FGF binding and FGF receptor activation by synthetic heparan-derived di- and trisaccharides.

Fibroblast growth factors (FGFs) require a polysaccharide cofactor, heparin or heparan sulfate (HS), for receptor binding and activation. To probe the molecular mechanism by which heparin or HS (heparin/HS) activates FGF, small nonsulfated oligosaccharides found within heparin/HS were assayed for activity. These synthetic and isomerically pure compounds can activate the FGF signaling pathway. The crystal structures of complexes between FGF and these heparin/HS oligosaccharides reveal several binding sites on FGF and constrain possible mechanisms by which heparin/HS can activate the FGF receptor. These studies establish a framework for the molecular design of compounds capable of modulating FGF activity.

Structural basis of chaperone function and pilus biogenesis.

Many Gram-negative pathogens assemble architecturally and functionally diverse adhesive pili on their surfaces by the chaperone-usher pathway. Immunoglobulin-like periplasmic chaperones escort pilus subunits to the usher, a large protein complex that facilitates the translocation and assembly of subunits across the outer membrane. The crystal structure of the PapD-PapK chaperone-subunit complex, determined at 2.4 angstrom resolution, reveals that the chaperone functions by donating its G(1) beta strand to complete the immunoglobulin-like fold of the subunit via a mechanism termed donor strand complementation. The structure of the PapD-PapK complex also suggests that during pilus biogenesis, every subunit completes the immunoglobulin-like fold of its neighboring subunit via a mechanism termed donor strand exchange.

Mutagenesis of the BH3 domain of BAX identifies residues critical for dimerization and killing.

The BCL-2 family of proteins is comprised of proapoptotic as well as antiapoptotic members (S. N. Farrow and R. Brown, Curr. Opin. Genet. Dev. 6:45-49, 1996). A prominent death agonist, BAX, forms homodimers and heterodimerizes with multiple antiapoptotic members. Death agonists have an amphipathic alpha helix, called BH3; however, the initial assessment of BH3 in BAX has yielded conflicting results. Our BAX deletion constructs and minimal domain constructs indicated that the BH3 domain was required for BAX homodimerization and heterodimerization with BCL-2, BCL-XL, and MCL-1. An extensive site-directed mutagenesis of BH3 revealed that substitutions along the hydrophobic face of BH3, especially charged substitutions, had the greatest affects on dimerization patterns and death agonist activity. Particularly instructive was the BAX mutant mIII-1 (L63A, G67A, L70A, and M74A), which replaced the hydrophobic face of BH3 with alanines, preserving its amphipathic nature. BAXmIII-1 failed to form heterodimers or homodimers by yeast two-hybrid or immunoprecipitation analysis yet retained proapoptotic activity. This suggests that BAX's killing function reflects mechanisms beyond its binding to BCL-2 or BCL-XL to inhibit them or simply displace other protein partners. Notably, BAXmIII-1 was found predominantly in mitochondrial membranes, where it was homodimerized as assessed by homobifunctional cross-linkers. This characteristic of BAXmIII-1 correlates with its capacity to induce mitochondrial dysfunction, caspase activation, and apoptosis. These data are consistent with a model in which BAX death agonist activity may require an intramembranous conformation of this molecule that is not assessed accurately by classic binding assays.

Chaperone-assisted pilus assembly and bacterial attachment.

Bacterial pili assembled by the chaperone-usher pathway can mediate microbial attachment, an early step in the establishment of an infection, by binding specifically to sugars present in host tissues. Recent work has begun to reveal the structural basis both of chaperone function in the biogenesis of these pili and of bacterial attachment.

Mass spectrometric and thermodynamic studies reveal the role of water molecules in complexes formed between SH2 domains and tyrosyl phosphopeptides.

BACKGROUND: SH2 domains have a fundamental role in signal transduction. These domains interact with proteins containing phosphorylated tyrosine residues and, in doing so, mediate the interactions of proteins involved in tyrosine kinase signalling. The issue of specificity in SH2 domain interactions is therefore of great interest in terms of understanding tyrosine kinase signal-transduction pathways and in the discovery of drugs to inhibit them. Water molecules are found at the interfaces of many complexes, however, to date little attention has been paid to their role in dictating specificity. RESULTS: Here we use a combination of nanoflow electrospray ionization mass spectrometry (ESI-MS), isothermal titration calorimetry and structural data to investigate the effect of water molecules in complexes formed between the SH2 domain of tyrosine kinase Src and tyrosyl phosphopeptides. Binding studies have been performed using a series of different peptides that were selected to allow changes in the water content at the complex interface and demonstrate changes in specificity. ESI-MS enables quantification of the number of water molecules that interact with a higher affinity than those generally found solvating the biomolecular complex. CONCLUSIONS: Comparing the interactions of different peptides, we show that an intricate network of water molecules have a key role in dictating specificity. The use of mass spectrometry to quantify tightly bound water molecules may prove of general use in structural biology, where an independent determination of the water molecules associated with a structure would be advantageous. Furthermore, the ability to assess whether given water molecules are important in high-affinity binding could make this method a precious tool in drug design.

Synthetic protein transduction domains: enhanced transduction potential in vitro and in vivo.

The protein transduction domain (PTD) embedded in the HIV TAT protein (amino acids 47-57) has been shown to successfully mediate the introduction of heterologous peptides and proteins in excess of Mr 100,000 into mammalian cells in vitro and in vivo. We report here that the modeled structure of the TAT PTD is a strong amphipathic helix. On the basis of this information, we synthesized a series of synthetic PTDs that strengthen the alpha-helical content and optimize the placement of arginine residues. Several PTD peptides possessed significantly enhanced protein transduction potential compared with TAT in vitro and in vivo. These optimized PTDs have the potential to deliver both existing and novel anticancer therapeutics.

Purification and characterization of two endo-beta-1,4-D-glucanases from Sclerotinia sclerotiorum.

The endo-beta-1,4-D-glucanase (EC 3.2.1.4) enzymes produced in vitro by Sclerotinia sclerotiorum consisted of numerous isoforms with pI ranging from 3.5 to 6.2. The two dominant isoforms, labelled EG1 and EG2, were purified. The pI of EG1 and EG2 were 6.2 and 3.7, respectively. Their molecular weights were, respectively, 48,000 and 34,000. EG1 and EG2 were both active towards carboxymethyl cellulose. However, EG1 was also active towards 4-methylumbelliferyl cellobioside. The amino acid compositions of EG1 and EG2 were different. The N-terminal amino acid sequences of the two enzymes showed little homology. However, the N-terminal sequence of EG1 showed considerable homology to the published N-terminal sequence of an endoglucanase (EG1) from Schizohyllum commune.

Purification of the beta-glucosidase from Sclerotinia sclerotiorum.

A beta-glucosidase (EC 3.2.1.21) has been isolated from culture filtrates of the fungus Sclerotinia sclerotiorum. The protein was purified by gel filtration on a column of Bio-Gel P-300 and by ion exchange chromatography on DEAE-Bio-Gel A. The molecular weight, determined by gel filtration, was 240,000. Km values for the enzyme towards p-nitrophenyl-beta-D-glucoside and cellobiose were respectively 0.10 mM and 1.23 mM. The beta-glucosidase activity was found to be strongly associated with a beta-xylosidase (EC 3.2.1.37) activity, suggesting that both activities could be represented in a single protein complex.

Purification and characterization of two endopolygalacturonases from Sclerotinia sclerotiorum.

The endopolygalacturonase (EC 3.2.1.15) enzyme produced in vitro by Sclerotinia sclerotiorum were found to consist of numerous isoforms covering a broad pI range. Two of the isoforms, labelled PG2 and PG3, were purified using gel-filtration chromatography, isoelectric focusing and anion-exchange chromatography. The pIs of PG2 and PG3 were, respectively, 4.8 and 4.9. Their molecular weights were similar. Both enzymes hydrolysed 0.9% of the bonds in reaching a 50% reduction in viscosity. However, their enzymic parameters were different. Their amino acid compositions differed only in the aspartic acid-asparagine content. The N-terminal sequences differed at the fourth amino acid only. PG2 and PG3 exhibited a high level of glycosylation compared to a similar enzyme isolated from Aspergillus niger. Antibody raised against PG3 was shown to crossreact with PG2, but not with the enzyme purified from Aspergillus niger.

The structure of myristoyl-CoA:protein N-myristoyltransferase.

Protein N-myristoylation is a covalent modification that occurs co-translationally in eukaryotes. Myristate, a rare 14 carbon saturated fatty acid (C14:0), is attached, via an amide linkage, to the N-terminal glycine of a subset of eukaryotic and viral proteins by myristoyl-CoA:protein N-myristoyltransferase (Nmt). Genetic and biochemical studies have established that Nmt is a target for development of a new class of fungicidal drugs. The enzyme is also a potential target for development of antiviral and antineoplastic agents. The structure of Saccharomyces cerevisiae Nmt1p has been determined recently with bound substrate analogs. The Nmt fold resembles the fold of members of the GCN5-related N-acetyltransferase superfamily. The structure reveals how Nmt's myristoyl-CoA and peptide substrates are recognized and bound, and what elements control the enzyme's ordered kinetic mechanism. Acyl transfer occurs through the nucleophilic addition-elimination reaction: an oxyanion hole formed by main chain atoms polarizes the thioester carbonyl and stabilizes the transition state while deprotonation of the ammonium of the Gly acceptor appears to be mediated by Nmt's C-terminal carboxylate. The use of main chain carboxylate atoms as general base catalyst is a novel feature.

Mutational investigation of the specificity determining region of the Src SH2 domain.

SH2 domains are protein modules which bind tyrosine phosphorylated sequences in many signaling pathways. These domains contain two regions with specialized functions: residues in one region form a deep pocket into which the phosphotyrosine of the target inserts, while the other region contains the so-called "specificity determining residues" which interact with the three residues C-terminal to the phosphotyrosine in the target. Here, titration calorimetry and site-directed mutagenesis have been used to probe the importance of eight specificity determining residues of the SH2 domain of the Src kinase involved in contacts with its tyrosine phosphorylated consensus peptide target (sequence pYEEI where pY indicates a phosphotyrosine). Mutating six of these eight residues to Ala individually, resulted in a threefold or less loss in binding affinity; hence the majority of the residues in the specificity determining region are by themselves of minimal importance for binding. Two residues were found to have significant effects on binding: Tyr betaD5 and Lys betaD3. Tyr betaD5 was the most crucial residue as evidenced by the 30-fold loss in affinity when Tyr betaD5 is mutated to Ile. However, while this mutation eliminated the specificity of the Src SH2 domain for the pYEEI peptide sequence, it was not sufficient to switch the specificity of the Src SH2 domain to that of a related SH2 domain which has an Ile at the betaD5 position. Mutation of Lys betaD3 to an Ala residue resulted in a modest reduction in binding affinity (sevenfold). It is interesting that this mutation resulted in a change of specificity affecting the selection of the +1 position residue C-terminal to the phosphotyrosine. Except for the Lys betaD3-+1 Glu interaction which is significantly coupled, only weak energetic coupling was observed across the binding interface, as assessed using double mutant cycles. The results of this study suggest that interactions involving the specificity determining region of SH2 domains may be insufficient by themselves to target single SH2 domains to particular phosphorylated sites.

Investigation of phosphotyrosine recognition by the SH2 domain of the Src kinase.

The binding of tyrosine phosphorylated targets by SH2 domains is required for propagation of many cellular signals in higher eukaryotes; however, the determinants of phosphotyrosine (pTyr) recognition by SH2 domains are not well understood. In order to identify the attributes of pTyr required for high affinity interaction with SH2 domains, the binding of the SH2 domain of the Src kinase (Src SH2 domain) to a dephosphorylated peptide, a phosphoserine-containing peptide, and the amino acid pTyr was studied using titration calorimetry and compared with the binding of a high affinity tyrosyl phosphopeptide. The dephosphorylated peptide and the phosphoserine containing peptide both bind extremely weakly to the Src SH2 domain (DeltaGo (dephosphorylated)=-3.6 kcal/mol, DeltaGo (phosphoserine) >-3.7 kcal/mol); however, the DeltaGo value of pTyr binding is more favorable (-4.7 kcal/mol, or 50 % of the entire binding free energy of a high affinity tyrosyl phosphopeptide). These results indicate that both the phosphate and the tyrosine ring of the pTyr are critical determinants of high affinity binding. Alanine mutagenesis was also used to evaluate the energetic contribution to binding of ten residues located in the pTyr-binding site. Mutation of the strictly conserved Arg betaB5 resulted in a large increase in DeltaGo (DeltaDeltaGo=3.2 kcal/mol) while elimination of the other examined residues each resulted in a significantly smaller (DeltaDeltaGo<1.4 kcal/mol) reduction in affinity, indicating that Arg betaB5 is the single most important determinant of pTyr recognition. However, mutation of Cys betaC3, a residue unique to the Src SH2 domain, surprisingly increased affinity by eightfold (DeltaDeltaGo=-1.1 kcal/mol). Using a double mutant cycle analysis, it was revealed that residues of the pTyr-binding pocket are not coupled to the peptide residues C-terminal to the pTyr. In addition, comparison of each residue's DeltaDeltaGo value upon mutation with that residue's sequence conservation among SH2 domains revealed only a modest correlation between a residue's energetic contribution to pTyr recognition and its conservation throughout evolution. The results of this investigation highlight the importance of a single critical interaction, the buried ionic bond between the phosphate of the pTyr and Arg betaB5 of the SH2 domain, driving the binding of SH2 domains to tyrosine phosphorylated targets.

Crystal structure of Escherichia coli thioredoxin reductase refined at 2 A resolution. Implications for a large conformational change during catalysis.

The crystal structures of three forms of Escherichia coli thioredoxin reductase have been refined: the oxidized form of the wild-type enzyme at 2.1 A resolution, a variant containing a cysteine to serine mutation at the active site (Cys138Ser) at 2.0 A resolution, and a complex of this variant with nicotinamide adenine dinucleotide phosphate (NADP+) at 2.3 A resolution. The enzyme mechanism involves the transfer of reducing equivalents from reduced nicotinamide adenine dinucleotide phosphate (NADPH) to a disulfide bond in the enzyme, via a flavin adenine dinucleotide (FAD). Thioredoxin reductase contains FAD and NADPH binding domains that are structurally similar to the corresponding domains of the related enzyme glutathione reductase. The relative orientation of these domains is, however, very different in the two enzymes: when the FAD domains of thioredoxin and glutathione reductases are superimposed, the NADPH domain of one is rotated by 66 degrees with respect to the other. The observed binding mode of NADP+ in thioredoxin reductase is non-productive in that the nicotinamide ring is more than 17 A from the flavin ring system. While in glutathione reductase the redox active disulfide is located in the FAD domain, in thioredoxin reductase it is in the NADPH domain and is part of a four-residue sequence (Cys-Ala-Thr-Cys) that is close in structure to the corresponding region of thioredoxin (Cys-Gly-Pro-Cys), with a root-mean-square deviation of 0.22 A for atoms in the disulfide bonded ring. There are no significant conformational differences between the structure of the wild-type enzyme and that of the Cys138Ser mutant, except that a disulfide bond is not present in the latter. The disulfide bond is positioned productively in this conformation of the enzyme, i.e. it stacks against the flavin ring system in a position that would facilitate its reduction by the flavin. However, the cysteine residues are relatively inaccessible for interaction with the substrate, thioredoxin. These results suggest that thioredoxin reductase must undergo conformational changes during enzyme catalysis. All three structures reported here are for the same conformation of the enzyme and no direct evidence is available as yet for such conformational changes. The simplest possibility is that the NADPH domain rotates between the conformation observed here and an orientation similar to that seen in glutathione reductase. This would alternately place the nicotinamide ring and the disulfide bond near the flavin ring, and expose the cysteine residues for reaction with thioredoxin in the hypothetical conformation.(ABSTRACT TRUNCATED AT 400 WORDS)

Structural basis for Syk tyrosine kinase ubiquity in signal transduction pathways revealed by the crystal structure of its regulatory SH2 domains bound to a dually phosphorylated ITAM peptide.

The Syk family of kinases, consisting of ZAP-70 and Syk, play essential roles in a variety of immune and non-immune cells. This family of kinases is characterized by the presence of two adjacent SH2 domains which mediate their localization to the membrane through receptor encoded tyrosine phosphorylated motifs. While these two kinases share many structural and functional features, the more ubiquitous nature of Syk has suggested that this kinase may accommodate a greater variety of motifs to mediate its function. We present the crystal structure of the tandem SH2 domain of Syk complexed with a dually phosphorylated ITAM peptide. The structure was solved by multiple isomorphous replacement at 3.0 A resolution. The asymmetric unit comprises six copies of the liganded protein, revealing a surprising flexibility in the relative orientation of the two SH2 domains. The C-terminal phosphotyrosine-binding site is very different from the equivalent region of ZAP-70, suggesting that in contrast to ZAP-70, the two SH2 domains of Syk can function as independent units. The conformational flexibility and structural independence of the SH2 modules of Syk likely provides the molecular basis for the more ubiquitous involvement of Syk in a variety of signal transduction pathways.

Crystallization of DsbA, an Escherichia coli protein required for disulphide bond formation in vivo.

DsbA is a 21 kDa protein that facilitates disulphide bond formation and is required for the correct folding and stability of a number of exported proteins in Escherichia coli. Crystals of oxidized DsbA have been obtained from polyethylene glycol 8000 (20 to 25%), 0.1 M-cacodylate buffer (pH 6.5) and 1% 2-methyl-2,4-pentanediol. Oxidation of the protein is critical for reproducibly obtaining high quality crystals. The resulting crystals diffract to 2 A and belong to the monoclinic space group C2 with cell dimensions a = 117.5 A, b = 65.0 A, c76.3 A, beta = 126.3 degrees with two molecules in the asymmetric unit.

Crystal structure of enteropeptidase light chain complexed with an analog of the trypsinogen activation peptide.

Enteropeptidase is a membrane-bound serine protease that initiates the activation of pancreatic hydrolases by cleaving and activating trypsinogen. The enzyme is remarkably specific and cleaves after lysine residues of peptidyl substrates that resemble trypsinogen activation peptides such as Val-(Asp)4-Lys. To characterize the determinants of substrate specificity, we solved the crystal structure of the bovine enteropeptidase catalytic domain to 2.3 A resolution in complex with the inhibitor Val-(Asp)4-Lys-chloromethane. The catalytic mechanism and contacts with lysine at substrate position P1 are conserved with other trypsin-like serine proteases. However, the aspartyl residues at positions P2-P4 of the inhibitor interact with the enzyme surface mainly through salt bridges with the Nzeta atom of Lys99. Mutation of Lys99 to Ala, or acetylation with acetic anhydride, specifically prevented the cleavage of trypsinogen or Gly-(Asp)4-Lys-beta-naphthylamide and reduced the rate of inhibition by Val-(Asp)4-Lys-chloromethane 22 to 90-fold. For these reactions, Lys99 was calculated to account for 1.8 to 2.5 kcal mol(-1) of the free energy of transition state binding. Thus, a unique basic exosite on the enteropeptidase surface has evolved to facilitate the cleavage of its physiological substrate, trypsinogen.

Crystal structures of a ddATP-, ddTTP-, ddCTP, and ddGTP- trapped ternary complex of Klentaq1: insights into nucleotide incorporation and selectivity.

The mechanism by which DNA polymerase I enzymes function has been the subject of extensive biochemical and structural studies. We previously determined the structure of a ternary complex of the large fragment of DNA polymerase I from Thermus aquaticus (Klentaq1) bound to a primer/template DNA and a dideoxycytidine 5'-triphosphate (ddCTP). In this report, we present the details of the 2.3-A resolution crystal structures of three additional ternary complexes of Klentaq1 bound to a primer/template DNA and a dideoxyguanosine 5'-triphosphate (ddGTP), a dideoxythymidine 5'-triphosphate (ddTTP), or a dideoxyadenosine 5'-triphosphate (ddATP). Comparison of the active site of the four ternary complexes reveals that the protein residues around the nascent base pair (that formed between the incoming dideoxynucleoside triphosphate [ddNTP] and the template base) form a snug binding pocket into which only a correct Watson-Crick base pair can fit. Except in the ternary complex bound to dideoxyguanosine 5'-triphosphate, there are no sequence specific contacts between the protein side chains and the nascent base pair, suggesting that steric constraints imposed by the protein onto the nascent base pair is the major contributor to nucleotide selectivity at the polymerase active site. The protein around the polymerase active site also shows plasticity, which may be responsible for the substrate diversity of the enzyme. Two conserved side chains, Q754 and R573, form hydrogen bonds with the N3 atom in the purine base and O2 atom in the pyrimidine base at the minor groove side of the base pair formed by the incorporated ddNMP and the corresponding template base in all the four ternary complexes. These hydrogen-bonding interactions may provide a means of detecting misincorporation at this position.

Crystal structures of the Klenow fragment of Thermus aquaticus DNA polymerase I complexed with deoxyribonucleoside triphosphates.

The crystal structures of the Klenow fragment of the Thermus aquaticus DNA polymerase I (Klentaq1) complexed with four deoxyribonucleoside triphosphates (dNTP) have been determined to 2.5 A resolution. The dNTPs bind adjacent to the O helix of Klentaq1. The triphosphate moieties are at nearly identical positions in all four complexes and are anchored by three positively charged residues, Arg659, Lys663, and Arg587, and by two polar residues, His639 and Gln613. The configuration of the base moieties in the Klentaq1/dNTP complexes demonstrates variability suggesting that dNTP binding is primarily determined by recognition and binding of the phosphate moiety. However, when superimposed on the Taq polymerase/blunt end DNA complex structure (Eom et al., 1996), two of the dNTP/Klentaq1 structures demonstrate appropriate stacking of the nucleotide base with the 3' end of the DNA primer strand, suggesting that at least in these two binary complexes, the observed dNTP conformations are functionally relevant.

pH-Dependent self-association of the Src homology 2 (SH2) domain of the Src homologous and collagen-like (SHC) protein.

The Src homologous and collagen-like (SHC) protein plays an essential role in signal transduction pathways in that it participates in the chain of events that leads to the activation of the protein Ras. The crystal structure of the SH2 domain of SHC has been determined using the method of multiple isomorphous replacement at a resolution of 2.5 A. The SH2 domain of SHC is similar in fold to other SH2 domains. The peptide-binding surfaces resemble that of the SH2 domain of Src in that a deep pocket is formed where the third amino acid C-terminal to the phosphotyrosine can insert. A novel feature of this structure is the observation of a disulfide bond and an extensive dimer interface between two symmetry-related molecules. Solution studies under reducing conditions using analytical centrifugation and PAGE suggest that the SH2 domain of SHC dimerizes in a pH-dependent manner where low pH conditions (approximately 4.5) are conducive to dimer formation. Dimerization of SHC may have important biological implications in that it may promote the assembly of large heteromultimeric signaling complexes.