- Email: [email protected]
Analysis of protein structure in solution using small angle X-ray scattering
S58 s39-3
s39-5
Structural Bases for the Regulation of Ets Transcription Factors by Auto-Inhibition and Phosphorylation. McIntosh LP, Dept. of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, BC V6T 123.
Phosphoenolpyruvate Carboxykinase; From Structure to Catalytic Mechanism Leslie W. Tari’, Allan Matte2, Hughes Goldie3 and Louis T. J. Delbaere3 ‘Department of Biological Sciences, University of Calgary, Calgary, AB T2N lN4, ‘Biotechnology Research Institute, 6100 Royalmount Dr. Montreal, QC, H4P 2R2, 3Departments of Biochemistry and Microbiology, University of Saskatchewan, Saskatoon, SK, S7N 5E.5
The ets family of transcription factors and oncoproteins is a paradigm for the study of eukaryotic gene expression. The activities of these proteins are dependent upon numerous protein-protein and protein-DNA interactions, as well as phosphorylation via signal transduction cascades, and thus form an ideal system for investigating the mechanisms of transcriptional regulation and its relationship to oncogenesis. The signature of the ets family is a conserved winged helixturn-helix DNA-bindin module called the ETS domain. The affinity of native Ets-I ?or DNA is repressed intramolecularly by sequences flanking the N- and C-terminus of this domain. Using NMR methods, we demonstrated that the two inhibitory sequences fold as cz-helices and pack along one side of the ETS domain to form an inhibitory module. Combined with biochemical data, we have developed a model in which DNA binding is coupled allosterically to the local unfolding of the Nterminal inhibitory sequence. We hypothesize that in viva regulation of efs transcription factors may occur though posttranslational modifications or protein-protein associations that disrupt the coupling of the inhibitory module with the ETS domain. Several ets proteins also contain a conserved Pointed domain and an adjacent MAP kinase phosphorylation site, implicated in protein-protein interactions and ras-dependent signaling, respectively. The Ets-I Pointed domain has a novel fold of a five-helix bundle, while the MAP kinase site is unstructured in solution. Phosphorylation neither alters the disordered character of the kinase substrate site nor causes a change in the structure or oligomerization state of the Pointed domain. These studies provide importavt, clues for defining h,oewmieEphorylatlon regulates the actlvltles of efs family
s39-4
Phosphoenolpyruvate carboxykinase (PCK; E. C. 4.1.1.49) is a key metabolic enzyme which catalyzes the reaction representing the first committed step in the diversion of tricarboxylic acid cycle intermediates towards the conversion of oxaloacetate to gluconeogenesis; phosphoenolpyruvate. Since there is evidence that this reaction is rate limiting with respect to glucose formation, mammalian PCK may be regarded as a prime candidate for metabolic regulation, particularly in the treatment of diabetes mellitus, where elevated levels of the enzyme are correlated with sustained hyperglycemia. The structures of several different metal-substrate complexes of PCK from Escherichia coli (I$ 59583, 540 residues) have been determined at high resolution by x-ray crystallography. One of the structures that will be presented offers a glimpse at an enzyme reaction intermediate, providing detailed insight into the reaction mechanism, while a second complex dis.lis;;~ how mixed metal clusters operate in enzyme Using x-ray transphosphorylations. crystallography, we have gained insights into the inner workings of this fascinating enzyme.
Symposium 40: The eye lens: function, evolution. Chair: J. Honvitz (USA)
structure,
development,
and
s40-1
Analvsis of motein structure in solution using small angle X-rav scatteriw. Brian Shilton Department of Biochemistry, University of Western Ontario, London, Ontario Canada N6A 5Cl; [email protected] In a small angle X-ray scattering (SAXS) experiment, the scattering of X-rays by protein in solution is measured. SAXS and X-ray crystallography use the same physical process scattering of X-rays by electrons - to derive structural information; however, SAXS provides a direct measurement of the solution conformation of a protein, and therefore yields structural information that may be difficult or impossible to obtain from crystallographic studies. SAXS is used to measure changes in protein conformation that result from ligand binding, and to compare the conformation of a protein in solution with that in a crystal. The bacterial periplasmic binding proteins will be used to illustrate how SAXS and crystallography can provide complementary structural information (Shilton et al (1996) J. Mol. Biol. 264~350-363 and 364-376). In cases where a crystal structure is not available, de now structural information is obtained by SAXS. At the very minimum, a SAXS study will provide the radius of gyration and maximal dimension of a protein, which together indicate its size and shape. More detailed structural information can be obtained using; recently developed mathematical methods to calculate a molecular envelope. SAXS was used in this way to derive a low resolution structure of the SecA ATPase (Shilton et al (1998) FEBS Lett. 436:277-282).
Gene sharing in the lens and cornea as an evolutionary
strategy.
Piatigorskv J Laboratory of Molecular and Developmental Biology, National Eye Institute, NIH. Bethesda, Maryland 20892-2730 The transparent lens and cornea of the eye function together to focus incident light onto the retinal photoreceptors. A diverse group of soluble proteins called crystallins are responsible for the transparency and refractive index of the cellular lens in vertebrates and invertebrates (i.e.. cephalopods. scallops. jellyfish). Surprisingly, many of the lens crystallins differ among species (i.e., are taxon-specific) and are identical or related to Imetabolic enzymes (i.e.. lactate dehydrogenasc, argininosuccinate Iyase, a-enolase) or stress proteins (i.e.. small heat shock proteins) used at lower concentrations in non-refractive tissues. The use of the same gene for tbese two completely different roles (refraction and metabolism) is called gene sharing, A few transcription factors including Pax-6 and retinoic acid receptors contribute to the high lens expression of a number of crystallin genes. Unlike the lens. the vcrtcbratc cornea has a stratified layer of anterior epithelial cells. a thick extracellular collagenous stroma containing keratocytes. and a single layer of posterior endothelial cells. Like the lens. the cornea1 epithelial cells accumulate unexpectedly high concentrations (20-50% of soluble proteins) of a few ubiquitiously expressed proteins in a taxonspecific manner. These include aldebyde dehydrogenase 3 and transketolase in mammals, peptidql-prolyl cis-trans isomerase in chichen, and gelsolin in zebrafish. This suggests that gcnc sharing contributes to cellular transparency in both the lens and cornea. We propose that the common strategy of gene sharing in lens and cornea treflects a much closer relationship between these two ectodermally derived ocular tissues than recopnircd previously.
s39-5
Structural Bases for the Regulation of Ets Transcription Factors by Auto-Inhibition and Phosphorylation. McIntosh LP, Dept. of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, BC V6T 123.
Phosphoenolpyruvate Carboxykinase; From Structure to Catalytic Mechanism Leslie W. Tari’, Allan Matte2, Hughes Goldie3 and Louis T. J. Delbaere3 ‘Department of Biological Sciences, University of Calgary, Calgary, AB T2N lN4, ‘Biotechnology Research Institute, 6100 Royalmount Dr. Montreal, QC, H4P 2R2, 3Departments of Biochemistry and Microbiology, University of Saskatchewan, Saskatoon, SK, S7N 5E.5
The ets family of transcription factors and oncoproteins is a paradigm for the study of eukaryotic gene expression. The activities of these proteins are dependent upon numerous protein-protein and protein-DNA interactions, as well as phosphorylation via signal transduction cascades, and thus form an ideal system for investigating the mechanisms of transcriptional regulation and its relationship to oncogenesis. The signature of the ets family is a conserved winged helixturn-helix DNA-bindin module called the ETS domain. The affinity of native Ets-I ?or DNA is repressed intramolecularly by sequences flanking the N- and C-terminus of this domain. Using NMR methods, we demonstrated that the two inhibitory sequences fold as cz-helices and pack along one side of the ETS domain to form an inhibitory module. Combined with biochemical data, we have developed a model in which DNA binding is coupled allosterically to the local unfolding of the Nterminal inhibitory sequence. We hypothesize that in viva regulation of efs transcription factors may occur though posttranslational modifications or protein-protein associations that disrupt the coupling of the inhibitory module with the ETS domain. Several ets proteins also contain a conserved Pointed domain and an adjacent MAP kinase phosphorylation site, implicated in protein-protein interactions and ras-dependent signaling, respectively. The Ets-I Pointed domain has a novel fold of a five-helix bundle, while the MAP kinase site is unstructured in solution. Phosphorylation neither alters the disordered character of the kinase substrate site nor causes a change in the structure or oligomerization state of the Pointed domain. These studies provide importavt, clues for defining h,oewmieEphorylatlon regulates the actlvltles of efs family
s39-4
Phosphoenolpyruvate carboxykinase (PCK; E. C. 4.1.1.49) is a key metabolic enzyme which catalyzes the reaction representing the first committed step in the diversion of tricarboxylic acid cycle intermediates towards the conversion of oxaloacetate to gluconeogenesis; phosphoenolpyruvate. Since there is evidence that this reaction is rate limiting with respect to glucose formation, mammalian PCK may be regarded as a prime candidate for metabolic regulation, particularly in the treatment of diabetes mellitus, where elevated levels of the enzyme are correlated with sustained hyperglycemia. The structures of several different metal-substrate complexes of PCK from Escherichia coli (I$ 59583, 540 residues) have been determined at high resolution by x-ray crystallography. One of the structures that will be presented offers a glimpse at an enzyme reaction intermediate, providing detailed insight into the reaction mechanism, while a second complex dis.lis;;~ how mixed metal clusters operate in enzyme Using x-ray transphosphorylations. crystallography, we have gained insights into the inner workings of this fascinating enzyme.
Symposium 40: The eye lens: function, evolution. Chair: J. Honvitz (USA)
structure,
development,
and
s40-1
Analvsis of motein structure in solution using small angle X-rav scatteriw. Brian Shilton Department of Biochemistry, University of Western Ontario, London, Ontario Canada N6A 5Cl; [email protected] In a small angle X-ray scattering (SAXS) experiment, the scattering of X-rays by protein in solution is measured. SAXS and X-ray crystallography use the same physical process scattering of X-rays by electrons - to derive structural information; however, SAXS provides a direct measurement of the solution conformation of a protein, and therefore yields structural information that may be difficult or impossible to obtain from crystallographic studies. SAXS is used to measure changes in protein conformation that result from ligand binding, and to compare the conformation of a protein in solution with that in a crystal. The bacterial periplasmic binding proteins will be used to illustrate how SAXS and crystallography can provide complementary structural information (Shilton et al (1996) J. Mol. Biol. 264~350-363 and 364-376). In cases where a crystal structure is not available, de now structural information is obtained by SAXS. At the very minimum, a SAXS study will provide the radius of gyration and maximal dimension of a protein, which together indicate its size and shape. More detailed structural information can be obtained using; recently developed mathematical methods to calculate a molecular envelope. SAXS was used in this way to derive a low resolution structure of the SecA ATPase (Shilton et al (1998) FEBS Lett. 436:277-282).
Gene sharing in the lens and cornea as an evolutionary
strategy.
Piatigorskv J Laboratory of Molecular and Developmental Biology, National Eye Institute, NIH. Bethesda, Maryland 20892-2730 The transparent lens and cornea of the eye function together to focus incident light onto the retinal photoreceptors. A diverse group of soluble proteins called crystallins are responsible for the transparency and refractive index of the cellular lens in vertebrates and invertebrates (i.e.. cephalopods. scallops. jellyfish). Surprisingly, many of the lens crystallins differ among species (i.e., are taxon-specific) and are identical or related to Imetabolic enzymes (i.e.. lactate dehydrogenasc, argininosuccinate Iyase, a-enolase) or stress proteins (i.e.. small heat shock proteins) used at lower concentrations in non-refractive tissues. The use of the same gene for tbese two completely different roles (refraction and metabolism) is called gene sharing, A few transcription factors including Pax-6 and retinoic acid receptors contribute to the high lens expression of a number of crystallin genes. Unlike the lens. the vcrtcbratc cornea has a stratified layer of anterior epithelial cells. a thick extracellular collagenous stroma containing keratocytes. and a single layer of posterior endothelial cells. Like the lens. the cornea1 epithelial cells accumulate unexpectedly high concentrations (20-50% of soluble proteins) of a few ubiquitiously expressed proteins in a taxonspecific manner. These include aldebyde dehydrogenase 3 and transketolase in mammals, peptidql-prolyl cis-trans isomerase in chichen, and gelsolin in zebrafish. This suggests that gcnc sharing contributes to cellular transparency in both the lens and cornea. We propose that the common strategy of gene sharing in lens and cornea treflects a much closer relationship between these two ectodermally derived ocular tissues than recopnircd previously.