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Disruption of the GDNF Binding Site in NCAM DissociatesLigand Binding and Homophilic Cell Adhesion
Division of Molecular Neurobiology, Department of Neuroscience, Karolinska Institute, S-171 77 Stockholm, Sweden.
Linköpings universitet, Institutionen för fysik, kemi och biologi, Bioinformatik. Linköpings universitet, Tekniska högskolan.
Division of Molecular Neurobiology, Department of Neuroscience, Karolinska Institute, S-171 77 Stockholm, Sweden.
Linköpings universitet, Institutionen för fysik, kemi och biologi, Bioinformatik. Linköpings universitet, Tekniska högskolan.
Vise andre og tillknytning
2007 (engelsk)Inngår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 282, nr 17, s. 12734-12740Artikkel i tidsskrift (Fagfellevurdert) Published
Abstract [en]

Most plasma membrane proteins are capable of sensing multiple cell-cell and cell-ligand interactions, but the extent towhich this functional versatility is founded on their modular design is less clear. We have identified the third immunoglobulin domain of the Neural Cell Adhesion Molecule (NCAM) as the necessary and sufficient determinant for its interaction with Glial Cell Line-derived Neurotrophic Factor (GDNF). Four charged contacts were identified by molecular modeling as the main contributors to binding energy. Their mutation abolished GDNF binding to NCAM but left intact the ability of NCAM tomediate cell adhesion, indicating that the two functions are genetically separable. The GDNF-NCAM interface allows complex formation with the GDNF family receptor α1, shedding light on the molecular architecture of a multicomponent GDNF receptor.

sted, utgiver, år, opplag, sider
Bethesda, MD: American Society for Biochemistry and Molecular Biology , 2007. Vol. 282, nr 17, s. 12734-12740
Emneord [en]
homology model, protein complex, interaction interface, mutagenesis
HSV kategori
Identifikatorer
URN: urn:nbn:se:liu:diva-21306DOI: 10.1074/jbc.M701588200OAI: oai:DiVA.org:liu-21306DiVA, id: diva2:241015
Tilgjengelig fra: 2009-09-30 Laget: 2009-09-30 Sist oppdatert: 2017-12-13bibliografisk kontrollert
Inngår i avhandling
1. Mutational effects on protein structure and function
Åpne denne publikasjonen i ny fane eller vindu >>Mutational effects on protein structure and function
2009 (engelsk)Doktoravhandling, med artikler (Annet vitenskapelig)
Abstract [en]

In this thesis several important proteins are investigated from a structural perspective. Some of the proteins are disease related while other have important but not completely characterised functions. The techniques used are general as demonstrated by applications on metabolic proteins (CYP21, CYP11B1, IAPP, ADH3), regulatory proteins (p53, GDNF) and a transporter protein (ANTR1).

When the protein CYP21 (steroid 21-hydroxylase) is deficient it causes CAH (congenital adrenal hyperplasia). For this protein, there are about 60 known mutations with characterised clinical phenotypes. Using manual structural analysis we managed to explain the severity of all but one of the mutations. By observing the properties of these mutations we could perform good predictions on, at the time, not classified mutations.

For the cancer suppressor protein p53, there are over thousand mutations with known activity. To be able to analyse such a large number of mutations we developed an automated method for evaluation of the mutation effect called PREDMUT. In this method we include twelve different prediction parameters including two energy parameters calculated using an energy minimization procedure. The method manages to differentiate severe mutations from non-severe mutations with 77% accuracy on all possible single base substitutions and with 88% on mutations found in breast cancer patients.

The automated prediction was further applied to CYP11B1 (steroid 11-beta-hydroxylase), which in a similar way as CYP21 causes CAH when deficient. A generalized method applicable to any kind of globular protein was developed. The method was subsequently evaluated on nine additional proteins for which mutants were known with annotated disease phenotypes. This prediction achieved 84% accuracy on CYP11B1 and 81% accuracy in total on the evaluation proteins while leaving 8% as unclassified. By increasing the number of unclassified mutations the accuracy of the remaining mutations could be increased on the evaluation proteins and substantially increase the classification quality as measured by the Matthews correlation coefficient. Servers with predictions for all possible single based substitutions are provided for p53, CYP21 and CYP11B1.

The amyloid formation of IAPP (islet amyloid polypeptide) is strongly connected to diabetes and has been studied using both molecular dynamics and Monte Carlo energy minimization. The effects of mutations on the amount and speed of amyloid formation were investigated using three approaches. Applying a consensus of the three methods on a number of interesting mutations, 94% of the mutations could be correctly classified as amyloid forming or not, evaluated with in vitro measurements.

In the brain there are many proteins whose functions and interactions are largely unknown. GDNF (glial cell line-derived neurotrophic factor) and NCAM (neural cell adhesion molecule) are two such neuron connected proteins that are known to interact. The form of interaction was studied using protein--protein docking where a docking interface was found mediated by four oppositely charged residues in respective protein. This interface was subsequently confirmed by mutagenesis experiments. The NCAM dimer interface upon binding to the GDNF dimer was also mapped as well as an additional interacting protein, GFRα1, which was successfully added to the protein complex without any clashes.

A large and well studied protein family is the alcohol dehydrogenase family, ADH. A class of this family is ADH3 (alcohol dehydrogenase class III) that has several known substrates and inhibitors. By using virtual screening we tried to characterize new ligands. As some ligands were already known we could incorporate this knowledge when the compound docking simulations were scored and thereby find two new substrates and two new inhibitors which were subsequently successfully tested in vitro.

ANTR1 (anion transporter 1) is a membrane bound transporter important in the photosynthesis in plants. To be able to study the amino acid residues involved in inorganic phosphate transportation a homology model of the protein was created. Important residues were then mapped onto the structure using conservation analysis and we were in this way able to propose roles of amino acid residues involved in the transportation of inorganic phosphate. Key residues were subsequently mutated in vitro and a transportation process could be postulated.

To conclude, we have used several molecular modelling techniques to find functional clues, interaction sites and new ligands. Furthermore, we have investigated the effect of muations on the function and structure of a multitude of disease related proteins.

 

sted, utgiver, år, opplag, sider
Linköping: Linköping University Electronic Press, 2009. s. 80
Serie
Linköping Studies in Science and Technology. Dissertations, ISSN 0345-7524 ; 1271
Emneord
Mutation, prediction, phenotypes, homology model, virtual screening, molecular dynamics, amyloid, cancer, membrane protein
HSV kategori
Identifikatorer
urn:nbn:se:liu:diva-50491 (URN)978-91-7393-539-9 (ISBN)
Disputas
2009-11-06, Planck, Fysikhuset, Campus Valla, Linköpings universitet, Linköping, 10:00 (engelsk)
Opponent
Veileder
Tilgjengelig fra: 2009-10-19 Laget: 2009-10-12 Sist oppdatert: 2009-10-19bibliografisk kontrollert

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