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Crystal structure of YegS, a homologue to the mammalian diacylglycerol kinases, reveals a novel regulatory metal binding site
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
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2007 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 282, no 27, 19644-19652 p.Article in journal (Refereed) Published
Abstract [en]

The human lipid kinase family controls cell proliferation, differentiation, and tumorigenesis and includes diacylglycerol kinases, sphingosine kinases, and ceramide kinases. YegS is an Escherichia coli protein with significant sequence homology to the catalytic domain of the human lipid kinases. We have solved the crystal structure of YegS and shown that it is a lipid kinase with phosphatidylglycerol kinase activity. The crystal structure reveals a two-domain protein with significant structural similarity to a family of NAD kinases. The active site is located in the interdomain cleft formed by four conserved sequence motifs. Surprisingly, the structure reveals a novel metal binding site composed of residues conserved in most lipid kinases.

Place, publisher, year, edition, pages
2007. Vol. 282, no 27, 19644-19652 p.
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
URN: urn:nbn:se:su:diva-25485DOI: 10.1074/jbc.M604852200ISI: 000247650600039OAI: oai:DiVA.org:su-25485DiVA: diva2:199818
Available from: 2008-09-22 Created: 2008-09-22 Last updated: 2017-12-13Bibliographically approved
In thesis
1. Structural studies of proteins in apoptosis and lipid signaling
Open this publication in new window or tab >>Structural studies of proteins in apoptosis and lipid signaling
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Signaling pathways control the fate of the cell. For example, they promote cell survival or commit the cell to death (apoptosis) in response to cell injury or developmental stimuli, decisions, which are vital for the proper development and functioning of metazoan. Tight control of such pathways is essential; dysregulation of apoptosis can disrupt the delicate balance between cell proliferation and cell death ending up in pathological processes, including cancer, autoimmunity diseases, inflammatory diseases, or degenerative disorders. We have used a structural genomic approach to study the structure and function of key proteins involved in apoptosis and lipid signaling: the antiapoptotic Bcl-2 family member Bfl-1 in complex with a Bim peptide, the BIR domains of the Inhibitor of Apoptosis (IAP) family members, cIAP2 and NAIP and the a lipid kinase YegS. The structural analysis of the apoptosis regulatory proteins has revealed important information on the structural determinants for recognition of interacting proteins, which can now assist in the development of therapeutic drugs for human diseases. The structural and complementing biochemical studies of the lipid kinase YegS have reveled the first detailed information on a lipid kinase and explained important aspects of its structure-function relationship.

Finally, one subject of this work aim to solve what is arguably the most challenging problem in structural projects – to obtain a high production level of proteins suitable for structural studies. We have developed a highthroughput protein solubility screening, the colony filtration (CoFi) blot, which allows soluble clones to be identified from large libraries of protein variants and now constitute a powerful tool for solving difficult protein production problems.

Place, publisher, year, edition, pages
Stockholm: Institutionen för biokemi och biofysik, 2008. 74 p.
Keyword
proteins, crystal structure, apoptosis, cancer, CoFi-Blot, kinases, lipid signaling
National Category
Structural Biology
Research subject
Structural Biology
Identifiers
urn:nbn:se:su:diva-8212 (URN)978-91-7155-703-2 (ISBN)
Public defence
2008-09-11, Nordenskiöldsalen, Geovetenskapens hus, Svante Arrhenius väg 8 C, Stockholm, 10:00 (English)
Opponent
Supervisors
Available from: 2008-09-22 Created: 2008-09-22 Last updated: 2010-01-13Bibliographically approved
2. Structural studies of three cell signaling proteins: crystal structures of EphB1, PTPA, and YegS
Open this publication in new window or tab >>Structural studies of three cell signaling proteins: crystal structures of EphB1, PTPA, and YegS
2007 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Kinases and phosphatases are key regulatory proteins in the cell. The disruption of their activities leads ultimately to the abolishment of the homeostasis of the cell, and is frequently correlated with cancer. EphB1 is a member of the largest family of receptor tyrosine kinases. It is associated with neurogenesis, angiogenesis, and cancer. The cytosolic part of the human EphB1 receptor is composed of two domains. Successful generation of soluble constructs, using a novel random construct screening approach, led to the structure determination of the kinase domain of this receptor. The native structure and the complex structure with an ATP analogue revealed novel features in the regulation of the Eph family of kinases.

The structure of PTPA, an activator of protein phosphatase 2 A, a tumor suppressor and a key phosphatase in the cell was solved. The structure revealed a novel fold containing a conserved cleft predicted to be involved in interaction with PP2A.

Finally, the structure of YegS, an Escherichia coli protein annotated as a putative diacylglycerol kinase, has been determined. Beside the elucidation of its atomic structure, a phosphatidylglycerol (PG) kinase activity, never seen before, has been assigned to YegS based on biochemical studies. The YegS structure shows resemblance to the fold previously seen in NAD kinases. The structure also revealed the existence of a novel metal site that could potentially play a regulatory role. The YegS structure has important implications for understanding related proteins in pathogenic organisms and is the first homologue of a human lipid kinase for which the structure has been elucidated.

Place, publisher, year, edition, pages
Stockholm: Institutionen för biokemi och biofysik, 2007. 74 p.
Keyword
protein production, structural genomics, cell signaling
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:su:diva-6577 (URN)91-7155-364-9 (ISBN)
Public defence
2007-02-23, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 12 A, Stockholm, 09:30
Opponent
Supervisors
Available from: 2007-02-01 Created: 2007-02-01 Last updated: 2011-02-11Bibliographically approved

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