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James H Geiger

Publications and source records attributed to James H Geiger.

17 recordsLinked to original sources
PubMed2006

X-ray crystallographic structure of the angiogenesis inhibitor, angiostatin, bound to a peptide from the group A streptococcal surface protein PAM.

The crystal structure of the human Pg-derived angiogenesis inhibitor, angiostatin, complexed to VEK-30, a peptide from the group A streptococcal surface protein, PAM, was determined and refined to 2.3 A resolution. This is the first structure of angiostatin bound to a ligand and provides a model of the interaction between Pg and streptococcal-derived pathogenic proteins. VEK-30 contains a "through-space isostere" for C-terminal lysine, wherein Arg and Glu side chains, separated by one helical turn, bind within the bipolar angiostatin kringle 2 (K2) domain lysine-binding site. VEK-30 also makes several contacts with K2 residues that exist outside of the canonical LBS and are not conserved among the other Pg kringles, thus providing a molecular basis for the selectivity of VEK-30 for K2. The structure also shows that Pg kringle domains undergo significant structural rearrangement relative to one another and reveals dimerization between two molecules of angiostatin and VEK-30 related by crystallographic symmetry. This dimerization, which exists only in the crystal structure, is consistent with the parallel coiled-coil full-length PAM dimer expected from sequence similarities and homology modeling.

Angiostatins↗
PubMed2006

The structure of Apo-wild-type cellular retinoic acid binding protein II at 1.4 A and its relationship to ligand binding and nuclear translocation.

CRABPII is a small, cytosolic protein that solubilizes and transfers retinoic acid (RA) to the nucleus while also enhancing its transcriptional activity. We have determined the first high-resolution structure of apo-wild type (WT) CRABPII at 1.35 A. Using three different data sets collected on apo-WT CRABPII we have shown that apo- and holo-CRABPII share very similar structures. Binding of RA appears to increase the overall rigidity of the structure, although the induced structural changes are not as pronounced as previously thought. The enhanced structural rigidity may be an important determinant for the enhanced nuclear localization of the RA-bound protein. Comparison of our apo-WT with a mutant apo-CRABPII structure shows that mutation of Arg111, a conserved residue of CRABPII and a key residue in RA binding, causes structural changes in the molecule. We further investigated the structural importance of conserved residues by determining the structure of the F15W mutant CRABPII (F15W-CRABPII). Our structures also demonstrate structural changes induced by crystal packing and show that a crystal can harbor demonstrative structural differences in the asymmetric unit.

Active Transport, Cell Nucleus↗
PubMed2006

The unorthodox SNAP50 zinc finger domain contributes to cooperative promoter recognition by human SNAPC.

Human small nuclear RNA gene transcription by RNA polymerases II and III depends upon promoter recognition by the SNAPC general transcription factor. DNA binding by SNAPC involves direct DNA contacts by the SNAP190 subunit in cooperation with SNAP50 and SNAP43. The data presented herein shows that SNAP50 plays an important role in DNA binding by SNAPC through its zinc finger domain. The SNAP50 zinc finger domain contains 15 cysteine and histidine residues configured in two potential zinc coordination arrangements. Individual alanine substitution of each cysteine and histidine residue demonstrated that eight sites are important for DNA binding by SNAPC. However, metal binding studies revealed that SNAPC contains a single zinc atom indicating that only one coordination site functions as a zinc finger. Of the eight residues critical for DNA binding, four cysteine residues were also essential for both U1 and U6 transcription by RNA polymerase II and III, respectively. Surprisingly, the remaining four residues, although critical for U1 transcription could support partial U6 transcription. DNA binding studies showed that defects in DNA binding by SNAPC alone could be suppressed through cooperative DNA binding with another member of the RNA polymerase III general transcription machinery, TFIIIB. These results suggest that these eight cysteine and histidine residues perform different functions during DNA binding with those residues involved in zinc coordination likely performing a dominant role in domain stabilization and the others involved in DNA binding. These data further define the unorthodox SNAP50 zinc finger region as an evolutionarily conserved DNA binding domain.

Amino Acid Sequence↗
PubMed2006

Engineering a rhodopsin protein mimic.

Due to the difficulties in handling and manipulating membrane-bound proteins, such as rhodopsin, and the lack of crystallographic information on the cone opsins, we have opted to engineer a protein mimic of the transmembrane G-protein coupled receptor. Human cellular retinoic acid binding protein (CRABPII), a well studied and characterized protein, has been reengineered into a protein that now will bind retinal as a protonated Schiff base with high binding affinity (Kd = 2 nM) mimicking that of rhodopsin.

Binding Sites↗
PubMed2006

Co-expression of multiple subunits enables recombinant SNAPC assembly and function for transcription by human RNA polymerases II and III.

Human small nuclear (sn) RNA genes are transcribed by either RNA polymerase II or III depending upon the arrangement of their core promoter elements. Regardless of polymerase specificity, these genes share a requirement for a general transcription factor called the snRNA activating protein complex or SNAP(C). This multi-subunit complex recognizes the proximal sequence element (PSE) commonly found in the upstream promoters of human snRNA genes. SNAP(C) consists of five subunits: SNAP190, SNAP50, SNAP45, SNAP43, and SNAP19. Previous studies have shown that a partial SNAP(C) composed of SNAP190 (1-514), SNAP50, and SNAP43 expressed in baculovirus is capable of PSE-specific DNA binding and transcription of human snRNA genes by RNA polymerases II and III. Expression in a baculovirus system yields active complex but the concentration of such material is insufficient for many bio-analytical methods. Herein, we describe the co-expression in Escherichia coli of a partial SNAP(C) containing SNAP190 (1-505), SNAP50, SNAP43, and SNAP19. The co-expressed complex binds DNA specifically and recruits TBP to U6 promoter DNA. Importantly, this partial complex functions in reconstituted transcription of both human U1 and U6 snRNA genes by RNA polymerases II and III, respectively. This co-expression system will facilitate the functional characterization of this unusual multi-protein transcription factor that plays an important early role for transcription by two different polymerases.

Cloning, Molecular↗
PubMed2006

The structure and mechanism of myo-inositol-1-phosphate synthase.

The first and rate-limiting step in the biosynthesis of myo-inositol is the conversion of D-glucose 6-phosphate to 1L-myo-inositol 1-phosphate catalyzed by 1L-myo-inositol 1-phosphate synthase (MIP synthase). MIP synthase has been identified in a wide variety of organisms from bacteria to humans and is relatively well-conserved throughout evolution. It is probably homotetrameric in most if not all cases and always requires NAD+ as a cofactor, with NADH being reconverted to NAD+ in the catalytic cycle. This review focuses on the structure and mechanism of MIP synthase, with a particular emphasis on the mechanistic insights that have come from several recent structures of the enzyme. These include the structure of the enzyme from Saccharomyces cerevisiae, Archeoglobus fulgidus and Mycobacterium tuberculosis.

Amino Acid Sequence↗
PubMed2005

Genetic perturbation of glycolysis results in inhibition of de novo inositol biosynthesis.

In a genetic screen for Saccharomyces cerevisiae mutants hypersensitive to the inositol-depleting drugs lithium and valproate, a loss of function allele of TPI1 was identified. The TPI1 gene encodes triose phosphate isomerase, which catalyzes the interconversion of dihydroxyacetone phosphate (DHAP) and glyceraldehyde 3-phosphate. A single mutation (N65K) in tpi1 completely abolished Tpi1p enzyme activity and led to a 30-fold increase in the intracellular DHAP concentration. The tpi1 mutant was unable to grow in the absence of inositol and exhibited the "inositol-less death" phenotype. Similarly, the pgk1 mutant, which accumulates DHAP as a result of defective conversion of 3-phosphoglyceroyl phosphate to 3-phosphoglycerate, exhibited inositol auxotrophy. DHAP as well as glyceraldehyde 3-phosphate and oxaloacetate inhibited activity of both yeast and human myo-inositol-3 phosphate synthase, the rate-limiting enzyme in de novo inositol biosynthesis. Implications for the pathology associated with TPI deficiency and responsiveness to inositol-depleting anti-bipolar drugs are discussed. This study is the first to establish a connection between perturbation of glycolysis and inhibition of de novo inositol biosynthesis.

Amino Acid Sequence↗
PubMed2005

Crystal structure of potato tuber ADP-glucose pyrophosphorylase.

ADP-glucose pyrophosphorylase catalyzes the first committed and rate-limiting step in starch biosynthesis in plants and glycogen biosynthesis in bacteria. It is the enzymatic site for regulation of storage polysaccharide accumulation in plants and bacteria, being allosterically activated or inhibited by metabolites of energy flux. We report the first atomic resolution structure of ADP-glucose pyrophosphorylase. Crystals of potato tuber ADP-glucose pyrophosphorylase alpha subunit were grown in high concentrations of sulfate, resulting in the sulfate-bound, allosterically inhibited form of the enzyme. The N-terminal catalytic domain resembles a dinucleotide-binding Rossmann fold and the C-terminal domain adopts a left-handed parallel beta helix that is involved in cooperative allosteric regulation and a unique oligomerization. We also report structures of the enzyme in complex with ATP and ADP-glucose. Communication between the regulator-binding sites and the active site is both subtle and complex and involves several distinct regions of the enzyme including the N-terminus, the glucose-1-phosphate-binding site, and the ATP-binding site. These structures provide insights into the mechanism for catalysis and allosteric regulation of the enzyme.

Adenosine Diphosphate↗
PubMed2003

The structure of the 1L-myo-inositol-1-phosphate synthase-NAD+-2-deoxy-D-glucitol 6-(E)-vinylhomophosphonate complex demands a revision of the enzyme mechanism.

1l-myo-inositol 1-phosphate (MIP) synthase catalyzes the conversion of d-glucose 6-phosphate to 1l-myo-inositol 1-phosphate, the first and rate-limiting step in the biosynthesis of all inositol-containing compounds. It involves an oxidation, enolization, intramolecular aldol cyclization, and reduction. Here we present the structure of MIP synthase in complex with NAD(+) and a high-affinity inhibitor, 2-deoxy-d-glucitol 6-(E)-vinylhomophosphonate. This structure reveals interactions between the enzyme active site residues and the inhibitor that are significantly different from that proposed for 2-deoxy-d-glucitol 6-phosphate in the previously published structure of MIP synthase-NAD(+)-2-deoxy-d-glucitol 6-phosphate. There are several other conformational changes in NAD(+) and the enzyme active site as well. Based on the new structural data, we propose a new and completely different mechanism for MIP synthase.

Binding Sites↗
PubMed2003

Structures of NAD(+)- and NADH-bound 1-l-myo-inositol 1-phosphate synthase.

1-l-myo-Inositol 1-phosphate synthase catalyzes the conversion of d-glucose 6-phosphate to 1-l-myo-inositol 1-phosphate, the first and rate-limiting step in the biosynthesis of all inositol-containing compounds. It involves an oxidation, an intramolecular aldol cyclization and a reduction. Here, the structure of the enzyme in its NAD(+)-bound, NADH-bound and apo forms is presented. These structures confirm that a significant portion of the active site is disordered in the absence of a small molecule, as none of the NAD(+)-bound forms of the enzyme have ordered active sites. On the other hand, the NADH-bound form contains two small molecules in the active site: a phosphate and glycerol. The entire active site is ordered in the presence of these two molecules, completely encapsulating them within the interior cavity. Significant changes in the structure of the active site are also seen, including repositioning of the nicotinamide ring and a motion of a loop region to accommodate the bound phosphate. These changes call into question the mechanism previously proposed for the enzyme. A comparison of the yeast and mycobacterial enzymes shows a surprisingly large change in the relative orientation of the catalytic and Rossmann-fold domains in the two enzymes.

Binding Sites↗
PubMed2003

NTP-driven translocation by human RNA polymerase II.

We report a "running start, two-bond" protocol to analyze elongation by human RNA polymerase II (RNAP II). In this procedure, the running start allowed us to measure rapid rates of elongation and provided detailed insight into the RNAP II mechanism. Formation of two bonds was tracked to ensure that at least one translocation event was analyzed. By using this method, RNAP II is stalled briefly at a defined template position before restoring the next NTP. Significantly, slow reaction steps are identified both before and after phosphodiester bond synthesis, and both of these steps can be highly dependent on the next templated NTP. The initial and final NTP-driven events, however, are not identical, because the slow step after chemistry, which includes translocation and pyrophosphate release, is regulated differently by elongation factors hepatitis delta antigen and transcription factor IIF. Because recovery from a stall and the processive transition from one bond to the next can be highly NTP-dependent, we conclude that translocation can be driven by the incoming substrate NTP, a model fully consistent with the RNAP II elongation complex structure.

Adenosine Triphosphate↗
PubMed2002

The X-ray crystallographic structure of Escherichia coli branching enzyme.

Branching enzyme catalyzes the formation of alpha-1,6 branch points in either glycogen or starch. We report the 2.3-A crystal structure of glycogen branching enzyme from Escherichia coli. The enzyme consists of three major domains, an NH(2)-terminal seven-stranded beta-sandwich domain, a COOH-terminal domain, and a central alpha/beta-barrel domain containing the enzyme active site. While the central domain is similar to that of all the other amylase family enzymes, branching enzyme shares the structure of all three domains only with isoamylase. Oligosaccharide binding was modeled for branching enzyme using the enzyme-oligosaccharide complex structures of various alpha-amylases and cyclodextrin glucanotransferase and residues were implicated in oligosaccharide binding. While most of the oligosaccharides modeled well in the branching enzyme structure, an approximate 50 degrees rotation between two of the glucose units was required to avoid steric clashes with Trp(298) of branching enzyme. A similar rotation was observed in the mammalian alpha-amylase structure caused by an equivalent tryptophan residue in this structure. It appears that there are two binding modes for oligosaccharides in these structures depending on the identity and location of this aromatic residue.

1,4-alpha-Glucan Branching Enzyme↗
PubMed2002

The X-ray crystallographic structure of the angiogenesis inhibitor angiostatin.

Angiogenesis inhibitors have gained much public attention recently as anti-cancer agents and several are currently in clinical trials, including angiostatin (Phase I, Thomas Jefferson University Hospital, Philadelphia, PA). We report here the bowl-shaped structure of angiostatin kringles 1-3, the first multi-kringle structure to be determined. All three kringle lysine-binding sites contain a bound bicine molecule of crystallization while the former of kringle 2 and kringle 3 are cofacial. Moreover, the separation of the kringle 2 and kringle 3 lysiner binding sites is sufficient to accommodate the alpha-helix of the 30 residue peptide VEK-30 found in the kringle 2/VEK-30 complex. Together the three kringles produce a central cavity suggestive of a unique domain where they may function in concert.

Amino Acid Sequence↗
PubMed2002

Crystallization of the Oct-1/SNAP190 peptide/DNA complex.

Crystals of the Oct-1 POU/SNAP190 peptide/DNA tertiary complex have been obtained by hanging-drop vapor diffusion at 293K in 20% 2-propanol, 20% PEG 4000 and 0.1M sodium citrate pH 5.6. The Oct-1 POU protein has two domains, one a homeodomain and the other a POU domain, which are connected by a flexible linker. The DNA used in the complex is slightly different in the octamer region compared with the two previously crystallized Oct-1 POU/DNA complexes. The DNA is 14 base pairs, with an octamer sequence of 5'-ATGTAGAT-3' and an overhang of one base on both strands. The SNAP190 peptide is 27 amino acids long (residues 884-910). The crystals diffract to 2.3 A (94.1% completeness) at the synchrotron under cryogenic (123K) conditions. The crystals are triclinic, space group P1, with unit-cell parameters a = 36.4, b = 54.9, c = 77.6A, alpha = 94.9, beta = 99.6, gamma = 109.2 degrees. This structure will provide insight into how Oct-1 interacts with SNAP190, a critical component of the small nuclear RNA-activating protein complex (SNAPc). Transcription of human snRNA genes is activated by these direct protein-protein interactions.

Bacterial Proteins↗
PubMed2002

Crystallization and preliminary X-ray diffraction studies of Escherichia coli branching enzyme.

Branching enzyme catalyzes the formation of the branch points in glycogen and starch by cleavage of the alpha-1,4 link and its subsequent transfer to the alpha-1,6 position. This paper reports the crystallization and preliminary structural studies of an amino-terminally truncated branching enzyme from Escherichia coli. High-resolution diffracting crystals were obtained and a complete native data set to a resolution of 2.3 A was collected. These crystals belong to the P2(1) space group, with unit-cell parameters a = 91.44, b = 102.58, c = 185.41 A, beta = 91.38 degrees. A native data set with 99.6% completeness, an overall R(merge) of 0.086 and I/sigma(I) of 10.43 was obtained.

1,4-alpha-Glucan Branching Enzyme↗
PubMed2002

The crystal structure and mechanism of 1-L-myo-inositol- 1-phosphate synthase.

1-l-myo-Inositol-1-phosphate synthase catalyzes the conversion of d-glucose 6-phosphate to 1-l-myo-inositol-1-phosphate (MIP), the first and rate-limiting step in the biosynthesis of all inositol-containing compounds. It involves an oxidation, intramolecular aldol cyclization, and reduction. We have determined the first crystal structure of MIP synthase. We present structures of both the NAD-bound enzyme and the enzyme bound to an inhibitor, 2-deoxy-glucitol-6-phosphate. While 58 amino acids are disordered in the unbound form of the enzyme in the vicinity of the active site, the inhibitor nucleates the folding of this domain in a striking example of induced fit, serving to completely encapsulate it within the enzyme. Three helices and a long beta-strand are formed in this process. We postulate a mechanism for the conversion based on the structure of the inhibitor-bound complex.

Crystallization↗