Crystallization and preliminary crystallographic characterization of an SH3 domain from the IB1 scaffold protein

Dar, Imran; Bonny, Christophe; Pedersen, Jan T.; Gajhede, Michael; Kristensen, Ole

doi:10.1107/s0907444903020304

Cited by 3 publications

(5 citation statements)

References 14 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Preparation of protein for crystallization followed standard procedures (see Supplementary data). IB1 SH3‐L was crystallized and data collected as reported previously (Dar et al , 2003). Crystals of the SeMet‐IB1 SH3‐S protein were obtained at 22°C by the hanging drop vapor diffusion method.…”

Section: Methodsmentioning

confidence: 99%

“…Binding of MKK7 was detected by autoradiography. (Dar et al, 2003), we prepared a construct optimized for crystallization (IB1 SH3-S, amino acids 494-553). 35 S-methionine-labeled IB1, truncated IB1, kinase domain of MLK3, MKK7a1 and MKK4, IB1 SH3 wt, IB1 SH3 R506A and IB1 SH3…”

Section: Dna Constructs and Mutagenesismentioning

confidence: 99%

“…All constructs used in this study were prepared by standard methods (Sambrook and Russell, 2001; see Supplementary data). In addition to a long rat IB1 SH3 variant (IB1 SH3-L, amino acids 489-559) (Dar et al, 2003), we prepared a construct optimized for crystallization (IB1 SH3-S, amino acids 494-553).…”

Section: Dna Constructs and Mutagenesismentioning

confidence: 99%

See 2 more Smart Citations

A unique set of SH3–SH3 interactions controls IB1 homodimerization

Kristensen

Guenat

Dar

et al. 2006

EMBO J

Self Cite

View full text Add to dashboard Cite

Islet-brain 1 (IB1 or JIP-1) is a scaffold protein that interacts with components of the c-Jun N-terminal kinase (JNK) signal-transduction pathway. IB1 is expressed at high levels in neurons and in pancreatic b-cells, where it controls expression of several insulin-secretory components and secretion. IB1 has been shown to homodimerize, but neither the molecular mechanisms nor the function of dimerization have yet been characterized. Here, we show that IB1 homodimerizes through a novel and unique set of Src homology 3 (SH3)-SH3 interactions. X-ray crystallography studies show that the dimer interface covers a region usually engaged in PxxP-mediated ligand recognition, even though the IB1 SH3 domain lacks this motif. The highly stable IB1 homodimer can be significantly destabilized in vitro by three individual point mutations directed against key residues involved in dimerization. Each mutation reduces IB1-dependent basal JNK activity in 293T cells. Impaired dimerization also results in a reduction in glucose transporter type 2 expression and in glucose-dependent insulin secretion in pancreatic b-cells. Taken together, these results indicate that IB1 homodimerization through its SH3 domain has pleiotropic effects including regulation of the insulin secretion process.

show abstract

Section: Methodsmentioning

confidence: 99%

Section: Dna Constructs and Mutagenesismentioning

confidence: 99%

See 1 more Smart Citation

A unique set of SH3–SH3 interactions controls IB1 homodimerization

Kristensen

Guenat

Dar

et al. 2006

EMBO J

Self Cite

View full text Add to dashboard Cite

show abstract

“…Crystallization of JIP1-SH3 and the variants Y526A, V517A, V517L, A541L and H493A JIP1-SH3 and its variants were concentrated to a final concentration of 20 mg ml −1 ( JIP1-SH3, JIP1-SH3(V517A), JIP1-SH3(A541L) and JIP1-SH3(V517L)), 10 mg ml −1 ( JIP1-SH3(Y526A)) and 4 mg ml −1 ( JIP1-SH3(H493A)) after size-exclusion chromatography by using Amicon Ultra-4 3.0-kDa centrifugal filters (Merck). All crystals were obtained in 0.1 M HEPES pH 7.5, 1-5% PEG 400 and 2-2.5 M ammonium sulfate at 20 °C by the hanging-drop vapour diffusion method in 24-well plates (Hampton research) 55 . Drops of 2-3 μl consisting of 1:1 or 2:1 parts of protein solution and reservoir solution were vapour-equilibrated against 500 μl of reservoir solution.…”

Section: Comparison Of Jip1-sh3 To Other Human Sh3 Domainsmentioning

confidence: 99%

Visualizing protein breathing motions associated with aromatic ring flipping

Pérez

Ielasi

Bessa

et al. 2022

Nature

View full text Add to dashboard Cite

Aromatic residues cluster in the core of folded proteins, where they stabilize the structure through multiple interactions. Nuclear magnetic resonance (NMR) studies in the 1970s showed that aromatic side chains can undergo ring flips—that is, 180° rotations—despite their role in maintaining the protein fold1–3. It was suggested that large-scale ‘breathing’ motions of the surrounding protein environment would be necessary to accommodate these ring flipping events1. However, the structural details of these motions have remained unclear. Here we uncover the structural rearrangements that accompany ring flipping of a buried tyrosine residue in an SH3 domain. Using NMR, we show that the tyrosine side chain flips to a low-populated, minor state and, through a proteome-wide sequence analysis, we design mutants that stabilize this state, which allows us to capture its high-resolution structure by X-ray crystallography. A void volume is generated around the tyrosine ring during the structural transition between the major and minor state, and this allows fast flipping to take place. Our results provide structural insights into the protein breathing motions that are associated with ring flipping. More generally, our study has implications for protein design and structure prediction by showing how the local protein environment influences amino acid side chain conformations and vice versa.

show abstract

“…Kristensen [19,20] reported two separated cases where deliberate hydrogen peroxide treatment of normal proteins, using 0.1% [19] or 0.3% hydrogen peroxide [20], yielded different crystal forms or improved crystal morphology. It is interesting to note that in both these two cases, the protein concentrations used for crystallization were low (2-4 mg/ml and 1.4-2.0 mg/ml, respectively).…”

Section: Resultsmentioning

confidence: 98%

Optimization of Crystals of an Inhibitory Antibody of Urokinase Plasminogen Activator Receptor (uPAR) with Hydrogen Peroxide and Low Protein Concentration

Shi¹,

Parry²,

Chen³

et al. 2005

PPL

View full text Add to dashboard Cite

Optimization of protein crystal formation is often a necessary step leading to diffraction-quality crystals to enable collection of a full X-ray data set. Typical protein crystal optimization involves screening different components, e.g., pH, precipitants, and additives of the precipitant solution. Here we present an example using an inhibitory antibody of urokinase plasminogen activator receptor (uPAR) where such procedures did not yield diffracting crystals. In contrast, it was the treatment of the protein with hydrogen peroxide incubation and the protein concentration reduction that were found to be key factors in obtaining diffracting crystals. Final crystals diffracted to 1.75 A, and belong to orthorhombic P2(1)2(1)2(1) space group with unit cell parameters a = 37.162 A, b = 84.474 A, c = 134.030 A, and contain one molecule of Fab fragment of anti-uro kinase receptor antibody in the asymmetric unit.

show abstract

Crystallization and preliminary crystallographic characterization of an SH3 domain from the IB1 scaffold protein

Cited by 3 publications

References 14 publications

A unique set of SH3–SH3 interactions controls IB1 homodimerization

A unique set of SH3–SH3 interactions controls IB1 homodimerization

Visualizing protein breathing motions associated with aromatic ring flipping

Optimization of Crystals of an Inhibitory Antibody of Urokinase Plasminogen Activator Receptor (uPAR) with Hydrogen Peroxide and Low Protein Concentration

Contact Info

Product

Resources

About