsolin/metabolism , Hydrogen Bonding , Hydrolysis , Invertebrates , Models, Molecular , Protein Conformation , Protein Structure, Secondary , Protein Subunits/chemistry , Saccharomyces cerevisiae
7.
Biochemistry ; 40(36): 10800-9, 2001 Sep 11.
Article in English | MEDLINE | ID: mdl-11535055

ABSTRACT

Adenine phosphoribosyltransferase (APRTase) is a widely distributed enzyme, and its deficiency in humans causes the accumulation of 2,8-dihydroxyadenine. It is the sole catalyst for adenine recycling in most eukaryotes. The most commonly expressed APRTase has subunits of approximately 187 amino acids, but the only crystal structure is from Leishmania donovani, which expresses a long form of the enzyme with 237 residues. Saccharomyces cerevisiae APRTase was selected as a representative of the short APRTases, and the structure of the apo-enzyme and sulfate bound forms were solved to 1.5 and 1.75 A, respectively. Yeast APRTase is a dimeric molecule, and each subunit is composed of a central five-stranded beta-sheet surrounded by five alpha-helices, a structural theme found in all known purine phosphoribosyltransferases. The structures reveal several important features of APRTase function: (i) sulfate ions bound at the 5'-phosphate and pyrophosphate binding sites; (ii) a nonproline cis peptide bond (Glu67-Ser68) at the pyrophosphate binding site in both apo-enzyme and sulfate-bound forms; and (iii) a catalytic loop that is open and ordered in the apo-enzyme but open and disordered in the sulfate-bound form. Alignment of conserved amino acids in short-APRTases from 33 species reveals 13 invariant and 15 highly conserved residues present in hinges, catalytic site loops, and the catalytic pocket. Mutagenesis of conserved residues in the catalytic loop, subunit interface, and phosphoribosylpyrophosphate binding site indicates critical roles for the tip of the catalytic loop (Glu106) and a catalytic site residue Arg69, respectively. Mutation of one loop residue (Tyr103Phe) increases k(cat) by 4-fold, implicating altered dynamics for the catalytic site loop.


Subject(s)
Adenine Phosphoribosyltransferase/chemistry , Adenine Phosphoribosyltransferase/metabolism , Saccharomyces cerevisiae/enzymology , Adenine Phosphoribosyltransferase/genetics , Amino Acid Sequence , Amino Acid Substitution , Animals , Apoenzymes/chemistry , Apoenzymes/metabolism , Bacteria/enzymology , Binding Sites , Cloning, Molecular , Dimerization , Drosophila/enzymology , Humans , Leishmania donovani/enzymology , Mice , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Protein Structure, Secondary , Protein Subunits , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Saccharomyces cerevisiae/genetics , Sequence Alignment , Sequence Homology, Amino Acid , Sulfates/metabolism
8.
Biochemistry ; 40(28): 8204-15, 2001 Jul 27.
Article in English | MEDLINE | ID: mdl-11444966

ABSTRACT

A structural genomics comparison of purine nucleoside phosphorylases (PNPs) indicated that the enzyme encoded by Mycobacterium tuberculosis (TB-PNP) resembles the mammalian trimeric structure rather than the bacterial hexameric PNPs. The crystal structure of M. tuberculosis PNP in complex with the transition-state analogue immucillin-H (ImmH) and inorganic phosphate was solved at 1.75 A resolution and confirms the trimeric structure. Binding of the inhibitor occurs independently at the three catalytic sites, unlike mammalian PNPs which demonstrate negative cooperativity in ImmH binding. Reduced subunit interface contacts for TB-PNP, compared to the mammalian enzymes, correlate with the loss of the cooperative inhibitor binding. Mammalian and TB-PNPs both exhibit slow-onset inhibition and picomolar dissociation constants for ImmH. The structure supports a catalytic mechanism of reactant destabilization by neighboring group electrostatic interactions, transition-state stabilization, and leaving group activation. Despite an overall amino acid sequence identity of 33% between bovine and TB-PNPs and almost complete conservation in active site residues, one catalytic site difference suggests a strategy for the design of transition-state analogues with specificity for TB-PNP. The structure of TB-PNP was also solved to 2.0 A with 9-deazahypoxanthine (9dHX), iminoribitol (IR), and PO(4) to reconstruct the ImmH complex with its separate components. One subunit of the trimer has 9dHX, IR, and PO(4) bound, while the remaining two subunits contain only 9dHX. In the filled subunit, 9dHX retains the contacts found in the ImmH complex. However, the region of IR that corresponds to the oxocarbenium ion is translocated in the direction of the reaction coordinate, and the nucleophilic phosphate rotates away from the IR group. Loose packing of the pieces of ImmH in the catalytic site establishes that covalent connectivity in ImmH is required to achieve the tightly bound complex.


Subject(s)
Enzyme Inhibitors/chemistry , Mycobacterium tuberculosis/enzymology , Purine-Nucleoside Phosphorylase/antagonists & inhibitors , Purine-Nucleoside Phosphorylase/chemistry , Pyrimidinones/chemistry , Pyrroles/chemistry , Actinomycetales/enzymology , Animals , Binding Sites , Catalysis , Cattle , Enzyme Stability , Escherichia coli/enzymology , Macromolecular Substances , Models, Molecular , Phosphates/chemistry , Protein Conformation , Purine Nucleosides
9.
J Mol Biol ; 310(2): 351-66, 2001 Jul 06.
Article in English | MEDLINE | ID: mdl-11428894

ABSTRACT

Fascin is an actin crosslinking protein that organizes actin filaments into tightly packed bundles believed to mediate the formation of cellular protrusions and to provide mechanical support to stress fibers. Using quantitative rheological methods, we studied the evolution of the mechanical behavior of filamentous actin (F-actin) networks assembled in the presence of human fascin. The mechanical properties of F-actin/fascin networks were directly compared with those formed by alpha-actinin, a prototypical actin filament crosslinking/bundling protein. Gelation of F-actin networks in the presence of fascin (fascin to actin molar ratio >1:50) exhibits a non-monotonic behavior characterized by a burst of elasticity followed by a slow decline over time. Moreover, the rate of gelation shows a non-monotonic dependence on fascin concentration. In contrast, alpha-actinin increased the F-actin network elasticity and the rate of gelation monotonically. Time-resolved multiple-angle light scattering and confocal and electron microscopies suggest that this unique behavior is due to competition between fascin-mediated crosslinking and side-branching of actin filaments and bundles, on the one hand, and delayed actin assembly and enhanced network micro-heterogeneity, on the other hand. The behavior of F-actin/fascin solutions under oscillatory shear of different frequencies, which mimics the cell's response to forces applied at different rates, supports a key role for fascin-mediated F-actin side-branching. F-actin side-branching promotes the formation of interconnected networks, which completely inhibits the motion of actin filaments and bundles. Our results therefore show that despite sharing seemingly similar F-actin crosslinking/bundling activity, alpha-actinin and fascin display completely different mechanical behavior. When viewed in the context of recent microrheological measurements in living cells, these results provide the basis for understanding the synergy between multiple crosslinking proteins, and in particular the complementary mechanical roles of fascin and alpha-actinin in vivo.


Subject(s)
Actinin/metabolism , Actins/chemistry , Actins/ultrastructure , Carrier Proteins/metabolism , Microfilament Proteins/metabolism , Actinin/ultrastructure , Actins/metabolism , Animals , Biopolymers/chemistry , Biopolymers/metabolism , Carrier Proteins/ultrastructure , Chickens , Elasticity , Humans , Kinetics , Light , Microfilament Proteins/ultrastructure , Microscopy, Confocal , Microscopy, Electron , Protein Binding , Protein Structure, Quaternary , Scattering, Radiation , Solutions
11.
Acta Crystallogr D Biol Crystallogr ; 57(Pt 6): 898-9, 2001 Jun.
Article in English | MEDLINE | ID: mdl-11375523

ABSTRACT

CTLA-4 is a dimeric T-cell surface receptor responsible for transducing signals that down-regulate activated T cells upon binding B7 ligands. The disulfide-linked homodimer of the extracellular segment of human CTLA-4 and the receptor-binding domain of human B7-2 were purified and cocrystallized. Diffraction from these crystals is consistent with the monoclinic space group P2(1) (unit-cell parameters a = 47.85, b = 54.56, c = 103.09 A, beta = 91.63); native data have been collected to 3.2 A resolution.


Subject(s)
Antigens, CD/chemistry , Antigens, Differentiation/chemistry , Immunoconjugates , Membrane Glycoproteins/chemistry , Abatacept , Antigens, CD/genetics , Antigens, Differentiation/genetics , B7-2 Antigen , CTLA-4 Antigen , Crystallization , Crystallography, X-Ray , Gene Deletion , Humans , Membrane Glycoproteins/genetics , Protein Conformation , Recombinant Proteins/chemistry
12.
Nature ; 410(6828): 604-8, 2001 Mar 29.
Article in English | MEDLINE | ID: mdl-11279501

ABSTRACT

Regulation of T-cell activity is dependent on antigen-independent co-stimulatory signals provided by the disulphide-linked homodimeric T-cell surface receptors, CD28 and CTLA-4 (ref. 1). Engagement of CD28 with B7-1 and B7-2 ligands on antigen-presenting cells (APCs) provides a stimulatory signal for T-cell activation, whereas subsequent engagement of CTLA-4 with these same ligands results in attenuation of the response. Given their central function in immune modulation, CTLA-4- and CD28-associated signalling pathways are primary therapeutic targets for preventing autoimmune disease, graft versus host disease, graft rejection and promoting tumour immunity. However, little is known about the cell-surface organization of these receptor/ligand complexes and the structural basis for signal transduction. Here we report the 3.2-A resolution structure of the complex between the disulphide-linked homodimer of human CTLA-4 and the receptor-binding domain of human B7-2. The unusual dimerization properties of both CTLA-4 and B7-2 place their respective ligand-binding sites distal to the dimer interface in each molecule and promote the formation of an alternating arrangement of bivalent CTLA-4 and B7-2 dimers that extends throughout the crystal. Direct observation of this CTLA-4/B7-2 network provides a model for the periodic organization of these molecules within the immunological synapse and suggests a distinct mechanism for signalling by dimeric cell-surface receptors.


Subject(s)
Antigens, CD/chemistry , Antigens, Differentiation/chemistry , Immunoconjugates , Membrane Glycoproteins/chemistry , T-Lymphocytes/immunology , Abatacept , Amino Acid Sequence , Antigens, CD/physiology , Antigens, Differentiation/physiology , B7-2 Antigen , Binding Sites , CTLA-4 Antigen , Crystallography, X-Ray , Dimerization , Humans , Lymphocyte Activation , Macromolecular Substances , Membrane Glycoproteins/physiology , Molecular Sequence Data , Protein Binding , Protein Conformation , Receptors, Antigen, T-Cell/metabolism , Recombinant Proteins/chemistry , Structure-Activity Relationship , T-Lymphocytes/chemistry , T-Lymphocytes/metabolism
13.
Biochemistry ; 40(4): 853-60, 2001 Jan 30.
Article in English | MEDLINE | ID: mdl-11170405

ABSTRACT

Immucillin-H [ImmH; (1S)-1-(9-deazahypoxanthin-9-yl)-1,4-dideoxy-1,4-imino-D-ribitol] is a 23 pM inhibitor of bovine purine nucleoside phosphorylase (PNP) specifically designed as a transition state mimic [Miles, R. W., Tyler, P. C., Furneaux, R. H., Bagdassarian, C. K., and Schramm, V. L. (1998) Biochemistry 37, 8615-8621]. Cocrystals of PNP and the inhibitor are used to provide structural information for each step through the reaction coordinate of PNP. The X-ray crystal structure of free ImmH was solved at 0.9 A resolution, and a complex of PNP.ImmH.PO(4) was solved at 1.5 A resolution. These structures are compared to previously reported complexes of PNP with substrate and product analogues in the catalytic sites and with the experimentally determined transition state structure. Upon binding, ImmH is distorted to a conformation favoring ribosyl oxocarbenium ion formation. Ribosyl destabilization and transition state stabilization of the ribosyl oxocarbenium ion occur from neighboring group interactions with the phosphate anion and the 5'-hydroxyl of the ribosyl group. Leaving group activation of hypoxanthine involves hydrogen bonds to O6, N1, and N7 of the purine ring. Ordered water molecules provide a proton transfer bridge to O6 and N7 and permit reversible formation of these hydrogen bonds. Contacts between PNP and catalytic site ligands are shorter in the transition state analogue complex of PNP.ImmH.PO(4) than in the Michaelis complexes of PNP.inosine.SO(4) or PNP.hypoxanthine.ribose 1-PO(4). Reaction coordinate motion is dominated by translation of the carbon 1' of ribose between relatively fixed phosphate and purine groups. Purine and pyrimidine phosphoribosyltransferases and nucleoside N-ribosyl hydrolases appear to operate by a similar mechanism.


Subject(s)
Purine-Nucleoside Phosphorylase/chemistry , Animals , Binding Sites , Catalysis , Cattle , Crystallography, X-Ray , Deuterium/chemistry , Electron Transport , Enzyme Inhibitors/chemistry , Hydrolysis , Inosine/chemistry , Macromolecular Substances , Motion , Phosphates/chemistry , Protein Conformation , Purine Nucleosides , Purine-Nucleoside Phosphorylase/antagonists & inhibitors , Pyrimidinones/chemistry , Pyrroles/chemistry
14.
J Biomol Struct Dyn ; 19(3): 405-18, 2001 Dec.
Article in English | MEDLINE | ID: mdl-11790140

ABSTRACT

Traditional approaches for macromolecular structure elucidation, including NMR, crystallography and cryo-EM have made significant progress in defining the structures of protein-protein complexes. A substantial number of macromolecular structures, however, have not been examined with atomic detail due to sample size and heterogeneity, or resolution limitations of the technique; therefore, the general applicability of each method is greatly reduced. Synchrotron footprinting attempts to bridge the gap in these methods by monitoring changes in accessible surface areas of discrete macromolecular moieties. As evidenced by our previous studies on RNA folding and DNA-protein interactions, the three-dimensional structure is probed by examining the reactions of these moieties with hydroxyl radicals generated by synchrotron X-rays. Here we report the application of synchrotron footprinting to the investigation of protein- protein interactions, as the novel technique has been utilized to successfully map the contact sites of gelsolin segment-1 in the gelsolin segment 1/actin complex. Footprinting results demonstrate that phenylalanine 104, located on the actin binding helix of gelsolin segment 1, is protected from hydroxyl radical modification in the presence of actin. This change in reactivity results from the specific protection of gelsolin segment-1, consistent with the substantial decrease in solvent accessibility of F104 upon actin binding, as calculated from the crystal structural of the gelsolin segment 1/actin complex. The results presented here establish synchrotron footprinting as a broadly applicable method to probe structural features of macromolecular complexes that are not amenable to conventional approaches.


Subject(s)
Protein Footprinting , Proteins/chemistry , Proteins/metabolism , Synchrotrons , Actins/chemistry , Actins/metabolism , Amino Acid Sequence , Animals , Aspartic Acid/chemistry , Binding Sites , Calcium/chemistry , Gelsolin/chemistry , Gelsolin/metabolism , Hydroxyl Radical/chemistry , Kinetics , Macromolecular Substances , Models, Molecular , Phenylalanine/chemistry , Protein Conformation , Protein Structure, Tertiary , Rabbits , X-Rays
15.
Science ; 290(5492): 816-9, 2000 Oct 27.
Article in English | MEDLINE | ID: mdl-11052947

ABSTRACT

The effective regulation of T cell responses is dependent on opposing signals transmitted through two related cell-surface receptors, CD28 and cytotoxic T lymphocyte-associated antigen 4 (CTLA-4). Dimerization of CTLA-4 is required for the formation of high-avidity complexes with B7 ligands and for transmission of signals that attenuate T cell activation. We determined the crystal structure of the extracellular portion of CTLA-4 to 2.0 angstrom resolution. CTLA-4 belongs to the immunoglobulin superfamily and displays a strand topology similar to Valpha domains, with an unusual mode of dimerization that places the B7 binding sites distal to the dimerization interface. This organization allows each CTLA-4 dimer to bind two bivalent B7 molecules and suggests that a periodic arrangement of these components within the immunological synapse may contribute to the regulation of T cell responsiveness.


Subject(s)
Antigens, Differentiation/chemistry , Antigens, Differentiation/immunology , Immunoconjugates , T-Lymphocytes/immunology , Abatacept , Amino Acid Sequence , Animals , Antigen-Presenting Cells/immunology , Antigens, CD , Antigens, Differentiation/metabolism , B7-1 Antigen/chemistry , B7-1 Antigen/metabolism , CD28 Antigens/immunology , CD28 Antigens/metabolism , CTLA-4 Antigen , Crystallography, X-Ray , Dimerization , Hydrogen Bonding , Ligands , Lymphocyte Activation , Mice , Models, Molecular , Molecular Sequence Data , Protein Binding , Protein Conformation , Protein Structure, Secondary , Protein Structure, Tertiary , Receptors, Antigen, T-Cell/metabolism , Signal Transduction
16.
J Immunol ; 165(8): 4494-504, 2000 Oct 15.
Article in English | MEDLINE | ID: mdl-11035089

ABSTRACT

Ag-specific T cell recognition is mediated through direct interaction of clonotypic TCRs with complexes formed between Ag-presenting molecules and their bound ligands. Although characterized in substantial detail for class I and class II MHC encoded molecules, the molecular interactions responsible for TCR recognition of the CD1 lipid and glycolipid Ag-presenting molecules are not yet well understood. Using a panel of epitope-specific Abs and site-specific mutants of the CD1b molecule, we showed that TCR interactions occur on the membrane distal aspects of the CD1b molecule over the alpha1 and alpha2 domain helices. The location of residues on CD1b important for this interaction suggested that TCRs bind in a diagonal orientation relative to the longitudinal axes of the alpha helices. The data point to a model in which TCR interaction extends over the opening of the putative Ag-binding groove, making multiple direct contacts with both alpha helices and bound Ag. Although reminiscent of TCR interaction with MHC class I, our data also pointed to significant differences between the TCR interactions with CD1 and MHC encoded Ag-presenting molecules, indicating that Ag receptor binding must be modified to accommodate the unique molecular structure of the CD1b molecule and the unusual Ags it presents.


Subject(s)
Antigen Presentation , Antigens, CD1/metabolism , Receptors, Antigen, T-Cell, alpha-beta/metabolism , Antibodies, Blocking/pharmacology , Antibodies, Monoclonal/pharmacology , Antigen Presentation/genetics , Antigens, CD1/blood , Antigens, CD1/genetics , Antigens, CD1/immunology , Cell Line , Clone Cells , Glycolipids/immunology , Glycolipids/metabolism , Humans , Macromolecular Substances , Major Histocompatibility Complex/genetics , Major Histocompatibility Complex/immunology , Models, Immunological , Mutagenesis, Site-Directed , T-Lymphocytes/immunology , T-Lymphocytes/metabolism
17.
J Cell Biol ; 150(4): 895-904, 2000 Aug 21.
Article in English | MEDLINE | ID: mdl-10953013

ABSTRACT

The actin monomer-binding protein, profilin, influences the dynamics of actin filaments in vitro by suppressing nucleation, enhancing nucleotide exchange on actin, and promoting barbed-end assembly. Profilin may also link signaling pathways to actin cytoskeleton organization by binding to the phosphoinositide PIP(2) and to polyproline stretches on several proteins. Although activities of profilin have been studied extensively in vitro, the significance of each of these activities in vivo needs to be tested. To study profilin function, we extensively mutagenized the Saccharomyces cerevisiae profilin gene (PFY1) and examined the consequences of specific point mutations on growth and actin organization. The actin-binding region of profilin was shown to be critical in vivo. act1-157, an actin mutant with an increased intrinsic rate of nucleotide exchange, suppressed defects in actin organization, cell growth, and fluid-phase endocytosis of pfy1-4, a profilin mutant defective in actin binding. In reactions containing actin, profilin, and cofilin, profilin was required for fast rates of actin filament turnover. However, Act1-157p circumvented the requirement for profilin. Based on the results of these studies, we conclude that in living cells profilin promotes rapid actin dynamics by regenerating ATP actin from ADP actin-cofilin generated during filament disassembly.


Subject(s)
Actins/metabolism , Microfilament Proteins/chemistry , Microfilament Proteins/metabolism , Contractile Proteins/chemistry , Contractile Proteins/metabolism , Kinetics , Microfilament Proteins/genetics , Models, Molecular , Mutagenesis, Site-Directed , Phosphatidylinositol 4,5-Diphosphate/metabolism , Point Mutation , Profilins , Protein Conformation , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Signal Transduction
18.
Biophys J ; 79(2): 1095-106, 2000 Aug.
Article in English | MEDLINE | ID: mdl-10920039

ABSTRACT

One of the central functions of actin cytoskeleton is to provide the mechanical support required for the establishment and maintenance of cell morphology. The mechanical properties of actin filament assemblies are a consequence of both the available polymer concentration and the actin regulatory proteins that direct the formation of higher order structures. By monitoring the displacement of well-dispersed microspheres via fluorescence microscopy, we probe the degree of spatial heterogeneity of F-actin gels and networks in vitro. We compare the distribution of the time-dependent mean-square displacement (MSD) of polystyrene microspheres imbedded in low- and high-concentration F-actin solutions, in the presence and absence of the F-actin-bundling protein fascin. The MSD distribution of a 2. 6-microM F-actin solution is symmetric and its standard deviation is similar to that of a homogeneous solution of glycerol of similar zero-shear viscosity. However, increasing actin concentration renders the MSD distribution wide and asymmetric, an effect enhanced by fascin. Quantitative changes in the shape of the MSD distribution correlate qualitatively with the presence of large heterogeneities in F-actin solutions produced by increased filament concentration and the presence of actin bundles, as detected by confocal microscopy. Multiple-particle tracking offers a new, quantitative method to characterize the organization of biopolymers in solution.


Subject(s)
Actins/chemistry , Actins/ultrastructure , Actins/physiology , Animals , Chickens , Cytoskeleton/physiology , Microscopy, Confocal/methods , Microscopy, Fluorescence/methods , Microspheres , Models, Molecular , Muscle, Skeletal/physiology , Protein Conformation , Solutions
19.
Biochemistry ; 39(28): 8171-9, 2000 Jul 18.
Article in English | MEDLINE | ID: mdl-10889023

ABSTRACT

Protein tyrosine phosphatase 1B (PTP1B) displays a preference for peptides containing acidic as well as aromatic/aliphatic residues immediately NH(2)-terminal to phosphotyrosine. The structure of PTP1B bound with DADEpYL-NH(2) (EGFR(988)(-)(993)) offers a structural explanation for PTP1B's preference for acidic residues [Jia, Z., Barford, D., Flint, A. J., and Tonks, N. K. (1995) Science 268, 1754-1758]. We report here the crystal structures of PTP1B in complex with Ac-ELEFpYMDYE-NH(2) (PTP1B.Con) and Ac-DAD(Bpa)pYLIPQQG (PTP1B.Bpa) determined to 1.8 and 1.9 A resolution, respectively. A structural analysis of PTP1B.Con and PTP1B.Bpa shows how aromatic/aliphatic residues at the -1 and -3 positions of peptide substrates are accommodated by PTP1B. A comparison of the structures of PTP1B.Con and PTP1B.Bpa with that of PTP1B.EGFR(988)(-)(993) reveals the structural basis for the plasticity of PTP1B substrate recognition. PTP1B is able to bind phosphopeptides by utilizing common interactions involving the aromatic ring and phosphate moiety of phosphotyrosine itself, two conserved hydrogen bonds between the Asp48 carboxylate side chain and the main chain nitrogens of the pTyr and residue 1, and a third between the main chain nitrogen of Arg47 and the main chain carbonyl of residue -2. The ability of PTP1B to accommodate both acidic and hydrophobic residues immediately NH(2)-terminal to pTyr appears to be conferred upon PTP1B by a single residue, Arg47. Depending on the nature of the NH(2)-terminal amino acids, the side chain of Arg47 can adopt one of two different conformations, generating two sets of distinct peptide binding surfaces. When an acidic residue is positioned at position -1, a preference for a second acidic residue is also observed at position -2. However, when a large hydrophobic group occupies position -1, Arg47 adopts a new conformation so that it can participate in hydrophobic interactions with both positions -1 and -3.


Subject(s)
Protein Tyrosine Phosphatases/chemistry , Arginine/metabolism , Crystallization , Enzyme Inhibitors/pharmacology , Kinetics , Models, Molecular , Peptides/chemistry , Peptides/metabolism , Protein Conformation , Protein Tyrosine Phosphatase, Non-Receptor Type 1 , Protein Tyrosine Phosphatases/antagonists & inhibitors , Protein Tyrosine Phosphatases/metabolism , Substrate Specificity
20.
Biochemistry ; 39(23): 6781-90, 2000 Jun 13.
Article in English | MEDLINE | ID: mdl-10841757

ABSTRACT

Giardia lamblia, the protozoan parasite responsible for giardiasis, requires purine salvage from its host for RNA and DNA synthesis. G. lamblia expresses an unusual purine phosphoribosyltransferase with a high specificity for guanine (GPRTase). The enzyme's sequence significantly diverges from those of related enzymes in other organisms. The transition state analogue immucillinGP is a powerful inhibitor of HGXPRTase from malaria [Li, C. M., et al. (1999) Nat. Struct. Biol. 6, 582-587] and is also a 10 nM inhibitor of G. lamblia GPRTase. Cocrystallization of GPRTase with immucillinGP led unexpectedly to a GPRTase.immucillinG binary complex with an open catalytic site loop. Diffusion of ligands into preformed crystals gave a GPRTase.immucillinGP.Mg(2+).pyrophosphate complex in which the open loop is stabilized by crystal contacts. G. lamblia GPRTase exhibits substantial structural differences from known purine phosphoribosyltransferases at positions remote from the catalytic site, but conserves most contacts to the bound inhibitor. The filled catalytic site with an open catalytic loop provides insight into ligand binding. One active site Mg(2+) ion is chelated to pyrophosphate, but the other is chelated to two conserved catalytic site carboxylates, suggesting a role for these amino acids. This arrangement of Mg(2+) and pyrophosphate has not been reported in purine phosphoribosyltransferases. ImmucillinG in the binary complex is anchored by its 9-deazaguanine group, and the iminoribitol is disordered. No Mg(2+) or pyrophosphate is detected; thus, the 5'-phosphoryl group is needed to immobilize the iminoribitol prior to magnesium pyrophosphate binding. Filling the catalytic site involves (1) binding the purine ring, (2) anchoring the 5'-phosphate to fix the ribosyl group, (3) binding the first Mg(2+) to Asp125 and Glu126 carboxyl groups and binding Mg(2+).pyrophosphate, and (4) closing the catalytic site loop and formation of bound (Mg(2+))(2). pyrophosphate prior to catalysis. Guanine specificity is provided by two peptide carbonyl oxygens hydrogen-bonded to the exocyclic amino group and a weak interaction to O6. Transition state formation involves N7 protonation by Asp129 acting as the general acid.


Subject(s)
Giardia lamblia/enzymology , Hypoxanthine Phosphoribosyltransferase/chemistry , Animals , Binding Sites , Crystallography, X-Ray , Dimerization , Enzyme Inhibitors/chemistry , Models, Molecular , Molecular Conformation , Pyrimidinones/chemistry , Pyrroles/chemistry , Recombinant Proteins/chemistry
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