Domain organization and sequence similarity

The CASKIN1 and CASKIN2 SH3 domains are located at the same position within each protein and are approximately 60 % similar (Fig. 1b). Following the SH3 domain, CASKIN1 and CASKIN2 diverge in sequence, the notable differences being the lack of a CASK interaction domain (CID) in CASKIN2, as well as the length and composition of a carboxy terminal proline-rich region. Thus, depending on the perspective, CASKIN1 and CASKIN2 are simultaneously similar and different, and therefore warrant some caution when making functional assumptions about one protein in the absence of knowledge from the other.

Structure of the CASKIN2 SH3 domain

The CASKIN2 SH3 domain peptide binding cleft

A typical SH3 domain presents a binding cleft comprised of six, nearly linearly arranged, hydrophobic amino acids. As shown in Fig. 1d, the six amino acids in CASKIN2 are K290, I341, F292, P338, R319, and Y336. Notably, there is strong deviation at the first and fifth positions, denoted by K290 and R319. Since the side chains of the SH3 domain binding cleft are surface exposed, K290 and R319 do not affect the protein fold, and presumably, this is also the case for CASKIN1. Given that the binding cleft deviates at two positions from the typical set of amino acids observed in PxxP type SH3 domains like Abl kinase [14] and RxxK type SH3 domains like STAM2 [13], we hypothesized that the CASKIN2 SH3 domain might bind an unconventional peptide ligand. To answer this question, we performed four successive rounds of panning a 6xHis₋-tagged CASKIN2 SH3 domain against a commercial bacteriophage 12-mer peptide display library (PHD-12; Novagen). A comparison of eight peptides from the last round of panning revealed a Pxx[L/M/W] motif in many of the candidates [presented in Additional file 1]. However, upon testing this motif with two chemically synthesized peptides and one reversed peptide as a control, we did not observe any binding in NMR based titrations suggesting that the library may have identified a very weak interaction. Since the phage display method failed to identify a ligand for the CASKIN2 SH3 domain, it led us to consider that the CASKIN2 SH3 domain might be a non-functional remnant of the protein.

Figure 1e presents a comparison of high resolution SH3 domain structures from the Protein Data Bank with an emphasis on representatives having peptide binding clefts that deviate from the usual complement of aliphatic and aromatic amino acids, and consequently, have no ligands reported for them. The Abl SH3 domain leads a set of sequences in Fig. 1e as a point of reference and prototype for the typical array of aromatic and aliphatic amino acids that populate the six positions of the binding cleft. Next to Abl in the presentation is the RUN/TBC1 domain containing protein-3 SH3 domain (PDB: 2YUO) that swaps an aromatic/aliphatic pair at positions 5 and 6 of the cleft with a compensatory aliphatic/aromatic pair. A survey of the remaining representatives in the figure demonstrate that it is possible for each of the six binding cleft positions to tolerate a hydrophilic or charged amino acid substitution. The Crk-II SH3(2) domain represents the most extreme case with substitutions at positions 1, 2, 5 and 6. Loss of ligand binding in the Crk-II SH3(2) domain in the context of the full adaptor protein is consistent with a stabilizing function rather than a regulatory function in this domain [15].

Substitution mutagenesis of the CASKIN2 peptide binding cleft

A CASKIN2 SH3 domain double mutant (K290Y, R319W) that we termed SH3-2x, was expressed to reintroduce a full complement of aromatic amino acids in the peptide binding cleft. Using circular dichroism (CD) spectroscopy, the SH3-2x protein demonstrated a 20 °C increase in its thermal denaturation midpoint (Table 3). This observation suggests that aromatics in these two positions contribute to the stability of the SH3 fold. Given the high degree of structural similarity to the STAM2 SH3 domain in complex with a UBPY peptide [16], we introduced four more substitutions into the SH3-2x framework to produce a full mimic of the STAM2 interaction in the CASKIN SH3 domain. Overall, this new mutant termed SH3-6x, contains two substitutions to reintroduce aromatics (K290Y, R319W), two minor substitutions that maintain hydrophobicity (Y336L, I341F) and two substitutions in the RT-loop (H296E, A300E) that provide important complementary ionic contacts to the UBPY peptide ligand in STAM2. Since the SH3-2x mutant was a subset of the SH3-6x mutant, the expressed protein had a similar thermal denaturation midpoint of 70 °C (Table 3). When tested against a Class-III UBPY peptide containing an RxxKP motif, high affinity binding (0.46 ± 0.11 μM) was observed (Fig. 2) in the same order of magnitude as another Class-III SH3 domain GADS with an SLP-76 peptide ligand [17]. As expected, the wild type CASKIN2 SH3 domain had an over 100 fold less affinity for the UBPY peptide. While ultralow affinity SH3 domains with biological relevance have been reported, there is no evidence to suggest if CASKIN2 is among this group [18, 19].

SH3	Mutation(s)	Tm (°C)
WT	none	50
2x	K290Y, R319W	70
6x	K290Y, H296E, A300E, R319W, Y336L, I341F	70

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 (T_m) 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 (¹⁵N_sw = 1450 Hz, ¹⁵N_pts = 28; ¹³C_sw, = 11309 Hz, ¹³C_pts = 40), HNCO and HNcaCO (¹⁵N_sw = 1450 Hz, ¹⁵N_pts = 28; ¹³C_sw, = 2262 Hz, ¹³C_pts = 28). Side chain directed experiments: HccoNH (¹⁵N_sw = 1450 Hz, ¹⁵N_pts = 28; ¹H_sw, = 6596 Hz, ¹H_pts = 80), hCcoNH (¹⁵N_sw = 1450 Hz, ¹⁵N_pts = 28; ¹³C_sw, = 11309 Hz, ¹³C_pts = 28), HCCH-TOCSY (¹³C_sw, = 11309 Hz, ¹³C_pts = 28), aromatic HBcbcgCD and HBcbcgcdCE (¹³C_sw, = 4524 Hz, ¹³C_pts = 32). Distance restrains were measures from peak volumes from a ¹⁵N-edited NOESY (¹⁵N_sw = 1450 Hz, ¹⁵N_pts = 28; ¹H_sw, = 6596 Hz, ¹H_pts = 80, 100 ms mixing time) and ¹³C-edited aliphatic (¹H_sw, = 6596 Hz, ¹H_pts = 80, ¹³C_sw, = 2800 Hz, ¹³C_pts = 36, 100 ms mixing time) and aromatic 3D-NOESYs aliphatic (¹H_sw, = 6596 Hz, ¹H_pts = 80, ¹³C_sw, = 2800 Hz, ¹³C_pts = 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.