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• 1.
Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
Rheumatology Unit, Department of Medicine, Center for Molecular Medicine L8:04, Karolinska Institutet, Stockholm, Sweden. Rheumatology Unit, Department of Medicine, Center for Molecular Medicine L8:04, Karolinska Institutet, Stockholm, Sweden. European Molecular Biology Laboratory, Grenoble Outstation, Grenoble, France. Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering. School of Molecular Bioscience, The University of Sydney, New South Wales, Australia. Rheumatology Unit, Department of Medicine, Center for Molecular Medicine L8:04, Karolinska Institutet, Stockholm, Sweden. Department of Medical Biochemistry and Biophysics, Protein Science Facility, Karolinska Institutet, Stockholm, Sweden. Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
Structure of a TRIM21 - UBE2El complex reveals the specificity of E2 and ubiquitin recognition by TRIM E3 RINGsManuscript (preprint) (Other academic)

TRIM21, a RlNG-containing E3 ubiquitin-ligase of the TRIM  $\tiny(\underline{tri}partite \ \underline{m}otif)$ protein family, is a major autoantigen in SLE and Sjögren's syndrome as well as a modifier of interferon regulatory factors, thereby regulating innate immune signalling. We herein report the 2.86 Å crystal structure ofhuman TRIM211-91 comprising the RING domain (residues 16-55), in complex with the human E2 conjugating UBE2El enzyme (also denoted UbcH6). The crystal structure, joint with analysis by NMR and SAXS as well as structure-directed mutations and functional assays provides a detailed view of the specificity-determining contacts that support specific E2 recognition in the TRIM family. A detailed comparison of our structure with known E2 bound ubiquitin complexes, supported by biochemical analyses, reveals the molecular basis for TRIM21 interactions with donor ubiquitin that activates catalytic ubiquitin transfer. Finally, our structure convincingly demonstrates the placement of the Ub-targeted Lys61 of the adjacent TRIM21$\small^,$1- 91 close to the catalytically active UBE2El cysteine, and how the Lys61 amide is activated fora nucleophilic attack by hydrogen-bondeffected deshielding by conserved acidic residues at the E2 active site. In all, our structural findings provide molecular details ofthe selectivity involved in TRIM21 interactions with its cognate UBE2E1 enzyme and how TRIM21 positions ubiquitin in a catalytic conformation for ubiquitin transfer, and presents a snapshot of the Ub ligation step on a specific target residue of TRIM211-91 as an auto-ubiquitinated pseudo-substrate at high concentration. Increased structural and functional understanding of the TRIM mediated ubiquitination will aid development ofnovel therapeutic approaches in the entire TRIM family ofproteins.

• 2.
Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology. Yokohama City University, Japan . Karolinska Institute, Sweden . Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology. Yokohama City University, Japan . University of Toronto, Canada . Karolinska Institute, Sweden . Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
High-resolution structure of TBP with TAF1 reveals anchoring patterns in transcriptional regulation2013In: Nature Structural & Molecular Biology, ISSN 1545-9993, E-ISSN 1545-9985, Vol. 20, no 8, p. 1008-+Article in journal (Refereed)

The general transcription factor TFIID provides a regulatory platform for transcription initiation. Here we present the crystal structure (1.97 angstrom) and NMR analysis of yeast TAF1 N-terminal domains TAND1 and TAND2 bound to yeast TBP, together with mutational data. We find that yeast TAF1-TAND1, which in itself acts as a transcriptional activator, binds TBPs concave DNA-binding surface by presenting similar anchor residues to TBP as does Mot1 but from a distinct structural scaffold. Furthermore, we show how TAF1-TAND2 uses an aromatic and acidic anchoring pattern to bind a conserved TBP surface groove traversing the basic helix region, and we find highly similar TBP-binding motifs also presented by the structurally distinct TFIIA, Mot1 and Brf1 proteins. Our identification of these anchoring patterns, which can be easily disrupted or enhanced, provides insight into the competitive multiprotein TBP interplay critical to transcriptional regulation.

• 3.
Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology . Linköping University, The Institute of Technology.
Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology . Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology . Linköping University, The Institute of Technology. Department of Medical Biochemistry and Biophysics, Karolinska Institutet, S171 77 Stockholm. Biacore AB, GE Healthcare Europe GmbH, Björkgatan 30, SE-571 25 Uppsala, Sweden. Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology . Linköping University, The Institute of Technology. Biacore AB, GE Healthcare Europe GmbH, Björkgatan 30, SE-571 25 Uppsala, Sweden. Department of Medical Biochemistry and Biophysics, Karolinska Institutet, S171 77 Stockholm. Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology . Linköping University, The Institute of Technology.
Molecular characterization of the interaction between the disordered c-Myc transactivation domain and the TATA-binding protein (TBP)Manuscript (preprint) (Other academic)

The proto-oncogene c-myc affects the occurrence, expansion, and evolution of numerous aggressive human cancers, and is often associated with the late-stage and/or poor prognostic disease. Regulation of target gene activity by c-Myc occurs through protein interactions with the c-Myc transactivation domain (TAD) which, in addition to binding the TATA-binding protein (TBP) also recruits a wide variety of co-activators and suppressor proteins. Here, we present a molecular model, based on NMR, X-ray crystallography and SPR measurements, which describes how the c-Myc TAD binds to TBP. Our model contributes to the understanding of how c-Myc can regulate individual genes as well as entire gene programs.

• 4.
Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Linköping University, The Institute of Technology.
Transient structure and dynamics in the disordered c-Myc transactivation domain affect Bin1 binding2012In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 40, no 13, p. 6353-6366Article in journal (Refereed)

The crucial role of Myc as an oncoprotein and as a key regulator of cell growth makes it essential to understand the molecular basis of Myc function. The N-terminal region of c-Myc coordinates a wealth of protein interactions involved in transformation, differentiation and apoptosis. We have characterized in detail the intrinsically disordered properties of Myc-1-88, where hierarchical phosphorylation of S62 and T58 regulates activation and destruction of the Myc protein. By nuclear magnetic resonance (NMR) chemical shift analysis, relaxation measurements and NOE analysis, we show that although Myc occupies a very heterogeneous conformational space, we find transiently structured regions in residues 22-33 and in the Myc homology box I (MBI; residues 45-65); both these regions are conserved in other members of the Myc family. Binding of Bin1 to Myc-1-88 as assayed by NMR and surface plasmon resonance (SPR) revealed primary binding to the S62 region in a dynamically disordered and multivalent complex, accompanied by population shifts leading to altered intramolecular conformational dynamics. These findings expand the increasingly recognized concept of intrinsically disordered regions mediating transient interactions to Myc, a key transcriptional regulator of major medical importance, and have important implications for further understanding its multifaceted role in gene regulation.

• 5.
Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology . Linköping University, The Institute of Technology.
Transient structure and intrinsic disorder in the c-Myc transactivation domain and its effects on ligand bindingManuscript (preprint) (Other academic)

The crucial role of c-Myc as an oncoprotein and as a key regulator of cell growth makes it essential to understand the molecular basis of c-Myc function. The transactivation domain of c-Myc coordinates a wealth of protein interactions involved in transformation, differentiation and apoptosis. We have characterized in detail the intrinsically disordered properties of c-Myc-1-88, where hierarchical phosphorylation of T58 and S62 regulates activation and destruction of the c-Myc protein. By NMR chemical shift analysis, relaxation measurements and NOE analysis, we show that both the MBI region (residues 45-65) and residues 22-33 are transiently structured regions, conserved also in other members of the Myc family. Binding of Bin1-SH3 to c-Myc-1-88 as assayed by NMR and SPR revealed primary binding to the S62 region, but also a dynamically disordered and multivalent complex in which intrinsic disorder of c-Myc-1-88 was retained while releasing transient intramolecular interactions. Our findings describe a novel mode of regulatory recognition of c-Myc that is in agreement with the increasingly recognized capability of intrinsically disordered regions to efficiently mediate transient interactions with a wide range of targets, with important implications towards understanding the unique multifaceted biological functions of c-Myc.

• 6.
Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology . Linköping University, The Institute of Technology.
Critical biophysical properties in the Pseudomonas aeruginosa efflux gene regulator MexR are targeted by mutations conferring multidrug resistance2010In: Protein Science, ISSN 0961-8368, E-ISSN 1469-896X, Vol. 19, no 4, p. 680-692Article in journal (Refereed)

The self-assembling MexA-MexB-OprM efflux pump system, encoded by the mexO operon, contributes to facile resistance of Pseudomonas aeruginosa by actively extruding multiple antimicrobials. MexR negatively regulates the mexO operon, comprising two adjacent MexR binding sites, and is as such highly targeted by mutations that confer multidrug resistance (MDR). To understand how MDR mutations impair MexR function, we studied MexR-wt as well as a selected set of MDR single mutants distant from the proposed DNA-binding helix. Although DNA affinity and MexA-MexB-OprM repression were both drastically impaired in the selected MexR-MDR mutants, MexR-wt bound its two binding sites in the mexO with high affinity as a dimer. In the MexR-MDR mutants, secondary structure content and oligomerization properties were very similar to MexR-wt despite their lack of DNA binding. Despite this, the MexR-MDR mutants showed highly varying stabilities compared with MexR-wt, suggesting disturbed critical interdomain contacts, because mutations in the DNA-binding domains affected the stability of the dimer region and vice versa. Furthermore, significant ANS binding to MexR-wt in both free and DNA-bound states, together with increased ANS binding in all studied mutants, suggest that a hydrophobic cavity in the dimer region already shown to be involved in regulatory binding is enlarged by MDR mutations. Taken together, we propose that the biophysical MexR properties that are targeted by MDR mutations stability, domain interactions, and internal hydrophobic surfaces are also critical for the regulation of MexR DNA binding.

• 7.
Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology . Linköping University, The Institute of Technology.
Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology . Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology . Linköping University, The Institute of Technology.
Structural and dynamic analysis of human glutaredoxin 3Manuscript (preprint) (Other academic)

Human glutaredoxin (Grx3) is an essential protein associated with biological functions including embryonic development and immune response, and is involved in human disease such as lung, colon cancer and cardiovascular disorder. Grx3 can harbour a [2Fe-2S]2+ cluster and is most likely involved in oxidative stress response. Grx3 consists of an N-terminal thioredoxin-like domain and two additional monothiol glutaredoxin domains, and is thus classified as a multidomain monothiol glutaredoxin. The Grx3 thioredoxin domain lacks both a characteristic active-site and catalytic activity, but is still essential in the yeast homologue and presumably functions together with its monothiol glutaredoxin domains. We have characterised the structures of the two Nterminal domains in Grx3, which have thioredoxin and glutaredoxin folds. We have analysed their dynamic and structural interdependence by analysing NMR relaxation data together with chemical shift changes between isolated and covalently linked domains. We find that although the two domains show interdomain mobility around a semi-flexible linker, there are indications for a preferred interaction surface between the two domains. Millisecond internal dynamics in a suggested ligand binding site in the isolated thioredoxin domain is dampened in the domain pair, suggesting that the two domains mutually affect each other on a profound level. Our results present a platform for further detailed studies of multidomain thioredoxin-glutaredoxin containing proteins, and their function in human cells.

• 8.
Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Applied Physics. Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology. Department of Medicine, Division of Infectious Diseases, Karolinska Institutet, Stockholm, Sweden. Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology. Department of Medicine, Division of Infectious Diseases, Karolinska Institutet, Stockholm, Sweden. Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
Molecular causes for deficient repression in multidrug resistant mutants in the Pseudomonas aeruginosa efflux gene regulator MexRManuscript (preprint) (Other academic)

n/a

• 9.
Department of Clinical Microbiology, Karolinska University Hospital, Karolinska Institutet, Sweden.
Chemistry Department, Princeton University, Princeton, NJ, United States. Department of Microbiology, Tumorbiology Center, Karolinska Institutet, Sweden. Karolinska Institutet. King Gustaf V Research Institute, Department of Medicine, Karolinska University Hospital, Sweden.
Open-ended assignments and student responsibility2007In: Biochemistry and molecular biology education, ISSN 1470-8175, E-ISSN 1539-3429, Vol. 35, no 3, p. 187-192Article in journal (Refereed)

An inquiry-based laboratory course was created in an effort to increase student responsibility in learning and to improve teaching in areas related to molecular medicine. Authentic medical cases with both scientific and clinical aspects formed the basis of a project-oriented course that also included student laboratory work focused on the disease-related proteins. Students used basic biochemical techniques to develop and test hypotheses relating their results to the clinical findings. The course also included patient demonstrations to personalize students' knowledge of case presentations, lectures on basic biochemical principles relevant to the molecular basis of the cases, and seminars by invited guests with expertise in translational medicine. Students developed proposals for future research as part of the final examination. An inquiry matrix was used to evaluate the degree of learning responsibility taken during the course. By allowing for openness in how to explore the case including choice of methods and interpretation of unexpected results, students gained confidence in their ability to solve problems, formulate and test hypotheses, and collaborate with both clinical and non-clinical professionals.

• 10.
Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
Linköping University, Department of Management and Engineering, Project management, Innovations and Entrepreneurship . Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology. Linköping University, Department of Science and Technology. Linköping University, The Institute of Technology.
To design a novel protein: A CDIO experience in Molecular Biotechnology at Linköping University2006Conference paper (Other academic)
• 11.
Rheumatology Unit, Department of Medicine, Karolinska Institute, Stockholm, Sweden.
Rheumatology Unit, Department of Medicine, Karolinska Institute, Stockholm, Sweden. Rheumatology Unit, Department of Medicine, Karolinska Institute, Stockholm, Sweden. Neuroimmunology Unit, Department of Clinical Neuroscience, Karolinska Institute, Stockholm, Sweden. Clinical Allergy Research Unit, Department of Medicine, Karolinska Institute, Stockholm, Sweden. Neuroimmunology Unit, Department of Clinical Neuroscience, Karolinska Institute, Stockholm, Sweden. Rheumatology Unit, Department of Medicine, Karolinska Institute, Stockholm, Sweden. Rheumatology Unit, Department of Medicine, Karolinska Institute, Stockholm, Sweden. Rheumatology Unit, Department of Medicine, Karolinska Institute, Stockholm, Sweden. Department of Woman and Child Health, Karolinska Institute, Stockholm, Sweden. Department of Woman and Child Health, Karolinska Institute, Stockholm, Sweden. Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Linköping University, The Institute of Technology. Hertie Institute for clinical Brain Research, University of Tübingen, Germany. Center for Neurological Diseases, Brigham and Women’s Hospital, Harvard Medical School, Boston, USA,. Center for Infectious Medicine, Department of Medicine, Karolinska Institutet, Stockholm, Sweden. Clinical Allergy Research Unit, Department of Medicine, Karolinska Institute, Stockholm, Sweden. Neuroimmunology Unit, Department of Clinical Neuroscience, Karolinska Institute, Stockholm, Sweden. Rheumatology Unit, Department of Medicine, Karolinska Institute, Stockholm, Sweden.
MHC Genes Determine Fetal Susceptibility in a Rat Model of Congenital Heart Block2010In: Scandinavian Journal of Immunology, ISSN 0300-9475, E-ISSN 1365-3083, Vol. 72, no 3, p. 269-269Article in journal (Other academic)

n/a

• 12.
Rheumatology Unit, Department of Medicine, CMM L8:04, Karolinska Institutet, SE-171 76 Stockholm, Sweden.
Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Linköping University, The Institute of Technology. Rheumatology Unit, Department of Medicine, CMM L8:04, Karolinska Institutet, SE-171 76 Stockholm, Sweden. Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering. Linköping University, Department of Clinical and Experimental Medicine, Division of Microbiology and Molecular Medicine. Linköping University, Faculty of Medicine and Health Sciences. Ontario Cancer Institute and Department of Medical Biophysics, University of Toronto, Canada. Department of Clinical Sciences at Danderyd Hospital, Karolinska Institute, Stockholm, Sweden. Ontario Cancer Institute and Department of Medical Biophysics, University of Toronto, Canada. Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Linköping University, The Institute of Technology. Rheumatology Unit, Department of Medicine, CMM L8:04, Karolinska Institutet, SE-171 76 Stockholm, Sweden.
Anti-Ro52 Autoantibodies from Patients with Sjögren's Syndrome Inhibit the Ro52 E3 Ligase Activity by Blocking the E3/E2 Interface2011In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 286, no 42, p. 36478-36491Article in journal (Refereed)

Ro52 (TRIM21) is an E3 ligase of the tripartite motif family that negatively regulates proinflammatory cytokine production by ubiquitinating transcription factors of the interferon regulatory factor family. Autoantibodies to Ro52 are present in patients with lupus and Sjögren's syndrome, but it is not known if these autoantibodies affect the function of Ro52. To address this question, the requirements for Ro52 E3 ligase activity were first analyzed in detail. Scanning a panel of E2 ubiquitin-conjugating enzymes, we found that UBE2D1–4 and UBE2E1–2 supported the E3 ligase activity of Ro52 and that the E3 ligase activity of Ro52 was dependent on its RING domain. We also found that the N-terminal extensions in the class III E2 enzymes affected their interaction with Ro52. Although the N-terminal extension in UBE2E3 made this E2 enzyme unable to function together with Ro52, the N-terminal extensions in UBE2E1 and UBE2E2 allowed for a functional interaction with Ro52. Anti-Ro52-positive patient sera and affinity-purified anti-RING domain autoantibodies inhibited the E3 activity of Ro52 in ubiquitination assays. Using NMR, limited proteolysis, ELISA, and Ro52 mutants, we mapped the interactions between Ro52, UBE2E1, and anti-Ro52 autoantibodies. We found that anti-Ro52 autoantibodies inhibited the E3 ligase activity of Ro52 by sterically blocking the E2/E3 interaction between Ro52 and UBE2E1. Our data suggest that anti-Ro52 autoantibodies binding the RING domain of Ro52 may be actively involved in the pathogenesis of rheumatic autoimmune disease by inhibiting Ro52-mediated ubiquitination.

• 13.
Karolinska Institutet, Stockholm.
Karolinska Institutet, Stockholm. Karolinska Institutet, Stockholm. Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology . Karolinska Institutet, Stockholm. University of Oulu, Finland. Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology . Karolinska Institutet, Stockholm. Karolinska Institutet, Stockholm.
A Novel Monothiol Glutaredoxin (Grx4) from Escherichia coli Can Serve as a Substrate for Thioredoxin Reductase2005In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 280, no 26, p. 24544-24552Article in journal (Refereed)

Glutaredoxins are ubiquitous proteins that catalyze the reduction of disulfides via reduced glutathione (GSH). Escherichia coli has three glutaredoxins (Grx1, Grx2, and Grx3), all containing the classic dithiol active site CPYC. We report the cloning, expression, and characterization of a novel monothiol E. coli glutaredoxin, which we name glutaredoxin 4 (Grx4). The protein consists of 115 amino acids (12.7 kDa), has a monothiol (CGFS) potential active site and shows high sequence homology to the other monothiol glutaredoxins and especially to yeast Grx5. Experiments with gene knock-out techniques showed that the reading frame encoding Grx4 was essential. Grx4 was inactive as a GSH-disulfide oxidoreductase in a standard glutaredoxin assay with GSH and hydroxyethyl disulfide in a complete system with NADPH and glutathione reductase. An engineered CGFC active site mutant did not gain activity either. Grx4 in reduced form contained three thiols, and treatment with oxidized GSH resulted in glutathionylation and formation of a disulfide. Remarkably, this disulfide of Grx4 was a direct substrate for NADPH and E. coli thioredoxin reductase, whereas the mixed disulfide was reduced by Grx1. Reduced Grx4 showed the potential to transfer electrons to oxidized E. coli Grx1 and Grx3. Grx4 is highly abundant (750–2000 ng/mg of total soluble protein), as determined by a specific enzyme-link immunosorbent assay, and most likely regulated by guanosine 3′,5′-tetraphosphate upon entry to stationary phase. Grx4 was highly elevated upon iron depletion, suggesting an iron-related function for the protein.

• 14.
Karolinska Institutet, Stockholm.
University of Padova, Italy. Karolinska Institutet, Stockholm. University of Padova, Italy. Karolinska Institutet, Stockholm. Karolinska Institutet, Stockholm. Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology .
Molecular Mapping of Functionalities in the Solution Structure of Reduced Grx4, a Monothiol Glutaredoxin from Escherichia coli2005In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 280, no 26, p. 24553-24561Article in journal (Refereed)
• 15.
Department of Medical Biochemistry and Biophysics Karolinska Institutet, Stockholm.
Molecular Biotechnology IFM, Linköping University. Department of Medical Biochemistry and Biophysics Karolinska Institutet, Stockholm. School of Molecular and Microbial Biosciences University of Sydney. Biacore AB, Uppsala. Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology .
N and C-terminal Sub-regions in the c-Myc Transactivation Region and their Joint Role in Creating Versatility in Folding and Binding2005In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 246, p. 175-189Article in journal (Refereed)
• 16.
University of Toronto.
University of Toronto. Linköping University, Department of Physics, Chemistry and Biology, Bioinformatics . Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology . Linköping University, The Institute of Technology. University of Toronto.
Structural and biochemical investigation of two Arabidopsis shikimate kinases: The heat-inducible isoform is thermostable2011In: Protein Science, ISSN 0961-8368, E-ISSN 1469-896X, Vol. 20, no 7, p. 1125-1136Article in journal (Refereed)

The expression of plant shikimate kinase (SK; EC 2.7.1.71), an intermediate step in the shikimate pathway to aromatic amino acid biosynthesis, is induced under specific conditions of environmental stress and developmental requirements in an isoform-specific manner. Despite their important physiological role, experimental structures of plant SKs have not been determined and the biochemical nature of plant SK regulation is unknown. The Arabidopsis thaliana genome encodes two SKs, AtSK1 and AtSK2. We demonstrate that AtSK2 is highly unstable and becomes inactivated at 37 degrees C whereas the heat-induced isoform, AtSK1, is thermostable and fully active under identical conditions at this temperature. We determined the crystal structure of AtSK2, the first SK structure from the plant kingdom, and conducted biophysical characterizations of both AtSK1 and AtSK2 towards understanding this mechanism of thermal regulation. The crystal structure of AtSK2 is generally conserved with bacterial SKs with the addition of a putative regulatory phosphorylation motif forming part of the adenosine triphosphate binding site. The heat-induced isoform, AtSK1, forms a homodimer in solution, the formation of which facilitates its relative thermostability compared to AtSK2. In silico analyses identified AtSK1 site variants that may contribute to AtSK1 stability. Our findings suggest that AtSK1 performs a unique function under heat stress conditions where AtSK2 could become inactivated. We discuss these findings in the context of regulating metabolic flux to competing downstream pathways through SK-mediated control of steady state concentrations of shikimate.

• 17.
Department of Medicine, Atherosclerosis Research Unit, King Gustaf V Research Institute, S-171 76 Stockholm, Sweden.
Department of Organic Chemistry, University of Padova, 35131 Padova, Italy. Department of Medicine, Atherosclerosis Research Unit, King Gustaf V Research Institute, S-171 76 Stockholm, Sweden. Department of Medicine, Atherosclerosis Research Unit, King Gustaf V Research Institute, S-171 76 Stockholm, Sweden. Department of Medicine, Atherosclerosis Research Unit, King Gustaf V Research Institute, S-171 76 Stockholm, Sweden. Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology . Department of Medicine, Atherosclerosis Research Unit, King Gustaf V Research Institute, S-171 76 Stockholm, Sweden, King Gustaf V Research Institute, Karolinska Hospital, S-171 76 Stockholm, Sweden.
Genetic and Structural Evaluation of Fatty Acid Transport Protein-4 in Relation to Markers of the Insulin Resistance Syndrome2004In: Journal of Clinical Endocrinology and Metabolism, ISSN 0021-972X, E-ISSN 1945-7197, Vol. 89, no 1, p. 392-399Article in journal (Refereed)

Disturbances in fatty acid metabolism are involved in the etiology of insulin resistance and the related dyslipidemia, hypertension, and procoagulant state. The fatty acid transport proteins (FATPs) are implicated in facilitated cellular uptake of nonesterified fatty acids (NEFAs), thus potentially regulating NEFA concentrations and metabolism. The aim of this study was to investigate polymorphic loci in the FATP4 gene with respect to associations with fasting and postprandial lipid and lipoprotein variables and markers of insulin resistance in 608 healthy, middle-aged Swedish men and to evaluate possible mechanisms behind any associations observed. Heterozygotes for a Gly209Ser polymorphism (Ser allele frequency 0.05) had significantly lower body mass index and, correcting for body mass index, significantly lower triglyceride concentrations, systolic blood pressure, insulin concentrations, and homeostasis model assessment index compared with common homozygotes. A three-dimensional model of the FATP4 protein based on structural and functional similarity with adenylate-forming enzymes revealed that the variable residue 209 is exposed in a region potentially involved in protein-protein interactions. Furthermore, the model indicated functional regions with respect to NEFA transport and acyl-coenzyme A synthase activity and membrane association. These findings propose FATP4 as a candidate gene for the insulin resistance syndrome and provide a structural basis for understanding FATP function in NEFA transport and metabolism.

• 18.
Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology. Princess Margaret Cancer Centre and Department of Medical Biophysics, University of Toronto, Canada. Princess Margaret Cancer Centre and Department of Medical Biophysics, University of Toronto, Canada. Department of Medical Sciences, Molecular Medicine and Science for Life Laboratory, Uppsala University, Sweden. Structural Genomics Consortium, University of Toronto, Canada. Princess Margaret Cancer Centre and Department of Medical Biophysics, University of Toronto, Canada. Princess Margaret Cancer Centre and Department of Medical Biophysics, University of Toronto, Canada. Structural Genomics Consortium, University of Toronto, Canada. Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
Basic Tilted Helix Bundle - A new protein fold in human FKBP25/FKBP3 and HectD12014In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 447, no 1, p. 26-31Article in journal (Refereed)

In this paper, we describe the structure of a N-terminal domain motif in nuclear-localized FKBP251-73, a member of the FKBP family, together with the structure of a sequence-related subdomain of the E3 ubiquitin ligase HectD1 that we show belongs to the same fold. This motif adopts a compact 5-helix bundle which we name the Basic Tilted Helix Bundle (BTHB) domain. A positively charged surface patch, structurally centered around the tilted helix H4, is present in both FKBP25 and HectD1 and is conserved in both proteins, suggesting a conserved functional role. We provide detailed comparative analysis of the structures of the two proteins and their sequence similarities, and analysis of the interaction of the proposed FKBP25 binding protein YY1. We suggest that the basic motif in BTHB is involved in the observed DNA binding of FKBP25, and that the function of this domain can be affected by regulatory YY1 binding and/or interactions with adjacent domains.

• 19.
Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
Pre-Anchoring of Pin1 to Unphosphorylated c-Myc in a Fuzzy Complex Regulates c-Myc Activity2015In: Structure, ISSN 0969-2126, E-ISSN 1878-4186, Vol. 23, no 12, p. 2267-2279Article in journal (Refereed)

Hierarchic phosphorylation and concomitant Pin1-mediated proline isomerization of the oncoprotein c-Myc controls its cellular stability and activity. However, the molecular basis for Pin1 recognition and catalysis of c-Myc and other multisite, disordered substrates in cell regulation and disease is unclear. By nuclear magnetic resonance, surface plasmon resonance, and molecular modeling, we show that Pin1 subdomains jointly pre-anchor unphosphorylated c-Myc1–88 in the Pin1 interdomain cleft in a disordered, or “fuzzy”, complex at the herein named Myc Box 0 (MB0) conserved region N-terminal to the highly conserved Myc Box I (MBI). Ser62 phosphorylation in MBI intensifies previously transient MBI-Pin1 interactions in c-Myc1–88 binding, and increasingly engages Pin1PPIase and its catalytic region with maintained MB0 interactions. In cellular assays, MB0 mutated c-Myc shows decreased Pin1 interaction, increased protein half-life, but lowered rates of Myc-driven transcription and cell proliferation. We propose that dynamic Pin1 recognition of MB0 contributes to the regulation of c-Myc activity in cells

• 20.
Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology . Linköping University, The Institute of Technology.
Linköping University, Department of Physics, Chemistry and Biology, Bioinformatics . Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology . Linköping University, The Institute of Technology. Gabriele von Bülow Gymnasium, Berlin. Karolinska Hospital. Linköping University, Department of Physics, Chemistry and Biology, Bioinformatics . Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology . Linköping University, The Institute of Technology.
The fellowship of the RING: The RING-B-box linker region (RBL) interacts with the RING in TRIM21/Ro52, contributes to an autoantigenic epitope in Sjögren's syndrome, and is an integral and conserved region in TRIM proteins2008In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 377, no 2, p. 431-449Article in journal (Refereed)

Ro52 is a major autoantigen that is targeted in the autoimmune disease Sjögren syndrome and belongs to the tripartite motif (TRIM) protein family. Disease-related antigenic epitopes are mainly found in the coiled-coil domain of Ro52, but one such epitope is located in the Zn2+-binding region, which comprises an N-terminal RING followed by a B-box, separated by a ∼40-residue linker peptide. In the present study, we extend the structural, biophysical, and immunological knowledge of this RING-B-box linker (RBL) by employing an array of methods. Our bioinformatic investigations show that the RBL sequence motif is unique to TRIM proteins and can be classified into three distinct subtypes. The RBL regions of all three subtypes are as conserved as their known flanking domains, and all are predicted to comprise an amphipathic helix. This helix formation is confirmed by circular dichroism spectroscopy and is dependent on the presence of the RING. Immunological studies show that the RBL is part of a conformation-dependent epitope, and its antigenicity is likewise dependent on a structured RING domain. Recombinant Ro52 RING-RBL exists as a monomer in vitro, and binding of two Zn2+ increases its stability. Regions stabilized by Zn2+ binding are identified by limited proteolysis and matrix-assisted laser desorption/ionization mass spectrometry. Furthermore, the residues of the RING and linker that interact with each other are identified by analysis of protection patterns, which, together with bioinformatic and biophysical data, enabled us to propose a structural model of the RING-RBL based on modeling and docking experiments. Sequence similarities and evolutionary sequence patterns suggest that the results obtained from Ro52 are extendable to the entire TRIM protein family.

• 21.
Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology . Linköping University, The Institute of Technology.
Bijvoet Center for Biomolecular Research, Science Faculty, Utrecht University, 3584 CH Utrecht, The Netherlands. Department of Informatics, Gabriele-von-B¨ulow Gymnasium, DE-13509 Berlin, Germany. Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology . Linköping University, The Institute of Technology. Bijvoet Center for Biomolecular Research, Science Faculty, Utrecht University, 3584 CH Utrecht, The Netherlands.

Background: MTMDAT is a recently developed program facilitating the analysis of mass spectrometry data of proteins and biomolecular complexes (Hennig et al, 2008), which were structurally probed by limited proteolysis. This can provide information about stable fragments of multidomain proteins, yields tertiary and quaternary structure data, and residue-wise origins of stability changes can be determined.

Results: A new feature allows for the direct identification of residues that are involved in complex formation bycomparing the mass spectra of bound and unbound proteins after proteolysis. If 3D structures of the unboundcomponents are available, this data can be used to restrain data-driven docking to calculate the structure ofthe complex. Here, we provide a new implementation of MTMDAT, with a pipeline to the data-driven dockingprogram HADDOCK (Dominguez et al., 2003; De Vries et al., 2007), streamlining the entire procedure. This,together with usability improvements in MTMDAT, enables direct high-throughput modelling of protein complexesfrom mass spectrometry data.

Conclusions: MTMDAT can be downloaded from http://cms.ifm.liu.se/chemistry/molbiotech/maria¯sunnerhagens¯group/mtmdat/ (windows- or unix-based, with or without HADDOCK pipeline) together withthe manual and example files. The program is free for academic/non-commercial purposes.

• 22.
Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
Utrecht University and Technical University München. Gabriele-von-Bülow Gymnasium, Berlin, Germany . University of Minnesota. University of Minnesota. Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering. Utrecht University.
MTMDAT-HADDOCK: high-throughput, protein complex structure modeling based on limited proteolysis and mass spectrometry2012In: BMC Structural Biology, ISSN 1472-6807, E-ISSN 1472-6807, Vol. 12, no 29Article in journal (Refereed)

Background

MTMDAT is a program designed to facilitate analysis of mass spectrometry data of proteins and biomolecular complexes that are probed structurally by limited proteolysis. This approach can provide information about stable fragments of multidomain proteins, yield tertiary and quaternary structure data, and help determine the origin of stability changes at the amino acid residue level. Here, we introduce a pipeline between MTMDAT and HADDOCK, that facilitates protein-protein complex structure probing in a high-throughput and highly automated fashion.

Results

A new feature of MTMDAT allows for the direct identification of residues that are involved in complex formation by comparing the mass spectra of bound and unbound proteins after proteolysis. If 3D structures of the unbound components are available, this data can be used to define restraints for data-driven docking to calculate a model of the complex. We describe here a new implementation of MTMDAT, which includes a pipeline to the data-driven docking program HADDOCK, thus streamlining the entire procedure. This addition, together with usability improvements in MTMDAT, enables high-throughput modeling of protein complexes from mass spectrometry data. The algorithm has been validated by using the protein-protein interaction between the ubiquitin-binding domain of proteasome component Rpn13 and ubiquitin. The resulting structural model, based on restraints extracted by MTMDAT from limited proteolysis and modeled by HADDOCK, was compared to the published NMR structure, which relied on twelve unambiguous intermolecular NOE interactions. The MTMDAT-HADDOCK structure was of similar quality to structures generated using only chemical shift perturbation data derived by NMR titration experiments.

Conclusions

The new MTMDAT-HADDOCK pipeline enables direct high-throughput modeling of protein complexes from mass spectrometry data. MTMDAT-HADDOCK can be downloaded from http://www.ifm.liu.se/chemistry/molbiotech/maria_sunnerhagens_group/mtmdat/webcitetogether with the manual and example files. The program is free for academic/non-commercial purposes.

• 23.
Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology . Linköping University, The Institute of Technology.
Department of Informatics, Gabriele-von-Bu¨ low Gymnasium, DE-13509 Berlin, Germany. Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology . Linköping University, The Institute of Technology.
MTMDAT: Automated analysis and visualization of mass spectrometry data for tertiary and quaternary structure probing of proteins2008In: Bioinformatics, ISSN 1367-4803, E-ISSN 1367-4811, Vol. 24, no 10, p. 1310-1312Article in journal (Refereed)

In structural biology and -genomics, nuclear magnetic resonance (NMR) spectroscopy and crystallography are the methods of choice, but sample requirements can be hard to fulfil. Valuable structural information can also be obtained by using a combination of limited proteolysis and mass spectrometry, providing not only knowledge of how to improve sample conditions for crystallization trials or NMR spectrosopy by gaining insight into subdomain identities but also probing tertiary and quaternary structure, folding and stability, ligand binding, protein interactions and the location of post-translational modifications. For high-throughput studies and larger proteins, however, this experimentally fast and easy approach produces considerable amounts of data, which until now has made the evaluation exceedingly laborious if at all manually possible. MTMDAT, equipped with a browser-like graphical user interface, accelerates this evaluation manifold by automated peak picking, assignment, data processing and visualization. © 2008 The Author(s).

• 24.
Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology . Linköping University, The Institute of Technology.
Department of Medicine, CMM L8:04, Karolinska Hospital, 5-171 76 Stockholm, Sweden. Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology . Linköping University, The Institute of Technology. Department of Medicine, CMM L8:04, Karolinska Hospital, 5-171 76 Stockholm, Sweden. Center for Neurological Diseases, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, United States. Department of Occupational and Environmental Medicine, Sahlgrenska Academy, Göteborg University, S-412 66 Göteborg, Sweden. Department of Medicine, CMM L8:04, Karolinska Hospital, 5-171 76 Stockholm, Sweden. Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology . Linköping University, The Institute of Technology.
Structural organization and Zn2+ -dependent subdomain interactions involving autoantigenic epitopes in the Ring-B-box-coiled-coil (RBCC) region of Ro522005In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 280, no 39, p. 33250-33261Article in journal (Refereed)

Ro52 is one of the major autoantigens targeted in the autoimmune disease Sjögren syndrome. By sequence similarity, Ro52 belongs to the RING-B-box-coiled-coil (RBCC) protein family. Disease-related antibodies bind Ro52 in a conformation-dependent way both in the coiled-coil region and in the Zn2+-binding Ring-B-box region. Primarily associated with Sjögren syndrome, Ro52 autoantibodies directed to a specific, partially structured epitope in the coiled-coil region may also induce a congenital heart block in the fetus of pregnant Ro52-positive mothers. To improve our understanding of the pathogenic effects of autoantibody binding to the Zn 2+-binding region, a multianalytical mapping of its structural, biophysical, and antigenic properties is presented. Structure content and ligand binding of subregions, dissected by peptide synthesis and subcloning, were analyzed by fluorescence and circular dichroism spectroscopy. A novel matrix-assisted laser desorption ionization time-of-flight mass spectrometry strategy for time-resolved proteolysis experiments of large protein domains was developed to facilitate analysis and to help resolve the tertiary arrangement of the entire RBCC subregion. The linker region between the RING and B-box motifs is crucial for full folding, and Zn2+ affinity of the RING-B-box region is further protected in the entire RBCC region and appears to interact with the coiled-coil region. Murine monoclonal antibodies raised toward the RING-B-box region were primarily directed toward the linker, further supporting a highly functional role for the linker in a cellular environment. Taken together with our previous analysis of autoantigenic epitopes in the coiled-coil region, localization of autoantigenic epitopes in Ro52 appears closely related to molecular functionalities. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc.

• 25.
Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Linköping University, The Institute of Technology.
Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Linköping University, The Institute of Technology.
AEDANS labeled MexR variants and their DNA interactionManuscript (preprint) (Other academic)

MexR is a transcriptional repressor of the multidrug efflux-pump operon in the bacterial pathogen Pseudomonas aeruginosa, and thus, malfunction in the DNA-binding of the MexR protein leads to multidrug resistance (Poole et al., 1996). lt has been shown that mutations in the MexR protein lead to lost DNA binding ability (Saito et al., 2003). X-ray studies of the MexR protein (Lim et al., 2002) show that the protein contains a winged helix-turn-helix motif and this type of motif is known to be involved in DNA-binding (Gajiwala & Burley, 2000), but no exact DNA-binding sequence in MexR has yet been determined.

This study attempts to identify regions in the MexR protein involved in DNA-binding using a molecular probe as mapping tool. Three cysteines naturally occur in the MexR protein, located in positions 30, 62 and 138, respectively, and we have used two of them to map the DNA-binding site. Preliminary results show that the fluorescence of an TAEDANS probe attached to cys 62 is affected by DNA-binding, while a probe at cys 30 is not affected. Further studies are required to analyse the implications of these results.

• 26.
King GustafV Research Institute Karolinska University Hospital, stockholm.
Department of Medicine Karolinska Institutet, Stockholm. Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology . King GustafV Research Institute Karolinska University Hospital, Stockholm.
The Ile128Thr polymorphism influences stability and ligand binding properties of the microsomal triglyceride transfer protein2006In: Journal of Lipid Research, ISSN 0022-2275, E-ISSN 1539-7262, Vol. 47, no 7, p. 1378-1385Article in journal (Refereed)

The microsomal triglyceride transfer protein (MTTP) is essential for the assembly of VLDLs. We recently observed that a polymorphism in the MTTP promoter (-493G>T), which is in allelic association with an isoleucine-to-theronine substitution at position 128 (Ile128Thr) in the expressed protein, confers an increased risk of coronary heart disease. Two variant proteins comprising amino acids 16-297 of intact MTTP, MTTPN-Ile128 and MTTP N-Thr128, had similar native secondary structure content, as judged by circular dichroism. However, the thermal stability of MTTPN-Thr128 was greatly reduced, and this protein was also more extensively cleaved in limited proteolysis experiments compared with MTTPN-Ile128, both of these findings support a less compact fold. On adding LDL, which includes natively folded apolipoprotein B (apoB), decreased stability of the MTTP N-Thr128-LDL complex was observed compared with that of the MTTP N-Ile128-LDL complex. In a refined model of the N-terminal domain of MTTP, residue 128 is located in a surface-exposed position, in the same region as an identified MTTP binding site in the homologous apoB protein. Thus, the Ile128Thr polymorphism confers reduced structural stability, leading to decreased binding of MTTP to LDL particles. Because the major MTTP binding target on LDL is apoB, the Ile128Thr polymorphism could target the MTTP-apoB interaction. Copyright © 2006 by the American Society for Biochemistry and Molecular Biology, Inc.

• 27.
University of Toronto.
University of Toronto. University of Toronto. University of Toronto. University of Toronto. Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology . Linköping University, The Institute of Technology. University of Toronto.
A novel strategy for NMR resonance assignment and protein structure determination2011In: JOURNAL OF BIOMOLECULAR NMR, ISSN 0925-2738, Vol. 49, no 1, p. 27-38Article in journal (Refereed)

The quality of protein structures determined by nuclear magnetic resonance (NMR) spectroscopy is contingent on the number and quality of experimentally-derived resonance assignments, distance and angular restraints. Two key features of protein NMR data have posed challenges for the routine and automated structure determination of small to medium sized proteins; (1) spectral resolution - especially of crowded nuclear Overhauser effect spectroscopy (NOESY) spectra, and (2) the reliance on a continuous network of weak scalar couplings as part of most common assignment protocols. In order to facilitate NMR structure determination, we developed a semi-automated strategy that utilizes non-uniform sampling (NUS) and multidimensional decomposition (MDD) for optimal data collection and processing of selected, high resolution multidimensional NMR experiments, combined it with an ABACUS protocol for sequential and side chain resonance assignments, and streamlined this procedure to execute structure and refinement calculations in CYANA and CNS, respectively. Two graphical user interfaces (GUIs) were developed to facilitate efficient analysis and compilation of the data and to guide automated structure determination. This integrated method was implemented and refined on over 30 high quality structures of proteins ranging from 5.5 to 16.5 kDa in size.

• 28.
Rheumatology Unit, Department of Medicine, Karolinska Inst., Karolinska H., Stockholm, Sweden.
Rheumatology Unit, Department of Medicine, Karolinska Inst., Karolinska H., Stockholm, Sweden. Rheumatology Unit, Department of Medicine, Karolinska Inst., Karolinska H., Stockholm, Sweden. Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology . Pathology Unit, Karolinska Institutet, Stockholm, Sweden. Erlandsson Harris, H., Rheumatology Unit, Department of Medicine, Karolinska Inst., Karolinska H., Stockholm, Sweden.
A pH-induced modification of CII increases its arthritogenic properties2004In: Journal of Autoimmunity, ISSN 0896-8411, E-ISSN 1095-9157, Vol. 23, no 2, p. 95-102Article in journal (Refereed)

Immunoreactivity to collagen type II (CII) has been implicated in the pathogenesis of rheumatoid arthritis. Patients have been described to have an acidic pH in their inflamed synovial tissue. It is known that protein structures are modified by environmental pH, thus it is plausible that changes in synovial pH could affect the conformation of proteins like CII. Posttranslational modifications could alter the biophysical properties of cartilage proteins leading to autoimmunity. In this study we investigated if arthritogenicity of CII was affected by changes in pH, and if so, this could be correlated to altered protein conformation. Immunisation with CII at neutral pH induced a milder disease than did CII at acidic pH. All animals elicited a humoral response to CII, although with a significantly higher IgG1/IgG2b-ratio in the pH 7.4 group. Analysis by circular dichroism and electron microscopy indicated less fibrillation of CII at low pH as compared to neutral pH. Our results suggest that CII is more immunogenic and arthritogenic in an acidic environment than in a neutral environment. We can correlate these findings to pH-induced conformational changes of CII. Hence, self-tolerance to CII might be affected by changes in pH leading to altered and increased arthritogenicity. © 2004 Elsevier Ltd. All rights reserved.

• 29.
Rheumatology Unit, Department of Medicine, Karolinska Institutet, Stockholm, Sweden.
Rheumatology Unit, Department of Medicine, Karolinska Institutet, Stockholm, Sweden. Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology . Linköping University, The Institute of Technology. Department of Women and Child Health, Karolinska Institutet, Stockholm, Sweden. Dörner, T., Department of Medicine, Rheumatol. and Clin. Immunology Unit, University Hospital Charité, Berlin, Germany. Department of Biochemistry, Radboud Univ. Nijmegen/ModiQuest BV, Nijmegen, Netherlands. Department of Neurology, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, United States. Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology . Linköping University, The Institute of Technology. Rheumatology Unit, Department of Medicine, Karolinska Institutet, Stockholm, Sweden, Department of Medicine, CMM L8:04, Karolinska Hospital, SE-171 76 Stockholm, Sweden.
Structurally derived mutations define congenital heart block-related epitopes within the 200-239 amino acid stretch of the Ro52 protein2005In: Scandinavian Journal of Immunology, ISSN 0300-9475, E-ISSN 1365-3083, Vol. 61, no 2, p. 109-118Article in journal (Refereed)

Congenital heart block is a passively transferred autoimmune condition, which affects the children of mothers with Ro/SSA autoantibodies. During pregnancy, the antibodies are transported across the placenta and affect the fetus. We have previously demonstrated that antibodies directed to the 200-239 amino acid (aa) stretch of the Ro52 component of the Ro/SSA antigen correlate with the development of congenital heart block. In this report, we investigated the antibody-antigen interaction of this target epitope in detail at a molecular and structural level. Peptides representing aa 200-239 (p200) with structurally derived mutations were synthesized to define the epitopes recognized by two Ro52 human monoclonal antibodies, S3A8 and M4H1, isolated from patient-derived phage display libraries. Analyses by ELISA, circular dichroism and MALDI-TOF-MS demonstrate that the antibody recognition is dependent on a partly a-helical fold within the putative leucine zipper of the 200-239 aa stretch and that the two human anti-p200 monoclonal antibodies, M4H1 and S3A8, recognize different epitopic structures within the p200 peptide. In addition, we investigated the representation of each fine specificity within the sera of mothers with children born with congenital heart block, and in such sera, antibodies of the S3A8 idiotype were more commonly detected and at higher levels than M4H1-like antibodies.

• 30.
Karolinska Institutet, Stockholm.
Karolinska Institutet, Stockholm. Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Karolinska Institutet, Stockholm. Rheumatology and Clinical Immunology Unit University Hospital Charité, Berlin, Germany. Department of Biochemistry Radboud University Nijmegen and ModiQuest, The Netherland. Center for Neurologic Deseases Brigham and Women´s Hospital and Harvard Medical School, Boston, USA. Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Department of Medicine Karolinska Institutet, Stockholm.
Structurally Derived Mutations Define Congenital Heart Block-Related Epitopes Within the 200-239 Amino Acid Stretch of the Ro52 Protein2004In: Scandinavian Journal of Immunology, ISSN 0300-9475, E-ISSN 1365-3083, Vol. 61, p. 109-118Article in journal (Refereed)
• 31.
Rheumatology Unit, Department of Medicine, CMM L8:04 Karolinska Institute.
Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology . Linköping University, The Institute of Technology. Rheumatology Unit, Department of Medicine, CMM L8:04 Karolinska Institute. Rheumatology Unit, Department of Medicine, CMM L8:04 Karolinska Institute. Rheumatology Unit, Department of Medicine, CMM L8:04 Karolinska Institute. Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology . Linköping University, The Institute of Technology.
Structural, functional and immunologic characterization of folded subdomains in the Ro52 protein targeted in Sjögren's syndrome2006In: Molecular Immunology, ISSN 0161-5890, E-ISSN 1872-9142, Vol. 43, no 6, p. 588-598Article in journal (Refereed)

Ro52, one of the major autoantigens in the rheumatic disease Sjögren's syndrome (SS), belongs to the tripartite motif (TRIM) or RING-B-box-coiled-coil (RBCC) protein family, thus comprising an N-terminal RING, followed by a B-box and a coiled-coil region. Several different proteomic functions have been suggested for Ro52, including DNA binding, protein interactions and Zn 2+-binding. To analyze the presence and/or absence of these functions and, in particular, map those to different subregions, the modular composition of the Ro52 protein was experimentally characterized. Two structured parts of Ro52 were identified, corresponding to the RING-B-box and the coiled-coil regions, respectively. Secondary structure analysis by circular dichroism (CD) spectroscopy indicated that the two subregions are independently structured. The entire RING-B-box region displayed Zn2+-dependent stabilization against proteolysis in the presence of Zn2+, indicating functional Zn2+-binding sites in both the RING and the B-box. However, no stabilization with DNA was detected, irrespective of Zn2+, thus suggesting that the RING-B-box region does not bind DNA. Oligomerization of the coiled-coil was investigated by analytical ultracentrifugation and in a mammalian two-hybrid system. Both methods show weak homodimer affinity, in parity with other coiled-coil domains involved in regulatory interactions. The C-terminal B30.2 region was rapidly degraded both during cellular expression and refolding, indicating a less stable structure. Immunologic analysis of the stable protein regions with sera from patients with Sjögren's syndrome shows that immunodominant epitopes to a large extent are localized in the structurally stable parts of Ro52. The results form a basis for further Ro52 functional studies on the proteome level. © 2005 Elsevier Ltd. All rights reserved.

• 32.
Karolinska Institutet.
Karolinska Institutet. University of Padova. Karolinska Institutet. Karolinska Institutet. Princeton University. Karolinska Institutet.
Biophysical properties of regions flanking the bHLH-Zip motif in the p22 Max protein2004In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 323, no 3, p. 750-759Article in journal (Refereed)

The Max protein is the central dimerization partner in the Myc-Max-Mad network of transcriptional regulators, and a founding structural member of the family of basic-helix-loop-helix (bHLH)-leucine zipper (Zip) proteins. Biologically important regions flanking its bHLH-Zip motif have been disordered or absent in crystal structures. The present study shows that these regions are resistant to proteolysis in both the presence and absence of DNA, and that Max dimers containing both flanking regions have significantly higher helix content as measured by circular dichroism than that predicted from the crystal structures. Nuclear magnetic resonance measurements in the absence of DNA also support the inferred structural order. Deletion of both flanking regions is required to achieve maximal DNA affinity as measured by EMSA. Thus, the previously observed functionalities of these Max regions in DNA binding, phosphorylation, and apoptosis are suggested to be linked to structural properties.

• 33.
Department of Molecular Medicine and Surgery, Center for Molecular Medicine, L8:02, Karolinska Institutet/Karolinska University Hospital, S-171 76 Stockholm, Sweden.
Linköping University, Department of Physics, Chemistry and Biology, Bioinformatics . Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology . Linköping University, The Institute of Technology. Department of Molecular Medicine and Surgery, Center for Molecular Medicine, L8:02, Karolinska Institutet/Karolinska University Hospital, S-171 76 Stockholm, Sweden. Linköping University, Department of Physics, Chemistry and Biology, Bioinformatics . Linköping University, The Institute of Technology.
Molecular Model of Human CYP21 Based onMammalian CYP2C5: Structural Features Correlatewith Clinical Severity of Mutations CausingCongenital Adrenal Hyperplasia2006In: Molecular Endocrinology, ISSN 0888-8809, E-ISSN 1944-9917, Vol. 20, no 11, p. 2946-2964Article in journal (Refereed)

Enhanced understanding of structure-function relationshipsof human 21-hydroxylase, CYP21, is requiredto better understand the molecular causesof congenital adrenal hyperplasia. To this end, astructural model of human CYP21 was calculatedbased on the crystal structure of rabbit CYP2C5.All but two known allelic variants of missense type,a total of 60 disease-causing mutations and sixnormal variants, were analyzed using this model. Astructural explanation for the corresponding phenotypewas found for all but two mutants for whichavailable clinical data are also discrepant with invitro enzyme activity. Calculations of protein stabilityof modeled mutants were found to correlateinversely with the corresponding clinical severity.Putative structurally important residues were identifiedto be involved in heme and substrate binding,redox partner interaction, and enzyme catalysisusing docking calculations and analysis of structurallydetermined homologous cytochrome P450s(CYPs). Functional and structural consequences ofseven novel mutations, V139E, C147R, R233G,T295N, L308F, R366C, and M473I, detected inScandinavian patients with suspected congenitaladrenal hyperplasia of different severity, were predictedusing molecular modeling. Structural featuresdeduced from the models are in good correlationwith clinical severity of CYP21 mutants,which shows the applicability of a modeling approachin assessment of new CYP21 mutations.

• 34.
Karolinska Institutet, Stockholm.
Karolinska Institutet, Stockholm. Karolinska Institutet, Stockholm. Karolinska Institutet, Stockholm. Karolinska Institutet, Stockholm. Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology . Harvard Medical School, Boston USA. Karolinska Institutet, Stockholm. Karolinska Institutet, Stockholm. Karolinska Institutet, Stockholm.
Ro/SSA autoantibodies directly bind cardiomyocytes, disturb calcium homeostasis, and mediate congenital heart block2005In: Journal of Experimental Medicine, ISSN 0022-1007, E-ISSN 1540-9538, Vol. 201, no 1, p. 11-17Article in journal (Refereed)
• 35.
University of Toronto.
University of Toronto. University of Toronto. University of Toronto. University of Toronto. University of Toronto. University of Toronto. Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology . University of Toronto. University of Toronto. York University. University of Toronto.
Molecular basis of Pirh2-mediated p53 ubiquitylation2008In: NATURE STRUCTURAL and MOLECULAR BIOLOGY, ISSN 1545-9985 , Vol. 15, no 12, p. 1334-1342Article in journal (Refereed)

Pirh2 (p53-induced RING-H2 domain protein; also known as Rchy1) is an E3 ubiquitin ligase involved in a negative-feedback loop with p53. Using NMR spectroscopy, we show that Pirh2 is a unique cysteine-rich protein comprising three modular domains. The protein binds nine zinc ions using a variety of zinc coordination schemes, including a RING domain and a left-handed beta-spiral in which three zinc ions align three consecutive small beta-sheets in an interleaved fashion. We show that Pirh2-p53 interaction is dependent on the C-terminal zinc binding module of Pirh2, which binds to the tetramerization domain of p53. As a result, Pirh2 preferentially ubiquitylates the tetrameric form of p53 in vitro and in vivo, suggesting that Pirh2 regulates protein turnover of the transcriptionally active form of p53.

• 36.
Karolinska Institute.
Karolinska Institute. Karolinska Institute. Karolinska Institute. Karolinska Institute. Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology . Linköping University, The Institute of Technology. University of Tubingen. Harvard University. Karolinska Institute. Karolinska Institute. Karolinska Institute. Karolinska Institute.
Maternal MHC Regulates Generation of Pathogenic Antibodies and Fetal MHC-Encoded Genes Determine Susceptibility in Congenital Heart Block2010In: JOURNAL OF IMMUNOLOGY, ISSN 0022-1767, Vol. 185, no 6, p. 3574-3582Article in journal (Refereed)

Congenital heart block develops in fetuses of anti-Ro52 Ab-positive women. A recurrence rate of 20%, despite the persistence of maternal autoantibodies, indicates that there are additional, yet unidentified, factors critical for development of congenital heart block. In this study, we demonstrate that besides the maternal MHC controlling Ab specificity, fetal MHC-encoded genes influence fetal susceptibility to congenital heart block. Using MHC congenic rat strains, we show that heart block develops in rat pups of three strains carrying MHC haplotype RT1(av1) (DA, PVG. AV1, and LEW.AV1) after maternal Ro52 immunization, but not in LEW rats (RT1(l)). Different anti-Ro52 Ab fine specificities were generated in RT1(av1) versus RT1(l) animals. Maternal and fetal influence was determined in an F-2 cross between LEW.AV1 and LEW strains, which revealed higher susceptibility in RT1(l) than RT1(av1) pups once pathogenic Ro52 Abs were present. This was further confirmed in that RT1(l) pups more frequently developed heart block than RT1(av1) pups after passive transfer of RT1(av1) anti-Ro52 sera. Our findings show that generation of pathogenic Ro52 Abs is restricted by maternal MHC, whereas the fetal MHC locus regulates susceptibility and determines the fetal disease outcome in anti-Ro52-positive pregnancies.

• 37.
Myc and its interactors take shape2015In: Biochimica et Biophysica Acta. Gene Regulatory Mechanisms, ISSN 1874-9399, E-ISSN 1876-4320, Vol. 1849, no 5, p. 469-483Article, review/survey (Refereed)

The Myc oncoprotein is a key contributor to the development of many human cancers. As such, understanding its molecular activities and biological functions has been a field of active research since its discovery more than three decades ago. Genome-wide studies have revealed Myc to be a global regulator of gene expression. The identification of its DNA-binding partner protein, Max, launched an area of extensive research into both the protein-protein interactions and protein structure of Myc. In this review, we highlight key insights with respect to Myc interactors and protein structure that contribute to the understanding of Mycs roles in transcriptional regulation and cancer. Structural analyses of Myc show many critical regions with transient structures that mediate protein interactions and biological functions. Interactors, such as Max, TRRAP, and PTEF-b, provide mechanistic insight into Mycs transcriptional activities, while others, such as ubiquitin ligases, regulate the Myc protein itself. It is appreciated that Myc possesses a large interactome, yet the functional relevance of many interactors remains unknown. Here, we discuss future research trends that embrace advances in genome-wide and proteome-wide approaches to systematically elucidate mechanisms of Myc action. This article is part of a Special Issue entitled: Myc proteins in cell biology and pathology. (C) 2014 Elsevier B.V. All rights reserved.

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