SBNet

SBNet - Research Reports 1999

Gerard J. Kleywegt

Position: Network Coordinator/"Forskare" (UU)

Projects: Structural Neurobiology / Protein Crystallography / Structural Bioinformatics

Personnel.

* Dr Gerard J Kleywegt, "Forskare", position funded by SBNet (Network Coordinator)

* Dr Kristina Bäckbro, post-doc ("Forskare"), funded through my SSF Junior Individual Grant

* Dr Dennis Madsen, post-doc, funded through Prof. T.A. Jones (Nov-Dec 1999), and through NFR (2000-2001)

* Emma Jakobsson, PhD student, funded by SBNet

Research progress.

* Structural studies of monoamine oxidase (MAO). Monoamine oxidase (MAO) is important in the metabolism of aromatic amines, including neurotransmitters such as serotonin, adrenaline, histamine and dopamine. Knowledge of the three-dimensional structures of MAO-A and B could have a significant impact on the rational (structure-based) design of novel inhibitors, potentially with therapeutic applications. We have worked with different systems to try and get large quantities of protein of a quality suitable for crystallisation. MAO-B was purified from bovine liver mitochondria using published protocols. It is possible to get large quantities but not of adequate quality due to aggregation problems. In collaboration with Prof. J.C. Shih and her group at USC, Los Angeles, we expressed a full length and a C-terminal truncated human MAO-A construct in Sf21 cells using the baculovirus system. However, these recombinant proteins also aggregate. In the absence of a crystal structure of MAO, we have constructed a homology model. This model has been used to design mutants with the goal to get a more soluble but still active protein. Cloning of these mutant enzymes is currently underway.

* Structural studies of Inhibitor of Apoptosis Proteins (IAPs). The aim of this work is to investigate the structure and function of NAIP (neuronal apoptosis inhibitory protein) and related IAPs (inhibitor of apoptosis protein). The IAPs have been shown to interact with TRAF proteins (tumour necrosis factor receptor associated factors) that are part of an apoptosis signalling cascade. All IAPs contain between one and three copies of a zinc-finger repeat called the BIR domain (baculoviral IAP repeat). The structure of one BIR domain was recently solved by NMR. However, the exact function of the IAPs in determining cell survival and death is not known. NAIP has turned out to be difficult to express in E.coli and our collaborators in Prof. Dan Lindholm's group (UU) are now expressing full-length NAIP, its BIR-3 domain, and XIAP in insect cells using the baculovirus expression system. Using two-hybrid system searches, they have also found that the BIR-3 domain of NAIP binds hippocalcin, a calcium-binding protein of unknown function. Hippocalcin is homologous to recoverin and several other calcium-binding proteins of the nervous system. We are expressing human hippocalcin as a GST fusion protein in E. coli. We are now trying to crystallise this protein alone but also hope to determine the structure of the complex of the BIR-3 domain of NAIP and hippocalcin in the future.

* Other experimental protein crystallographic projects.

- I have solved the crystal structure of a CMC-ase and refined it to a resolution of 1.8 Å (1.2 Å synchrotron data has been collected but not yet processed). This project is a collaboration with Dr Jerry Ståhlberg (SLU) and others.

- We have begun a new collaboration with Prof. Lars Hellman (UU). We will work on KRAB domains and on an inactive form of rat mast-cell protease (RMCP) 2. Both projects are currently at the stage of protein expression and purification. However, in initial crystallisation screens with RMCP-2 small spherulites have already been obtained.

- We will begin a collaboration with the group of Prof. Bengt Mannervik (UU) on mutant glutathione S-transferases early in the year 2000.

- At least one other new protein crystallographic project is expected to get underway in the year 2000.

* Structural Bioinformatics. During 1999, the program package SPASM (for spatial motif recognition in protein structures) has been made available to the community, and a paper describing the method and its applications has been published. The major weakness of the program used to be its relatively poor sensitivity (i.e., often a lot of false positives were retrieved). This problem has been remedied by writing a post-processing program that does an atom-by-atom least-squares superpositioning of the motif and each potential hit (not a trivial task, since different residue types may be matched with one another). Furthermore, an illustrated tutorial on the use of SPASM has been prepared and made available through the web. Finally, Dr Dennis Madsen has written a web interface to the SPASM package, making it extremely easy and fast to use this powerful tool. Dr Madsen has also written a web interface for DEJAVU, a program we developed a few years ago that can be used to recognise the fold of newly determined protein structures. Dr Madsen will continue to work in my group as an NFR-funded post-doctoral fellow. In the future new and improved tools for the alignment of two or more protein sequences (in particular for cases where the degree of sequence similarity is low) will be developed, using a priori information as constraints, and evolving profiles (or Hidden Markov Models) for multiple sequence alignment. Another new project will entail the development of an expert system to detect (and correct) errors in intermediate protein crystallographic models. The expert system is intended to be tightly coupled to Prof. T. Alwyn Jones' program O.

Miscellaneous events during 1999.

* January-February: principal author of a proposal to the Knut & Alice Wallenberg Foundation to fund the Linnaeus Centre for Bioinformatics in Uppsala. This proposal was funded with 60 MSEK. An earlier proposal to SSF for the same centre was funded with 7.5 MSEK.

* 3 June 1999: member of the PhD thesis evaluation committee of Enrique Carredano, Swedish University of Agricultural Sciences. Thesis title: "Crystallographic studies of phospholipase A2 and naphtalene 1,2-dioxygenase: implications for phospholipase activity and dioxygenase substrate specificity."

* June: Organised the Third Annual Conference of the Structural Biology Network, and chaired the session on Methodology.

* June/July: Co-organised an EMBO practical course in Bioinformatics at the Linnaeus Centre for Bioinformatics in Uppsala (http://xray.bmc.uu.se/embo). The course was attended by 20 students from 17 different countries.

* August: invited lecturer in the 1999 IUCr Crystallographic Computing School, Hinxton (UK).

* September: invited lecturer in the Protein Crystallography Summer School, Barcelona.

* October: invited lecturer in the PhD course in Protein Crystallography, Copenhagen University.

* December: NFR support obtained for the years 2000 and 2001 for a project in the area of bioinformatics (one post-doctoral fellow; Dr Dennis Madsen).

* Refereed 10 papers for Acta Crystallographica and the Journal of Molecular Biology.

* External reviewer of a grant proposal for the BBSRC Bioinformatics initiative.

* External reviewer of a national initiative for the Danish Natural Sciences Research Council.

* Paper alerts abstracter for the journal Current Opinion in Structural Biology.

* Member of the executive committee of the Linnaeus Centre for Bioinformatics in Uppsala.

Web sites.

I continue to maintain three WWW sites with information, software and services relevant to the structural biology community:

* USF, at http://xray.bmc.uu.se/usf/, the Uppsala Software Factory (38,000 visits during 1999, with 163,000 pages served)

* SBNet, at http://xray.bmc.uu.se/sbnet/, the Swedish Structural Biology Network (16,000 visits during 1999, with 61,000 pages served)

* HIC-Up, at http://xray.bmc.uu.se/hicup/, the "Hetero-compound Information Centre - Uppsala" (75,000 visits during 1999, with 176,000 pages served)

Publications.

[1] G J Kleywegt (1999). Recognition of spatial motifs in protein structures. J. Mol. Biol. 285, 1187-1197.

[2] B N Chaudhuri, G J Kleywegt, J Björkman, L D Lehman-McKeeman, J D Oliver & T A Jones (1999). The crystal structures of [[alpha]]2u-globulin and its complex with a hyaline droplet inducer. Acta Cryst. D55, 753-762.

[3] G J Kleywegt & T A Jones (1999). Software for handling macromolecular envelopes. Acta Cryst. D55, 941-944.

[4] J Y Zou, G J Kleywegt, J Ståhlberg, H Drigues, W Nerinckx, M Claeysens, A Koivula, T Teeri & T A Jones (1999). Crystallographic evidence for substrate ring distortion and protein conformational changes during catalysis in cellobiohydrolase Cel6A from Trichoderma reesei. Structure Fold. Des. 7, 1035-1045.

[5] T A Jones & G J Kleywegt (1999). CASP3 Comparative Modelling Evaluation. Proteins: Struct. Funct. Genet. Suppl. 3, 30-46.

[6] B N Chaudhuri, G J Kleywegt, I Broutin-L'Hermite, T Bergfors, H Senn, P Le Motte, O Partouche & T A Jones (1999). Crystal structures of cellular retinoic acid binding proteins I and II in complex with synthetic retinoids. Acta Cryst. D55, 1850-1857.

[7] G J Kleywegt (1999). Experimental assessment of differences between related protein crystal structures. Acta Cryst. D55, 1878-1884.

[8] G J Kleywegt (2000). Validation of protein crystal structures. Acta Cryst. D56, 249-265.

[9] G J Kleywegt (2000). Structure Validation I. Chapter 21.1, International Tables for Crystallography, Volume F, In press.

[10] G J Kleywegt, J Y Zou, M Kjeldgaard & T A Jones (2000). Around O. Chapter 17.1, International Tables for Crystallography, Volume F, In press.

Lectures.

* "Hetero-compound dictionaries for structure refinement", lecture in the SBNet Workshop on CNS, Uppsala, February 2000.

* "Playing with Protein Structures", invited lecture, Linnaeus Centre for Bioinformatics, Uppsala, November 1999.

* "Playing with Protein Structures", invited seminar, AstraZeneca Structural Chemistry Laboratory, Gothenburg, October 1999.

* "Validation in Protein Crystallography", invited seminar, AstraZeneca Structural Chemistry Laboratory, Gothenburg, October 1999.

* "Validation in Protein Crystallography", lecture in the PhD course in Protein Crystallography, Copenhagen University, October 1999.

* "Playing with Protein Structures", invited seminar, Copenhagen University, October 1999.

* "Playing with Protein Structures", invited seminar, Department of Molecular Biophysics, Lund University, October 1999.

* "Validation in Protein Crystallography", invited seminar, Department of Molecular Biophysics, Lund University, October 1999.

* "Map Interpretation", lecture in the Protein Crystallography Summer School, Barcelona, September 1999.

* "Validation", lecture in the Protein Crystallography Summer School, Barcelona, September 1999.

* "Validation", lecture in the 1999 IUCr Crystallographic Computing School, Hinxton (UK), August 1999.

* "Structure determination and validation", lecture in the EMBO practical course on "Bioinformatics", Uppsala (S), July 1999.

Citations.

Citations were counted using ISI's BIBSYS system on 5 December, 1999 (in square brackets are the corresponding counts on 16 December, 1998, and on 9 December, 1997, respectively). On this date, 48 [43, 39] of my papers had been cited, with a total of 1691 [1134, 752] citations, yielding an impact factor (citations per publication, not corrected for self-citation) of 35 [26, 19]. More detailed statistics are available from my home page (http://xray.bmc.uu.se/gerard).

Other SSF items.

3.1d total budget

3.1e sources of funding

- SBNet (salary plus 50 kSEK consumables)

- SSF Junior Individual Grant (500,000 SEK p.a. 1997, 1998, 1999)

- SBNet support for Emma Jakobsson (salary plus 50 kSEK consumables)

- NFR "ramanslag" (~200,000 SEK p.a. 1999, 2000)

5.1 indicate any true collaborations with Swedish industry, the health care sector, etc. were any formal agreements signed ?

not applicable

6.1 indicate any true international collaborations (name, affiliation). were any of these part of international programs (e.g. EU) ?

- Prof Jean Shih, California (MAO-A)

- Prof Creed Abell, Texas (MAO-B)

6.2 list visiting foreign post-docs, students, senior scientists. list research/study stays abroad by programme researchers/students

- Emma Jakobsson visited the lab of Prof. Shih (March, 1999)


SBNet Latest update at 3 April, 2000.