Cloning, expression and protein purification
The human CASKIN2 SH3 domain (aa. 284–348; Uniprot Q8WXE0) was amplified by PCR from a human cDNA and inserted into the BamHI and XhoI restriction sites of pGEX4T2 (GE Life Sciences) followed by transformation into E. coli BL21:DE3 to produce a glutathione S-transferase (GST) tagged fusion protein with intervening thrombin protease cleavage site. Two CASKIN2 SH3 domain mutants, termed 2x and 6x, contained two aromatic substitutions to reconstitute a typical hydrophobic binding cleft and six substitutions mimicking the canonical binding cleft of the STAM2 SH3 domain. These mutants were made using Quikchange method (Agilent). All constructs were verified by sequencing at the York University Core Facility. Isotopic labeling of wild type GST-CASKIN2 SH3 domain and mutants for NMR spectroscopy was achieved by performing a 1.5 L fermentation in a minimal medium containing 1 g of 15NH4Cl as the sole nitrogen source and/or 3 g of 13C-glucose as the sole carbon source. The cell pellet was suspended in T300 buffer (20 mM Tris-HCl, 300 mM NaCl, 0.05 % NaN3) and lysed by French press. Purification of the GST-CASKIN2 SH3 protein was achieved by glutathione affinity chromatography (GE Biosciences). After an 8-h digestion with activated human thrombin (Sigma-Aldrich), the SH3 domain was resolved from the GST carrier protein by gel filtration chromatography (Sephacryl-100, HiLoad 16/60; GE Life Sciences). The final buffer for all analyses was phosphate buffered saline (PBS; 20 mM sodium phosphate, pH 7.8, 0.15 M NaCl, 0.05 % (w/v) sodium azide.
Circular dichroism
Far UV spectra (190–260 nm) of CASKIN SH3 proteins at 20 μM in PBS were obtained on a Jasco J-810 spectropolarimeter and processed with Spectra Analysis 1.54.04 software. A midpoint thermal denaturation curve (Tm) was generated by monitoring the ellipticity signal at 220 nm as the temperature was ramped from 20 to 90 °C at a rate of 1 °C/min.
Peptide binding
A solution of peptide YTPMVRENKPP at > 90 % purity (Canpeptide; Montreal, QC) corresponding to the UBPY ligand in the STAM2 SH3 domain structure (PDB: 1UJ0) was titrated into 1 μM wild type CASKIN2 SH3 domain, a 6x CASKIN-SH3 domain mutant, or a reference cell containing buffer only (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.05 % sodium azide). Upon excitation at 280 nm, intrinsic fluorescence emission was measured at 340 nm corresponding to one partially buried tryptophan in the CASKIN2 SH3 domain. Each measurement was made three times and averaged.
NMR spectroscopy and structure determination
All experiments were performed on a uniformly 13C,15N labeled sample of the CASKIN2 SH3 domain sample at 0.6 mM plus 10 % D2O. A conventional heteronuclear, triple-resonance strategy was employed with all experiments being acquired on an Agilent 600 MHz spectrometer equipped with a 5 mm cryoprobe. Backbone directed experiments : HNCACB and CBCAcoNH (15Nsw = 1450 Hz, 15Npts = 28; 13Csw, = 11309 Hz, 13Cpts = 40), HNCO and HNcaCO (15Nsw = 1450 Hz, 15Npts = 28; 13Csw, = 2262 Hz, 13Cpts = 28). Side chain directed experiments: HccoNH (15Nsw = 1450 Hz, 15Npts = 28; 1Hsw, = 6596 Hz, 1Hpts = 80), hCcoNH (15Nsw = 1450 Hz, 15Npts = 28; 13Csw, = 11309 Hz, 13Cpts = 28), HCCH-TOCSY (13Csw, = 11309 Hz, 13Cpts = 28), aromatic HBcbcgCD and HBcbcgcdCE (13Csw, = 4524 Hz, 13Cpts = 32). Distance restrains were measures from peak volumes from a 15N-edited NOESY (15Nsw = 1450 Hz, 15Npts = 28; 1Hsw, = 6596 Hz, 1Hpts = 80, 100 ms mixing time) and 13C-edited aliphatic (1Hsw, = 6596 Hz, 1Hpts = 80, 13Csw, = 2800 Hz, 13Cpts = 36, 100 ms mixing time) and aromatic 3D-NOESYs aliphatic (1Hsw, = 6596 Hz, 1Hpts = 80, 13Csw, = 2800 Hz, 13Cpts = 24, 100 ms mixing time). Datasets were processed with NMRpipe [20] and interpreted with NMRView [21]. Distance restraints were obtained using CYANA. Backbone torsion angles were predicted from backbone chemical shift data with TALOS+ [22]. From an initial set of 500 structures calculated with CYANA 2.0 [23], the top 25 structures were selected with no NOE violations > 0.3 Å and no torsion angle violations < 5°. This ensemble was then subjected to additional refinement in explicit solvent [24] with XPLOR-NIH 2.30 [25] and 15 final lowest energy structures were selected for deposition to the Protein Data Bank.