Biomedical subjects

G W Zhou

Publications and source records attributed to G W Zhou.

At least 19 recordsLinked to original sources

PubMed2001

Structural analysis of regulatory protein domains using GST-fusion proteins.

The glutathione S-transferase (GST) fusion protein expression system has been used extensively to generate a large quantity of proteins for structural studies. To avoid the inter-domain flexibility introduced by the GST segment, GST-fusion proteins are normally cleaved with proteases to release the GST moiety prior to crystallization. Recently, several reports have shown that GST-fusion proteins can also be used as a vehicle to determine the crystal structures of the attached small peptides and biological regulatory domains. In comparison with the standard method, GST-fusion proteins are more easily crystallized under similar conditions. In addition, the structure of the desired protein or peptide can be determined using the molecular replacement method with the help of the GST structure. Thus, GST-fusion proteins can be used as a new technique for structural determination of small regulatory domains, especially of small peptides. Here, we review the recent progress on this technique, known as GST-driven crystallization. We have summarized and compared different methods of protein preparation and crystallization used by different groups. We have also compared the three-dimensional structures, especially those of the fused peptide segments. Finally, we have discussed the potential effects of the crystal packing on the crystal structure.

Crystallization↗

PubMed2001

Activation of the Akt-related cytokine-independent survival kinase requires interaction of its phox domain with endosomal phosphatidylinositol 3-phosphate.

Protein kinases of the Akt and related serum- and glucocorticoid-regulated kinase (SGK) families are major downstream mediators of phosphatidylinositol (PI) 3-kinase signaling to many cellular processes including metabolic flux, membrane trafficking, and apoptosis. Activation of these kinases is thought to occur at the plasma membrane through their serine and threonine phosphorylation by the phosphoinositide-dependent kinase 1 (PDK1) protein kinase, which interacts with membrane 3'-polyphosphoinositides through its pleckstrin homology (PH) domain. Here, we demonstrate that the SGK family member cytokine-independent survival kinase (CISK) binds strongly and selectively to the monophosphoinositide PI(3)P through its phox homology (PX) domain. Comparing native green fluorescent protein-CISK (EGFP-CISK) to a mutant EGFP-CISK (Y51A) that displays attenuated binding to PI(3)P reveals that this interaction is both necessary and sufficient for its localization to early endosome antigen (EEA1)-positive endosomes. Furthermore, early endosome association of expressed epitope-tagged CISK in COS cells directed by binding of its PX domain to PI(3)P is required for activation of the CISK protein kinase by both insulin-like growth factor-1 and epidermal growth factor. Taken together, these results reveal a critical role of endosomal PI(3)P in the signal transmission mechanism whereby this survival kinase is activated in response to PI3-kinase stimulation by growth factors.

Animals↗

PubMed2001

Phox homology domains specifically bind phosphatidylinositol phosphates.

The recruitment of specific cytosolic proteins to intracellular membranes through binding phosphorylated derivatives of phosphatidylinositol (PtdIns) controls such processes as endocytosis, regulated exocytosis, cytoskeletal organization, and cell signaling. Protein modules such as FVYE domains and PH domains that bind specifically to PtdIns 3-phosphate (PtdIns-3-P) and polyphosphoinositides, respectively, can direct such membrane targeting. Here we show that two representative Phox homology (PX) domains selectively bind to specific phosphatidylinositol phosphates. The PX domain of Vam7p selectively binds PtdIns-3-P, while the PX domain of the CPK PI-3 kinase selectively binds PtdIns-4,5-P(2). In contrast, the PX domain of Vps5p displays no binding to any PtdInsPs that were tested. In addition, the double mutant (Y42A/L48Q) of the PX domain of Vam7p, reported to cause vacuolar trafficking defects in yeast, has a dramatically decreased level of binding to PtdIns-3-P. These data reveal that the membrane targeting function of the Vam7p PX domain is based on its ability to associate with PtdIns-3-P, analogous to the function of FYVE domains.

Amino Acid Motifs↗

PubMed2001

Assay of protein tyrosine phosphatases by using matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

A nonradioactive assay for protein tyrosine phosphatases (PTPs), employing a tyrosine-phosphorylated peptide as a substrate, has been developed and applied to analyze purified enzymes, cell extracts, and immunoprecipitates. The reaction was followed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) in a linear and positive ion mode with delayed extraction. MALDI-TOF MS detects a loss of peptide mass by 80 Da as a result of dephosphorylation and, more importantly, it yields phospho-peptide to dephosphorylated product peak intensity ratios proportional to their concentration ratios. A strong bias of the MALDI-TOF MS toward detection of the non-phospho-peptide allows accurate detection of small fractions of dephosphorylation. The method is highly sensitive and reproducible. It can be applied to general assays of protein phosphatases with various phospho-peptides as substrates.

Oligopeptides↗

PubMed2001

Relation between the flexibility of the WPD loop and the activity of the catalytic domain of protein tyrosine phosphatase SHP-1.

The conserved WPD loop of protein tyrosine phosphatases play an important role in the catalytic activity and the invariant aspartate residue acts as a general acid/base catalyst in the dephosphorylation reaction. In our previous report, we have demonstrated that the catalytic activities of the PTPs are influenced by the flexibility and stability of the WPD loop in its active "open" conformation [Yang et al., 1998]. Phosphatases with a more flexible WPD loop generally have higher specific activity. In this report, we modify the WPD loop of SHP-1 by alanine-scan mutation of the residues flanking the loop and measure their effects on the catalytic activity of the phosphatase. We show that the S418A, V424A, S426A, E427A, and P428A mutants increase the phosphatase activity, possibly due to the increased flexibility of the WPD loop, whereas the L417A, L417G and P425A mutants decrease its phosphatase activity. In addition, we propose that the two-proline residues in the WPD loop (Pro(420) and Pro(425) in SHP-1) work as pivotal points through a conserved hydrophobic network and allows residues between the pivotal points to have maximum flexibility in enhancing the phosphatase activity. Furthermore, our data suggest that the hydrolysis of the phosphoryl-cysteine intermediate, not its formation, is the rate-limiting step with p-nitrophenyl phosphate as the substrate while both the steps are rate-limiting with phosphotyrosine as the substrate.

Alanine↗

PubMed2001

[Esterification of octanoic acid with 1-octanol catalyzed by lipase in W/O microemulsions and in microemulsion-based organogels].

The esterification reactions of octanoic acid with 1-octanol catalyzed by Candida lypolytical (CL) lipase was studied in water-in-oil microemulsions formed by water/bis-(2-ethylhexyl)sulfosuccinate sodium (AOT)/isooctane. The results of kinetic study showed that the reaction follows a Ping-Pong Bi-Bi mechanism. The values of apparent kinetic parameters were determined. Lipase has also been immobilized in gelatin-containing AOT microemulsion-based organogels (MBGs) for retention of catalytic activity. These lipase-containing MBGs proved to be a solid-phase catalysts for use in apolar organic solvents, retaining its higher activity after many runs of esterification reactions.

1-Octanol↗

PubMed2000

Structural basis for substrate specificity of protein-tyrosine phosphatase SHP-1.

The substrate specificity of the catalytic domain of SHP-1, an important regulator in the proliferation and development of hematopoietic cells, is critical for understanding the physiological functions of SHP-1. Here we report the crystal structures of the catalytic domain of SHP-1 complexed with two peptide substrates derived from SIRPalpha, a member of the signal-regulatory proteins. We show that the variable beta5-loop-beta6 motif confers SHP-1 substrate specificity at the P-4 and further N-terminal subpockets. We also observe a novel residue shift at P-2, the highly conserved subpocket in protein- tyrosine phosphatases. Our observations provide new insight into the substrate specificity of SHP-1.

Animals↗

PubMed1999

Crystal structure of the nuclear matrix targeting signal of the transcription factor acute myelogenous leukemia-1/polyoma enhancer-binding protein 2alphaB/core binding factor alpha2.

Transcription factors of the acute myelogenous leukemia (AML)/polyoma enhancer-binding protein (PEBP2alpha)/core-binding factor alpha (CBFA) class are key transactivators of tissue-specific genes of the hematopoietic and bone lineages. AML-1/PEBP2alphaB/CBFA2 proteins participating in transcription are associated with the nuclear matrix. This association is solely dependent on a highly conserved C-terminal protein segment, designated the nuclear matrix targeting signal (NMTS). The NMTS of AML-1 is physically distinct from the nuclear localization signal, operates autonomously, and supports transactivation. Our data indicate that the related AML-3 and AML-2 proteins are also targeted to the nuclear matrix in situ by analogous C-terminal domains. Here we report the first crystal structure of an NMTS in an AML-1 segment fused to glutathione S-transferase. The model of the NMTS consists of two loops connected by a flexible U-shaped peptide chain.

Amino Acid Sequence↗

PubMed1999

Kinetic comparison of the catalytic domains of SHP-1 and SHP-2.

The phosphatase activity of SH2-containing protein tyrosine phosphatase (SHP) is inhibited by its SH2 domains and C-terminal tail. In order to determine the inhibitory effects of the SH2 domains and C-terminal tail, we have expressed and purified the catalytic domains of SHP-1 and SHP-2, and the SH2 domain truncated SHP-1 and SHP-2. We have then measured their kinetic parameters using p-nitrophenyl phosphate (p-NPP) and phosphotyrosine (pY) as substrates under the same experimental conditions. The results indicate that the pH-dependent profiles of SHP-1 and SHP-2 are mainly determined by their catalytic domains. Both enzymes have maximum activity at pH 5.0. In addition, the phosphatase activity of different forms of SHP-1 and SHP-2 decreases as the salt concentration increases. Without SH2 domains, both SHP-1 and SHP-2 are no longer inhibited by their C-terminal tails. However, the C-terminal tail of SHP-1 can further prevent the salt inhibition of the phosphatase activity. Under the same experimental conditions, the catalytic domain of SHP-1 is two times more active than the catalytic domain of SHP-2.

Binding Sites↗

PubMed1998

Crystal structure of the catalytic domain of protein-tyrosine phosphatase SHP-1.

The crystal structures of the protein-tyrosine phosphatase SHP-1 catalytic domain and the complex it forms with the substrate analogue tungstate have been determined and refined to crystallographic R values of 0.209 at 2.5 A resolution and 0.207 at 2.8 A resolution, respectively. Despite low sequence similarity, the catalytic domain of SHP-1 shows high similarity in secondary and tertiary structures with other protein-tyrosine phosphatases (PTPs). In contrast to the conformational changes observed in the crystal structures of PTP1B and Yersinia PTP, the WPD loop (Trp419-Pro428) in the catalytic domain of SHP-1 moves away from the substrate binding pocket after binding the tungstate ion. Sequence alignment and structural analysis suggest that the residues in the WPD loop, especially the amino acid following Asp421, are critical for the movement of WPD loop on binding substrates and the specific activity of protein-tyrosine phosphatases. Our mutagenesis and kinetic measurements have supported this hypothesis.

Amino Acid Sequence↗

PubMed1998

Preliminary crystallographic study of glutathione S-transferase fused with the nuclear matrix targeting signal of the transcription factor AML-1/CBF-alpha2.

A glutathione S-transferase fused with the nuclear matrix targeting signal (GST-NMTS) of AML-1/CBF-alpha2 has been crystallized by the vapor diffusion method using polyethylene glycol (PEG) as the precipitant. The NMTS is a 31-amino-acid signal peptide that can target the AML-1/CBF-alpha2 protein to the nuclear matrix. The crystal belongs to tetragonal space group P43212 with unit cell dimensions a = b = 93.4 A, c = 57.6 A. There is one GST-fusion protein per asymmetric unit. Crystals diffracted to at least 2.7 A and are appropriate for structure determination.

Animals↗

PubMed1997

Expression, purification, and crystallization of the catalytic domain of protein tyrosine phosphatase SHP-1.

The catalytic domain of SHP-1, a SH2-domain containing protein tyrosine phosphatase, has been crystallized by the vapor diffusion method using polyethylene glycol as the precipitant. The crystals belong to the monoclinic space group P21 with unit cell dimensions a = 42.12 A, b = 87.94 A, c = 43.22 A, alpha = 90.0 degrees, beta = 120.12 degrees, and gamma = 90.0 degrees. There is one catalytic domain of SHP-1 per asymmetric unit. X-ray was diffracted to at least 2.5 A and the crystals are appropriate for high-resolution structure determination.

Binding Sites↗

PubMed1995

Mechanistically different catalytic antibodies obtained from immunization with a single transition-state analog.

The variable-region peptide sequence and steady-state kinetic behavior are compared for a family of catalytic antibodies that arose from the same immune response to a transition-state analog. The crystal structure of the most catalytically active member of the family (17E8) has been solved to 2.5 A resolution and shows that the antibody active site contains a SerH99-HisH35 (H = heavy chain) catalytic dyad analogous to the Ser-His-Asp catalytic triad of serine proteases. The variable-region peptide sequence of the next most active antibody (29G11) differs from that of 17E8 by nine heavy-chain point mutations, and results from computer modeling suggest that the three-dimensional structure of 29G11 is similar to that of 17E8. In addition, 29G11 is an efficient catalytic antibody; it possesses 26% of the hydrolytic activity of 17E8. There is one active-site mutation in 29G11 compared to 17E8; position 99 of the heavy chain of 29G11 contains a glycine residue in place of the nucleophilic serine at this position in 17E8. Consistent with this mutation, results from pH-rate studies and hydroxylamine partitioning experiments indicate that in contrast to the catalytic mechanism of 17E8, the mechanism of 29G11-catalyzed esterolysis does not feature nucleophilic catalysis.

Amino Acid Sequence↗

PubMed1994

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.

Amino Acid Sequence↗

PubMed1991

Prediction of a crystallization pathway for Z-DNA hexanucleotides.

Crystallization of macromolecules for structural studies has long been a hit-or-miss process. The crystallization of hexanucleotides as Z-DNA was studied, and it was shown that the cation concentration for crystal formation could be predicted from solvation free energy (SFE) calculations. Solution studies on the conformation and solubilities of the hexanucleotides showed that a critical concentration of the DNA in the Z-conformation must be present in solution to effect crystallization. The SFE calculations therefore predict the propensity of the hexanucleotides to adopt the left-handed conformation and the driving force required to reach this critical concentration relative to the intrinsic solubility of Z-DNA for crystallization.

Base Sequence↗

PubMed1991

Neural network optimization for E. coli promoter prediction.

Methods for optimizing the prediction of Escherichia coli RNA polymerase promoter sequences by neural networks are presented. A neural network was trained on a set of 80 known promoter sequences combined with different numbers of random sequences. The conserved -10 region and -35 region of the promoter sequences and a combination of these regions were used in three independent training sets. The prediction accuracy of the resulting weight matrix was tested against a separate set of 30 known promoter sequences and 1500 random sequences. The effects of the network's topology, the extent of training, the number of random sequences in the training set and the effects of different data representations were examined and optimized. Accuracies of 100% on the promoter test set and 98.4% on the random test set were achieved with the optimal parameters.

Artificial Intelligence↗

PubMed1990

Stabilization of Z-DNA by demethylation of thymine bases: 1.3-A single-crystal structure of d(m5CGUAm5CG).

Methylation of cytosine bases at the C5 position has been known to stabilize Z-DNA. We had previously predicted from calculations of solvent-accessible surfaces that the methyl group at the same position of thymine has a destabilizing effect on Z-DNA. In the current studies, the sequence d(m5CGUAm5CG) has been crystallized and its structure solved as Z-DNA to 1.3-A resolution. A well-defined octahedral hexaaquomagnesium complex was observed to bridge the O4 oxygens of the adjacent uridine bases at the major groove surface, and four well-structured water molecules were found in the minor groove crevice at the d(UA) dinucleotide. These solvent interactions were not observed in the previously published Z-DNA structure of the analogous d(m5CGTAm5CG) sequence. A comparison of the thymine and uridine structures supports our prediction that demethylation of thymine bases helps to stabilize Z-DNA. A comparison of this d(UA)-containing Z-DNA structure with the analogous d(TA) structure shows that access of the O4 position is hindered by the C5 methyl of thymine due to steric and hydrophobic inhibition. In the absence of the methyl group, a magnesium-water complex binds to and slightly affects the structure of the Z-DNA major groove surface. This perturbation of the solvent structure at the major groove surface is translated into a much larger 1.41-A widening of the minor groove crevice, thereby allowing the specific binding of two water molecules at well-defined sites of each internal d(UA) base pair. Possible mechanisms by which modifications at the major groove surface of Z-DNA can affect the solvent properties of the minor groove crevice are discussed.

5-Methylcytosine↗

PubMed1990

Polarized electronic spectra of Z-DNA single crystals.

Polarized electronic absorption spectra of the (100) face of single crystals of the Z-form double helical duplex of d(m5CGUAm5CG) have been obtained from Kramers-Kronig analysis of reflection data. The c crystallographic axis is parallel to the helix axis and shows but weak absorption. The b axis is perpendicular to the helix axis and shows a structureless absorption band centered at 270 nm with an oscillator strength of 0.26. Calculations of the crystal spectra utilizing available transition moment data for the individual chromophores are carried through using the oriented gas model (no interbase interactions) and, again, employing all base-base interactions (point dipole) in the duplex. The calculated hypochromism of the 270 nm band is much less than the experimental value obtained from the crystal data. The crystal spectra appear to be representative of Z-form double helices of essentially infinite length and not of a collection of twelve base duplexes. No evidence for n pi* transitions polarized parallel to the helix axis is found.

DNA↗