The Three-Dimensional Structure of Aspergillus niger Pectin Lyase B at 1.7-Å Resolution1

The three-dimensional structure of Aspergillus niger pectin lyase B (PLB) has been determined by crystallographic techniques at a resolution of 1.7 Å. The model, with all 359 amino acids and 339 water molecules, refines to a final crystallographic R factor of 16.5%. The polypeptide backbone folds into a large right-handed cylinder, termed a parallel β helix. Loops of various sizes and conformations protrude from the central helix and probably confer function. The largest loop of 53 residues folds into a small domain consisting of three antiparallel β strands, one turn of an α helix, and one turn of a 310 helix. By comparison with the structure of Erwinia chrysanthemi pectate lyase C (PelC), the primary sequence alignment between the pectate and pectin lyase subfamilies has been corrected and the active site region for the pectin lyases deduced. The substrate-binding site in PLB is considerably less hydrophilic than the comparable PelC region and consists of an extensive network of highly conserved Trp and His residues. The PLB structure provides an atomic explanation for the lack of a catalytic requirement for Ca2+ in the pectin lyase family, in contrast to that found in the pectate lyase enzymes. Surprisingly, however, the PLB site analogous to the Ca2+ site in PelC is filled with a positive charge provided by a conserved Arg in the pectin lyases. The significance of the finding with regard to the enzymatic mechanism is discussed.

BeF\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{3}^{-}}}\end{equation*}\end{document} acts as a phosphate analog in proteins phosphorylated on aspartate: Structure of a BeF\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{3}^{-}}}\end{equation*}\end{document} complex with phosphoserine phosphatase

Protein phosphoaspartate bonds play a variety of roles. In response regulator proteins of two-component signal transduction systems, phosphorylation of an aspartate residue is coupled to a change from an inactive to an active conformation. In phosphatases and mutases of the haloacid dehalogenase (HAD) superfamily, phosphoaspartate serves as an intermediate in phosphotransfer reactions, and in P-type ATPases, also members of the HAD family, it serves in the conversion of chemical energy to ion gradients. In each case, lability of the phosphoaspartate linkage has hampered a detailed study of the phosphorylated form. For response regulators, this difficulty was recently overcome with a phosphate analog, BeF\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{3}^{-}}}\end{equation*}\end{document}, which yields persistent complexes with the active site aspartate of their receiver domains. We now extend the application of this analog to a HAD superfamily member by solving at 1.5-Å resolution the x-ray crystal structure of the complex of BeF\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{3}^{-}}}\end{equation*}\end{document} with phosphoserine phosphatase (PSP) from Methanococcus jannaschii. The structure is comparable to that of a phosphoenzyme intermediate: BeF\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{3}^{-}}}\end{equation*}\end{document} is bound to Asp-11 with the tetrahedral geometry of a phosphoryl group, is coordinated to Mg2+, and is bound to residues surrounding the active site that are conserved in the HAD superfamily. Comparison of the active sites of BeF\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{3}^{-}}}\end{equation*}\end{document}⋅PSP and BeF\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{3}^{-}}}\end{equation*}\end{document}⋅CeY, a receiver domain/response regulator, reveals striking similarities that provide insights into the function not only of PSP but also of P-type ATPases. Our results indicate that use of BeF\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{3}^{-}}}\end{equation*}\end{document} for structural studies of proteins that form phosphoaspartate linkages will extend well beyond response regulators.

The fifth epidermal growth factor-like domain of thrombomodulin does not have an epidermal growth factor-like disulfide bonding pattern.

The disulfide bonding pattern of the fourth and fifth epidermal growth factor (EGF)-like domains within the smallest active fragment of thrombomodulin have been determined. In previous work, this fragment was expressed and purified to homogeneity, and its cofactor activity, as measured by Kcat for thrombin activation of protein C, was the same as that for full-length thrombomodulin. CNBr cleavage at the single methionine in the connecting region between the domains and subsequent deglycosylation yielded the individual EGF-like domains. The disulfide bonds were mapped by partial reduction with tris(2-carboxyethyl)phosphine according to the method of Gray [Gray, W. R. (1993) Protein Sci. 2, 1732-1748], which provides unambiguous results. The disulfide bonding pattern of the fourth EGF-like domain was (1-3, 2-4, 5-6), which is the same as that found previously in EGF and in a synthetic version of the fourth EGF-like domain. Surprisingly, the disulfide bonding pattern of the fifth domain was (1-2, 3-4, 5-6), which is unlike that found in EGF or in any other EGF-like domain analyzed so far. This result is in line with an earlier observation that the (1-2, 3-4, 5-6) isomer bound to thrombin more tightly than the EGF-like (1-3, 2-4, 5-6) isomer. The observation that not all EGF-like domains have an EGF-like disulfide bonding pattern reveals an additional element of diversity in the structure of EGF-like domains.

The structure of bovine F1-ATPase complexed with the peptide antibiotic efrapeptin.

In the previously determined structure of mitochondrial F1-ATPase determined with crystals grown in the presence of adenylyl-imidodiphosphate (AMP-PNP) and ADP, the three catalytic beta-subunits have different conformations and nucleotide occupancies. AMP-PNP and ADP are bound to subunits beta TP and beta DP, respectively, and the third beta-subunit (beta E) has no bound nucleotide. The efrapeptins are a closely related family of modified linear peptides containing 15 amino acids that inhibit both ATP synthesis and hydrolysis by binding to the F1 catalytic domain of F1F0-ATP synthase. In crystals of F1-ATPase grown in the presence of both nucleotides and inhibitor, efrapeptin is bound to a unique site in the central cavity of the enzyme. Its binding is associated with small structural changes in side chains of F1-ATPase around the binding pocket. Efrapeptin makes hydrophobic contacts with the alpha-helical structure in the gamma-subunit, which traverses the cavity, and with subunit beta E and the two adjacent alpha-subunits. Two intermolecular hydrogen bonds could also form. Intramolecular hydrogen bonds probably help to stabilize efrapeptin's two domains (residues 1-6 and 9-15, respectively), which are connected by a flexible region (beta Ala-7 and Gly-8). Efrapeptin appears to inhibit F1-ATPase by blocking the conversion of subunit beta E to a nucleotide binding conformation, as would be required by an enzyme mechanism involving cyclic interconversion of catalytic sites.

Structure of the catalytic fragment of poly(AD-ribose) polymerase from chicken.

The crystal structures of the catalytic fragment of chicken poly(ADP-ribose) polymerase [NAD+ ADP-ribosyltransferase; NAD+:poly(adenosine-diphosphate-D-ribosyl)-acceptor ADP-D-ribosyltransferase, EC 2.4.2.30] with and without a nicotinamide-analogue inhibitor have been elucidated. Because this enzyme is involved in the regulation of DNA repair, its inhibitors are of interest for cancer therapy. The inhibitor shows the nicotinamide site and also suggests the adenosine site. The enzyme is structurally related to bacterial ADP-ribosylating toxins but contains an additional alpha-helical domain that is suggested to relay the activation signal issued on binding to damaged DNA.

Mutation of Pro-258 in transmembrane domain 6 constitutively activates the G protein-coupled alpha-factor receptor.

The alpha-factor pheromone receptor stimulates MATa yeast cells to undergo conjugation. The receptor contains seven transmembrane domains that function in ligand binding and in transducing a signal to the cytoplasmic receptor sequences to mediate G protein activation. A genetic screen was used to isolate receptor mutations that constitutively signal in the absence of alpha-factor. The Pro-258-->Leu (P258L) mutation caused constitutive receptor signaling that was equivalent to about 45% of the maximum level observed in wild-type cells stimulated with alpha-factor. Mutations of both Pro-258 and the adjacent Ser-259 to Leu increased constitutive signaling to > or = 90% of the maximum level. Since Pro-258 occurs in the central portion of transmembrane domain 6, and since proline residues are expected to cause a kink in alpha-helical domains, the P258L mutation is predicted to alter the structure of transmembrane domain 6. The P258L mutation did not result in a global distortion of receptor structure because alpha-factor bound to the mutant receptors with high affinity and induced even higher levels of signaling. These results suggest that sequences surrounding Pro-258 may be involved in ligand activation of the receptor. Conformational changes in transmembrane domain 6 may effect a change in the adjacent sequences in the third intracellular loop that are thought to function in G protein activation. Greater than 90% of all G protein-coupled receptors contain a proline residue at a similar position in transmembrane domain 6, suggesting that this aspect of receptor activation may be conserved in other receptors.

Minimizing a binding domain from protein A.

We present a systematic approach to minimizing the Z-domain of protein A, a three-helix bundle (59 residues total) that binds tightly (Kd = 10 nM) to the Fc portion of an immunoglobin IgG1. Despite the fact that all the contacts seen in the x-ray structure of the complex with the IgG are derived from residues in the first two helices, when helix 3 is deleted, binding affinity is reduced > 10(5)-fold (Kd > 1 mM). By using structure-based design and phage display methods, we have iteratively improved the stability and binding affinity for a two-helix derivative, 33 residues in length, such that it binds IgG1, with a Kd of 43 nM. This was accomplished by stepwise selection of random mutations from three regions of the truncated Z-peptide: the 4 hydrophobic residues from helix 1 and helix 2 that contacted helix 3 (the exoface), followed by 5 residues between helix 1 and helix 2 (the intraface), and lastly by 19 residues at or near the interface that interacts with Fc (the interface). As selected mutations from each region were compiled (12 in total), they led to progressive increases in affinity for IgG, and concomitant increases in alpha-helical content reflecting increased stabilization of the two-helix scaffold. Thus, by sequential increases in the stability of the structure and improvements in the quality of the intermolecular contacts, one can reduce larger binding domains to smaller ones. Such mini-protein binding domains are more amenable to synthetic chemistry and thus may be useful starting points for the design of smaller organic mimics. Smaller binding motifs also provide simplified and more tractable models for understanding determinants of protein function and stability.

Structure and conformational changes of DNA topoisomerase II visualized by electron microscopy.

Type II DNA topoisomerases, which create a transient gate in duplex DNA and transfer a second duplex DNA through this gate, are essential for topological transformations of DNA in prokaryotic and eukaryotic cells and are of interest not only from a mechanistic perspective but also because they are targets of agents for anticancer and antimicrobial chemotherapy. Here we describe the structure of the molecule of human topoisomerase II [DNA topoisomerase (ATP-hydrolyzing), EC 5.99.1.3] as seen by scanning transmission electron microscopy. A globular approximately 90-angstrom diameter core is connected by linkers to two approximately 50-angstrom domains, which were shown by comparison with genetically truncated Saccharomyces cerevisiae topoisomerase II to contain the N-terminal region of the approximately 170-kDa subunits and that are seen in different orientations. When the ATP-binding site is occupied by a nonhydrolyzable ATP analog, a quite different structure is seen that results from a major conformational change and consists of two domains approximately 90 angstrom and approximately 60 angstrom in diameter connected by a linker, and in which the N-terminal domains have interacted. About two-thirds of the molecules show an approximately 25 A tunnel in the apical part of the large domain, and the remainder contain an internal cavity approximately 30 A wide in the large domain close to the linker region. We propose that structural rearrangements lead to this displacement of an internal tunnel. The tunnel is likely to represent the channel through which one DNA duplex, after capture in the clamp formed by the N-terminal domains, is transferred across the interface between the enzyme's subunits. These images are consistent with biochemical observations and provide a structural basis for understanding the reaction of topoisomerase II.

Three-dimensional structure of human protein kinase C interacting protein 1, a member of the HIT family of proteins.

The three-dimensional structure of protein kinase C interacting protein 1 (PKCI-1) has been solved to high resolution by x-ray crystallography using single isomorphous replacement with anomalous scattering. The gene encoding human PKCI-1 was cloned from a cDNA library by using a partial sequence obtained from interactions identified in the yeast two-hybrid system between PKCI-1 and the regulatory domain of protein kinase C-beta. The PKCI-1 protein was expressed in Pichia pastoris as a dimer of two 13.7-kDa polypeptides. PKCI-1 is a member of the HIT family of proteins, shown by sequence identity to be conserved in a broad range of organisms including mycoplasma, plants, and humans. Despite the ubiquity of this protein sequence in nature, no distinct function has been shown for the protein product in vitro or in vivo. The PKCI-1 protomer has an alpha+beta meander fold containing a five-stranded antiparallel sheet and two helices. Two protomers come together to form a 10-stranded antiparallel sheet with extensive contacts between a helix and carboxy terminal amino acids of a protomer with the corresponding amino acids in the other protomer. PKCI-1 has been shown to interact specifically with zinc. The three-dimensional structure has been solved in the presence and absence of zinc and in two crystal forms. The structure of human PKCI-1 provides a model of this family of proteins which suggests a stable fold conserved throughout nature.

Structure and function in rhodopsin: correct folding and misfolding in point mutants at and in proximity to the site of the retinitis pigmentosa mutation Leu-125-->Arg in the transmembrane helix C.

L125R is a mutation in the transmembrane helix C of rhodopsin that is associated with autosomal dominant retinitis pigmentosa. To probe the orientation of the helix and its packing in the transmembrane domain, we have prepared and studied the mutations E122R, I123R, A124R, S127R, L125F, and L125A at, and in proximity to, the above mutation site. Like L125R, the opsin expressed in COS-1 cells from E122R did not bind 11-cis-retinal, whereas those from I123R and S127R formed the rhodopsin chromophore partially. A124R opsin formed the rhodopsin chromophore (lambda max 495 nm) in the dark, but the metarhodopsin II formed on illumination decayed about 6.5 times faster than that of the wild type and was defective in transducin activation. The mutant opsins from L125F and L125A bound 11-cis-retinal only partially, and in both cases, the mixtures of the proteins produced were separated into retinal-binding and non-retinal-binding (misfolded) fractions. The purified mutant rhodopsin from L125F showed lambda max at 500 nm, whereas that from L125A showed lambda max at 503 nm. The mutant rhodopsin L125F showed abnormal bleaching behavior and both mutants on illumination showed destabilized metarhodopsin II species and reduced transducin activation. Because previous results have indicated that misfolding in rhodopsin is due to the formation of a disulfide bond other than the normal disulfide bond between Cys-110 and Cys-187 in the intradiscal domain, we conclude from the misfolding in mutants L125F and L125A that the folding in vivo in the transmembrane domain is coupled to that in the intradiscal domain.

Crystal structure of the T state of allosteric yeast chorismate mutase and comparison with the R state.

The crystal structure of the tyrosine-bound T state of allosteric yeast Saccharomyces cerevisiae chorismate mutase was solved by molecular replacement at a resolution of 2.8 angstroms using a monomer of the R-state structure as the search model. The allosteric inhibitor tyrosine was found to bind in the T state at the same binding site as the allosteric activator tryptophan binds in the R state, thus defining one regulatory binding site for each monomer. Activation by tryptophan is caused by the larger steric size of its side chain, thereby pushing apart the allosteric domain of one monomer and helix H8 of the catalytic domain of the other monomer. Inhibition is caused by polar contacts of tyrosine with Arg-75 and Arg-76 of one monomer and with Gly-141, Ser-142, and Thr-145 of the other monomer, thereby bringing the allosteric and catalytic domains closer together. The allosteric transition includes an 8 degree rotation of each of the two catalytic domains relative to the allosteric domains of each monomer (domain closure). Alternatively, this transition can be described as a 15 degree rotation of the catalytic domains of the dimer relative to each other.

Swiveling-domain mechanism for enzymatic phosphotransfer between remote reaction sites.

The crystal structure of pyruvate phosphate dikinase, a histidyl multiphosphotransfer enzyme that synthesizes adenosine triphosphate, reveals a three-domain molecule in which the phosphohistidine domain is flanked by the nucleotide and the phosphoenolpyruvate/pyruvate domains, with the two substrate binding sites approximately 45 angstroms apart. The modes of substrate binding have been deduced by analogy to D-Ala-D-Ala ligase and to pyruvate kinase. Coupling between the two remote active sites is facilitated by two conformational states of the phosphohistidine domain. While the crystal structure represents the state of interaction with the nucleotide, the second state is achieved by swiveling around two flexible peptide linkers. This dramatic conformational transition brings the phosphocarrier residue in close proximity to phosphoenolpyruvate/pyruvate. The swiveling-domain paradigm provides an effective mechanism for communication in complex multidomain/multiactive site proteins.

The crystal structure of human glycosylation-inhibiting factor is a trimeric barrel with three 6-stranded beta-sheets.

Glycosylation-inhibiting factor (GIF) is a cytokine that is involved in the regulation of IgE synthesis. The crystal structure of recombinant human GIF was determined by the multiple isomorphous replacement method. The structure was refined to an R factor of 0.168 at 1.9 angstrom resolution. The overall structure is seen to consist of three interconnected subunits forming a barrel with three 6-stranded beta-sheets on the inside and six alpha-helices on the outside. There is a 5-angstrom-diameter "hole" through the middle of the barrel. The barrel structure of GIF in part resembles other "trefoil" cytokines such as interleukin 1 and fibroblast growth factor. Each subunit has a new class of alpha + beta sandwich structure consisting of two beta-alpha-beta motifs. These beta-alpha-beta motifs are related by a pseudo-twofold axis and resemble both interleukin 8 and the peptide binding domain of major histocompatibility complex protein, although the topology of the polypeptide chain is quite different.

The wing of the enhancer-binding domain of Mu phage transposase is flexible and is essential for efficient transposition.

A tetramer of the Mu transposase (MuA) pairs the recombination sites, cleaves the donor DNA, and joins these ends to a target DNA by strand transfer. Juxtaposition of the recombination sites is accomplished by the assembly of a stable synaptic complex of MuA protein and Mu DNA. This initial critical step is facilitated by the transient binding of the N-terminal domain of MuA to an enhancer DNA element within the Mu genome (called the internal activation sequence, IAS). Recently we solved the three-dimensional solution structure of the enhancer-binding domain of Mu phage transposase (residues 1-76, MuA76) and proposed a model for its interaction with the IAS element. Site-directed mutagenesis coupled with an in vitro transposition assay has been used to assess the validity of the model. We have identified five residues on the surface of MuA that are crucial for stable synaptic complex formation but dispensable for subsequent events in transposition. These mutations are located in the loop (wing) structure and recognition helix of the MuA76 domain of the transposase and do not seriously perturb the structure of the domain. Furthermore, in order to understand the dynamic behavior of the MuA76 domain prior to stable synaptic complex formation, we have measured heteronuclear 15N relaxation rates for the unbound MuA76 domain. In the DNA free state the backbone atoms of the helix-turn-helix motif are generally immobilized whereas the residues in the wing are highly flexible on the pico- to nanosecond time scale. Together these studies define the surface of MuA required for enhancement of transposition in vitro and suggest that a flexible loop in the MuA protein required for DNA recognition may become structurally ordered only upon DNA binding.

Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain.

A long-term goal in the field of restriction-modification enzymes has been to generate restriction endonucleases with novel sequence specificities by mutating or engineering existing enzymes. This will avoid the increasingly arduous task of extensive screening of bacteria and other microorganisms for new enzymes. Here, we report the deliberate creation of novel site-specific endonucleases by linking two different zinc finger proteins to the cleavage domain of Fok I endonuclease. Both fusion proteins are active and under optimal conditions cleave DNA in a sequence-specific manner. Thus, the modular structure of Fok I endonuclease and the zinc finger motifs makes it possible to create "artificial" nucleases that will cut DNA near a predetermined site. This opens the way to generate many new enzymes with tailor-made sequence specificities desirable for various applications.