Crystal structure of a catalytic antibody with a serine protease active site.

The three-dimensional structure of an unusually active hydrolytic antibody with a phosphonate transition state analog (hapten) bound to the active site has been solved to 2.5 A resolution. The antibody (17E8) catalyzes the hydrolysis of norleucine and methionine phenyl esters and is selective for amino acid esters that have the natural alpha-carbon L configuration. A plot of the pH-dependence of the antibody-catalyzed reaction is bell-shaped with an activity maximum at pH 9.5; experiments on mechanism lend support to the formation of a covalent acyl-antibody intermediate. The structural and kinetic data are complementary and support a hydrolytic mechanism for the antibody that is remarkably similar to that of the serine proteases. The antibody active site contains a Ser-His dyad structure proximal to the phosphorous atom of the bound hapten that resembles two of the three components of the Ser-His-Asp catalytic triad of serine proteases. The antibody active site also contains a Lys residue to stabilize oxyanion formation, and a hydrophobic binding pocket for specific substrate recognition of norleucine and methionine side chains. The structure identifies active site residues that mediate catalysis and suggests specific mutations that may improve the catalytic efficiency of the antibody. This high resolution structure of a catalytic antibody-hapten complex shows that antibodies can converge on active site structures that have arisen through natural enzyme evolution.

Domain separation in the activation of glycogen phosphorylase a.

The crystal structure of glycogen phosphorylase a complexed with its substrates, orthophosphate and maltopentaose, has been determined and refined at a resolution of 2.8 angstroms. With oligosaccaride bound at the glycogen storage site, the phosphate ion binds at the catalytic site and causes the regulatory and catalytic domains to separate with the loss of stabilizing interactions between them. Homotropic cooperativity between the active sites of the allosteric dimer results from rearrangements in isologous contacts between symmetry-related helices in the subunit interface. The conformational changes in the core of the interface are correlated with those observed on covalent activation by phosphorylation at Ser14 (phosphorylase b----a).

A protein phosphorylation switch at the conserved allosteric site in GP.

A phosphorylation-initiated mechanism of local protein refolding activates yeast glycogen phosphorylase (GP). Refolding of the phosphorylated amino-terminus was shown to create a hydrophobic cluster that wedges into the subunit interface of the enzyme to trigger activation. The phosphorylated threonine is buried in the allosteric site. The mechanism implicates glucose 6-phosphate, the allosteric inhibitor, in facilitating dephosphorylation by dislodging the buried covalent phosphate through binding competition. Thus, protein phosphorylation-dephosphorylation may also be controlled through regulation of the accessibility of the phosphorylation site to kinases and phosphatases. In mammalian glycogen phosphorylase, phosphorylation occurs at a distinct locus. The corresponding allosteric site binds a ligand activator, adenosine monophosphate, which triggers activation by a mechanism analogous to that of phosphorylation in the yeast enzyme.

High-resolution chromosome sorting and DNA spot-blot analysis assign McArdle's syndrome to chromosome 11.

A rapid gene-mapping system uses a high-resolution, dual-laser sorter to identify genes from separate human chromosomes prepared with a new stain combination. This system was used to sort 21 unique chromosome types onto nitrocellulose filter papers. Several labeled gene probes hybridized to the sorted chromosomal DNA types predicted by their previous chromosome assignments. The skeletal muscle glycogen phosphorylase gene was then mapped to a portion of chromosome 11 by spot blotting normal and translocated chromosomes.

Structural basis for the activation of glycogen phosphorylase b by adenosine monophosphate.

The three-dimensional structure of the activated state of glycogen phosphorylase (GP) as induced by adenosine monophosphate (AMP) has been determined from crystals of pyridoxalpyrophosphoryl-GP. The same quaternary changes relative to the inactive conformation as those induced by phosphorylation are induced by AMP, although the two regulatory signals function through different local structural mechanisms. Moreover, previous descriptions of the phosphorylase active state have been extended by demonstrating that, on activation, the amino- and carboxyl-terminal domains of GP rotate apart by 5 degrees, thereby increasing access of substrates to the catalytic site. The structure also reveals previously unobserved interactions with the nucleotide that accounts for the specificity of the nucleotide binding site for AMP in preference to inosine monophosphate.

Hormone-dependent coactivator binding to a hydrophobic cleft on nuclear receptors.

The ligand-binding domain of nuclear receptors contains a transcriptional activation function (AF-2) that mediates hormone-dependent binding of coactivator proteins. Scanning surface mutagenesis on the human thyroid hormone receptor was performed to define the site that binds the coactivators, glucocorticoid receptor-interacting protein 1 (GRIP1) and steroid receptor coactivator 1 (SRC-1). The residues involved encircle a small surface that contains a hydrophobic cleft. Ligand activation of transcription involves formation of this surface by folding the carboxyl-terminal alpha helix against a scaffold of three other helices. These features may represent general ones for nuclear receptors.

The three-dimensional structure of Asn102 mutant of trypsin: role of Asp102 in serine protease catalysis.

The structure of the Asn102 mutant of trypsin was determined in order to distinguish whether the reduced activity of the mutant at neutral pH results from an altered active site conformation or from an inability to stabilize a positive charge on the active site histidine. The active site structure of the Asn102 mutant of trypsin is identical to the native enzyme with respect to the specificity pocket, the oxyanion hole, and the orientation of the nucleophilic serine. The observed decrease in rate results from the loss of nucleophilicity of the active site serine. This decreased nucleophilicity may result from stabilization of a His57 tautomer that is unable to accept the serine hydroxyl proton.

Phosphorylase: a biological transducer.

A transducer is a device that receives energy from one system and transmits it, often in a different form, to another. Glycogen phosphorylase receives information from the cell or organism in the form of metabolic signals. The energy associated with the binding of these ligand signals is integrated and transmitted at an atomic level, allowing precise adjustment of the enzymatic activity. Understanding this elegant allosteric control has required several different approaches, but the structural requirements of allostery are being defined.

Engineered metalloregulation in enzymes.

Protein engineering of metal-dependent enzyme activity is now possible due to the wealth of information available about metalloproteins. The results emerging from these studies provide insight into our understanding of the chemistry of metals in macromolecular environments as well as the biology of metal-protein interactions.

Molecular analysis of GPH1, the gene encoding glycogen phosphorylase in Saccharomyces cerevisiae.

In yeast cells, the activity of glycogen phosphorylase is regulated by cyclic AMP-mediated phosphorylation of the enzyme. We have previously cloned the gene for glycogen phosphorylase (GPH1) in Saccharomyces cerevisiae. To assess the role of glycogen and phosphorylase-catalyzed glycogenolysis in the yeast life cycle, yeast strains lacking a functional GPH1 gene or containing multiple copies of the gene were constructed. GPH1 was found not to be an essential gene in yeast cells. Haploid cells disrupted in GPH1 lacked phosphorylase activity and attained higher levels of intracellular glycogen but otherwise were similar to wild-type cells. Diploid cells homozygous for the disruption were able to sporulate and give rise to viable ascospores. Absence of functional GPH1 did not impair cells from synthesizing and storing trehalose. Increases in phosphorylase activity of 10- to 40-fold were detected in cells carrying multiple copies of GPH1-containing 2 microns plasmid. Northern (RNA) analysis indicated that GPH1 transcription was induced at the late exponential growth phase, almost simultaneous with the onset of intracellular glycogen accumulation. Thus, the low level of glycogen in exponential cells was not primarily maintained through regulating the phosphorylation state of a constitutive amount of phosphorylase. GPH1 did not appear to be under formal glucose repression, since transcriptional induction occurred well in advance of glucose depletion from the medium.

Nucleotide switches in molecular motors: structural analysis of kinesins and myosins.

Recent breakthroughs in the structural biology of cytoskeletal motor proteins show that two distinct families of motors--kinesins and myosins - use a similar mechanism of conformational switching for converting small structural changes in their nucleotide-binding sites into larger movements to provide force generation and motion. This mechanism is found to be similar to that employed by G proteins, the well-known molecular switches that regulate protein-protein interactions in many biological systems.

Distinct phosphorylation signals converge at the catalytic center in glycogen phosphorylases.

BACKGROUND: Glycogen phosphorylases (GPs) catalyze the conversion of the storage form of carbohydrate (glycogen) to the readily usable form (glucose-1-phosphate) to provide cellular energy. Members of this enzyme family have evolved diverse regulatory mechanisms that control a conserved catalytic function. The mammalian and yeast GPs are expressed as inactive forms requiring phosphorylation for activation. Phosphorylation of yeast GP occurs at a distinct site from that of mammalian GP. This work addresses the structural basis by which distinct activation signals relay to the conserved catalytic site in yeast and mammalian GPs. Such knowledge may help understand the principles by which diverse biological regulation evolves. RESULTS: We have compared the crystal structures of the unphosphorylated and phosphorylated forms of yeast GP and propose a relay which links phosphorylation to enzyme activation. Structural components along the activation relay becomes more conserved within the GP family downstream along the relay, towards the catalytic center. Despite distinct upstream activation signals, a response element downstream of the relay leading to the catalytic center is conserved in all GPs. The response element consists of ten hydrophobic residues dispersed over two subunits of the homodimer. Phosphorylation induces hydrophobic condensation of these residues via structural rearrangement, which triggers conformation change of the active site GATE loop, leading to enzyme activation. CONCLUSIONS: Members of the GP family with diverse activation mechanisms have evolved from a constitutively active ancestral enzyme which has the TOWER hydrophobic response element in the active position. Diverse regulation evolved as a result of evolutionary constraint on the downstream response element in the active state, coupled with flexibility and variability in elements of the upstream relays.

The evolution of an allosteric site in phosphorylase.

BACKGROUND: Glycogen phosphorylases consist of a conserved catalytic core onto which different regulatory sites are added. By comparing the structures of isozymes, we hope to understand the structural principles of allosteric regulation in this family of enzymes. Here, we focus on the differences in the glucose 6-phosphate (Glc-6-P) binding sites of two isozymes. RESULTS: We have refined the structure of Glc-6-P inhibited yeast phosphorylase b to 2.6 A and compared it with known structures of muscle phosphorylase. Glc-6-P binds in a novel way, interacting with a distinct set of secondary elements. Structural links connecting the Glc-6-P binding sites and catalytic sites are conserved, although the specific contacts are not. CONCLUSIONS: Our comparison reveals that the Glc-6-P binding site was modified over the course of evolution from yeast to vertebrates to become a bi-functional switch. The additional ability of muscle phosphorylase to be activated by AMP required the recruitment of structural elements into the binding site and sequence changes to create a binding subsite for adenine, whilst maintaining links to the catalytic site.

A target within the target: probing cruzain's P1' site to define structural determinants for the Chagas' disease protease.

BACKGROUND: Cysteine proteases of the papain superfamily are present in nearly all groups of eukaryotes and play vital roles in a wide range of biological processes and diseases, including antigen and hormone processing, bacterial infection, arthritis, osteoporosis, Alzheimer's disease and cancer-cell invasion. Because they are critical to the life-cycle progression of many pathogenic protozoa, they represent potential targets for selective inhibitors. Chagas' disease, the leading cause of death due to heart disease in Latin American countries, is transmitted by Trypanosoma cruzi. Cruzain is the major cysteine protease of T cruzi and has been the target of extensive structure-based drug design. RESULTS: High-resolution crystal structures of cruzain bound to a series of potent phenyl-containing vinyl-sulfone, sulfonate and sulfonamide inhibitors have been determined. The structures show a consistent mode of interaction for this family of inhibitors based on a covalent Michael addition formed at the enzyme's active-site cysteine, hydrophobic interactions in the S2 substrate-binding pocket and a strong constellation of hydrogen bonding in the S1' region. CONCLUSIONS: The series of vinyl-sulfone-based inhibitors examined in complex with cruzain was designed to probe recognition and binding potential of an aromatic-rich region of the enzyme. Analysis of the interactions formed shows that aromatic interactions play a less significant role, whereas the strength and importance of hydrogen bonding in the conformation adopted by the inhibitor upon binding to the enzyme was highlighted. A derivative of one inhibitor examined is currently under development as a therapeutic agent against Chagas' disease.

Purification and crystallization of bovine liver phosphorylase.

We have purified and crystallized bovine liver phosphorylase a. Starting from 2.5 kg of liver, we obtain 250 mg of phosphorylase a, with a specific activity of 90 units/mg, representing 15% recovery. SDS polyacrylamide gels show three bands, a 95 kDa band with the same mobility as muscle phosphorylase, and two smaller bands of 55 kDa and 40 kDa, which are probably proteolytic fragments. These fragments remain associated and have native conformation and catalytic activity. Crystals which diffract to 2.8 A resolution, were prepared by the hanging drop method using polyethylene glycol PEG 4000 as precipitant. The crystals were prepared in the presence of activators maltotriose and phosphite and crack when placed in solutions containing the inhibitors glucose and caffeine. This suggests phosphorylase is present in an active conformation.

Substitution of lysine for arginine at position 199 of a hypoxanthine phosphoribosyltransferase interferes with binding of the primary substrate to the active site.

Lysine was substituted for a conserved arginine at position 199 of the schistosomal hypoxanthine phosphoribosyltransferase (HPRT). This resulted in a > or = 35-fold increase in the K(M) for binding phosphoribosyl-pyrophosphate (PRPP). The possible functional role of R199 in tertiary structure, as well as in the binding of PRPP, is interpreted in the context of the reported three dimensional structure for the human HPRT.

Expression of the protease inhibitor ecotin and its co-crystallization with trypsin.

We have expressed the serine protease inhibitor ecotin to high levels (greater than 400 mg/l of cell culture) in its natural mileau, the Escherichia coli periplasm, using the endogenous signal peptide and the heterologous tac promoter. After induction, functional, soluble ecotin comprises 15% of total cellular protein. This expression system has facilitated initiation of a crystallographic study to determine the structural basis for inhibition of the pancreatic serine proteases by ecotin. Ecotin was co-crystallized with rat trypsin mutant D102N. Preliminary crystallographic analysis of co-crystals showed that they diffract to at least 2.7 A, and indicate that they belong to the monoclinic space group, P21. The cell constants are a = 52.0 A, b = 93.3 A, c = 160.7 A, and beta = 96 degrees. Four molecules each of trypsin and ecotin are found in the asymmetric unit.

Purification and crystallization of glycogen phosphorylase from Saccharomyces cerevisiae.

Glycogen phosphorylase from Saccharomyces cerevisiae is activated by the covalent phosphorylation of a single threonine residue in the N terminus of the protein. We have hypothesized that the structural features that effect activation must be distinct from those characterized in rabbit muscle phosphorylase because the two enzymes have unrelated phosphorylation sites located in dissimilar protein contexts. To understand this potentially novel mechanism of activation by phosphorylation, we require information at atomic resolution of the phosphorylated and unphosphorylated forms of the enzyme. To this end, we have purified, characterized and crystallized glycogen phosphorylase from S. cerevisiae. The enzyme was isolated from a phosphorylase-deficient strain harboring a multicopy plasmid containing the phosphorylase gene under the control of its own promoter. One liter of cultured cells yields 12 mg of crystallizable material. The purified protein was not phosphorylated and had an activity of 4.7 units/mg in the presence of saturating amounts of substrate. Yeast phosphorylase was crystallized in four different crystal forms, only one of which is suitable for diffraction studies at high resolution. The latter belongs to space group P4(1)2(1)2 with unit cell constants of a = 161.1 A and c = 175.5 A Based on the density of the crystals, the solvent content is 49.7%, indicating that the asymmetric unit contains the functional dimer of yeast phosphorylase.

Crystallographic analysis of trypsin-G226A. A specificity pocket mutant of rat trypsin with altered binding and catalysis.

The crystal structure of trypsin-G226A has been determined, in the presence of benzamidine, to a resolution of 1.75 A with an R-factor of 14.6%. The mutation was designed to alter substrate specificity by disrupting arginine binding, but was previously found to disrupt catalysis to a greater extent than binding. The arginine analog, benzamidine, has rotated 40 degrees and 49 degrees and translated 1.1 A in the specificity pocket, relative to the position in wild-type trypsin. The salt-bridge between the amidinium group of benzamidine and the carboxylate of D189 as well as four other hydrogen bonds have been replaced by a set of six new hydrogen bonds. Based on these interactions, computer modeling of an arginine substrate demonstrates that arginine terminal nitrogen atoms can occupy the new benzamidine nitrogen positions with torsion angle adjustments and without short contacts. In the secondary orientation, arginine substrates appear to be forced out of alignment with the active site. This may account for the larger drop in kcat with arginine relative to lysine substrates. A second possible cause of the altered activity is a change of the enzyme structure with concomitant loss of activity. No evidence of such a change is seen in the co-ordinates or temperature factors of the trypsin-G226A-benzamidine complex. A226 disrupts mainly the co-ordinates of amino acids with which it has direct contacts such that the effects of the mutation are absorbed locally.

Crystal structure of rat trypsin-S195C at -150 degrees C. Analysis of low activity of recombinant and semisynthetic thiol proteases.

The X-ray crystal structure of trypsin-S195C, a rat anionic trypsin mutant in which the active site serine has been replaced by cysteine, was determined at -150 degrees C and room temperature to 1.6 A resolution, R = 15.4% and 1.8 A resolution, R = 15.0%, respectively. Cryo-crystallography was employed to improve the quality of the diffraction data and the resulting structure by eliminating radiation damage and decreasing atomic thermal motion. The average temperature factor decreased by 10 A2 relative to that of the room temperature structure. No radiation-induced decay of the data was detected. The side-chains of the catalytic cysteine and histidine of trypsin-S195C are found with 25% occupancy in secondary orientations rotated 104 degrees and 90 degrees out of the active site, respectively. These alterations, as well as more subtle changes in the active site may be caused by the oxidation of the catalytic sulfur to sulfenic acid. The position of the carbonyl carbon of the tetrahedral intermediate analog, p-amidinophenylpyruvic acid, modeled into trypsin-S195C, is 1.1 A from the catalytic sulfur. The large size and altered approach of the catalytic sulfur to substrates could account for the observed low catalytic activity relative to wild-type trypsin. In addition to the benzamidine in the specificity pocket, two additional binding sites for benzamidine are characterized. One of these mediates an intermolecular contact that appears to maintain the crystal lattice.