Journal: Communications Biology

Article Title: Competitive binding of actin and SH3 domains at proline-rich regions of Las17/WASP regulates actin polymerisation

doi: 10.1038/s42003-025-08188-4

Figure Lengend Snippet: A Schematic diagram outlining one possible arrangement of Sla1 SH3 domains bound to the N-terminal PPR of Las17 (amino acids 300-422). SH3 domains of Sla1 are numbered #1, #2 and #3. The PPR-N of Las17 is the region shown to bind SH3 domains. PP in pink boxes indicate poly-proline tracts in this region. Other regions of Las17 are included but not to scale. B Representative pyrene actin assays show inhibition of Las17-Arp2/3 mediated actin polymerisation by Sla1-SH3 domains when added as separate domains (orange #1, green #2 or cyan #3) or on a single peptide (red). Actin; 3 µM, Las17; 300 nM, and Sla1 SH3 domains; 300 nM each. C Biolayer Interferometry measurements of the affinity of Sla1 SH3 domains for Las17 (aa 300-422). Sla1 SH3#1 (aa 3-68), SH3#3 (aa 354-413), SH3#1-2 (aa 5-131), SH3#1-3 (aa 5-413).

Article Snippet: Membrane dot blots carrying overlapping 12-mer peptides of Las17 over the region 300-536 were purchased from Intavis Celluspots.

Techniques: Inhibition

Journal: Communications Biology

Article Title: Competitive binding of actin and SH3 domains at proline-rich regions of Las17/WASP regulates actin polymerisation

doi: 10.1038/s42003-025-08188-4

Figure Lengend Snippet: A Schematic diagram of Las17. The PPR from residues 300-535 is subdivided into PPR-N (300-422) and PPR-C (423-535). The amino acid sequence is shown for PPR-N with three polyproline (PP) motifs indicated. Lines above the sequence are SH3 domain binding consensus sequences with green indicating type-I SH3 domain binding motifs, and blue type-II SH3 domain binding motifs. Underscored arginine residues in red text motifs have previously been identified as important in G-actin binding. Pink text indicates a predicted new ABS region. B A region of the HSQC spectrum from the titration of 15 N-labelled Sla1 SH3 domains 1 and 2 with peptide 3 is shown. For clarity only the titrations with 0, 1, 2, 3, 5 and 8 peptide equivalents are shown (red, orange, yellow, green, blue, purple respectively). C Chemical shift changes in protein signals were fitted to standard equations (Williamson 2013) to estimate binding affinity. The estimated affinities clearly fell into two groups: signals from SH3#1 (top panel) fitted to a common affinity of 24 μM, while signals from SH3#2 (bottom panel) fitted to a common affinity of 190 μM. The signals shown are (top) 15 N shifts for I38 (blue), W42 (red) and W41 (purple) (bottom) 1 H shifts for W108 (blue), G124 (red) and N85 (purple). D Chemical shift changes on addition of each peptide (PP1, PP2, PP3) are shown as the weighted chemical shift changes for 1 H and 15 N, [Δδ H 2 + (0.14Δδ N 2 )] 1/2 . The three peptides bind at similar locations, although peptide 1 binds approximately three times more weakly. The approximate affinities for PP1, PP2 and PP3 obtained from these data are respectively 70, 22 and 24 μM for SH3#1 and 550, 160 and 190 μM for SH3#2. E The binding site on the protein for peptide 3. Chemical shift changes on addition of peptide 3 were used to calculate the mean and standard deviation weighted shift change for each protein residue. Residues with shift changes larger than (mean + sd) are indicated in orange for domain 1 and red for domain 2, and comprise V11, Y12, Y14, S36, I38, D39, W41 and W42 (domain 1), and D105, A106 and W108 (domain 2). The figure shows two views rotated by 180°.

Article Snippet: Membrane dot blots carrying overlapping 12-mer peptides of Las17 over the region 300-536 were purchased from Intavis Celluspots.

Techniques: Sequencing, Binding Assay, Titration, Standard Deviation, Residue

Journal: Communications Biology

Article Title: Competitive binding of actin and SH3 domains at proline-rich regions of Las17/WASP regulates actin polymerisation

doi: 10.1038/s42003-025-08188-4

Figure Lengend Snippet: A A representative pyrene assay comparing the impact of Las17 fragment (300–633) with the minimal fragment (342–392) on actin polymerisation (in the absence of Arp2/3). 300 nM each fragment; 3 µM actin. B MST was used to measure binding of different concentrations of Las17 (wild type or with mutations in the actin binding sites (ABS)) to labelled actin. Error bars are standard error of mean. Green trace shows the impacts of all three sites mutagenized; dark blue is with no sites mutagenized.

Article Snippet: Membrane dot blots carrying overlapping 12-mer peptides of Las17 over the region 300-536 were purchased from Intavis Celluspots.

Techniques: Binding Assay

Journal: Communications Biology

Article Title: Competitive binding of actin and SH3 domains at proline-rich regions of Las17/WASP regulates actin polymerisation

doi: 10.1038/s42003-025-08188-4

Figure Lengend Snippet: All residues are shown as a cartoon ribbon except for arginines that flank the polyproline sequence of the ABS sites, and key interacting actin residues. Yeast G-actin is shown in green (PDB: 1YAG). A Structures for four of the top ten HPEPDOCK predictions of ABS3 docking show interactions in the barbed end groove of the actin monomer. Peptides are shown in different colours. B All four of these structures coordinate actin residue E334 via the double arginine pair (R6 and R7 in the docked peptide). C A structure illustrating one of the top ten structure predictions for ABS1 shows how arginines at each end of the peptide of Las17 may interact simultaneously with acidic residues flanking the barbed end groove of actin (E334, and E361/364). D All three docked peptide structures can be modelled to illustrate binding of three actin monomers along the Las17 peptide. The ABS sequences are shown in red whilst the adjoining sequences that were part of the modelling in pink. The dashed pink line indicates parts of Las17 with no structural information.

Article Snippet: Membrane dot blots carrying overlapping 12-mer peptides of Las17 over the region 300-536 were purchased from Intavis Celluspots.

Techniques: Sequencing, Residue, Binding Assay

Journal: Communications Biology

Article Title: Competitive binding of actin and SH3 domains at proline-rich regions of Las17/WASP regulates actin polymerisation

doi: 10.1038/s42003-025-08188-4

Figure Lengend Snippet: A Microscale thermophoresis showing binding of Las17 to actin in the presence (red) or absence (blue) of Sla1 SH3 domains #1–3. The concentration of actin (50 nM) and Sla1 (4.5 µM) were kept constant throughout the experiment, and the concentration of Las17 300–422 was varied between 0.29 and 9.5 µM. Error bars are standard deviation. B Liposome co-sedimentation assay shows that GST-Sla1 SH3#1-3 co-precipitates with liposomes prepared from bovine brain extract, whereas GST alone does not. C Quantification of liposome co-sedimentation assays. Šídák’s multiple comparisons test P < 0.0001 ( n = 6). D Representative pyrene actin assay showing alleviation of Sla1 inhibition (red) by Sec4 (pink). Actin only (black); Actin + Las17 (blue). E Alphafold prediction of an interaction of Sec4 with a region of Las17 primarily between 327 and 333 which lies at the C-terminal end of the first Las17 PP motif. Shown is surface representation of Sec4 in green and ribbon depiction of Las17 in blue with predicted interacting surface residues within 3.5 Å in red.

Article Snippet: Membrane dot blots carrying overlapping 12-mer peptides of Las17 over the region 300-536 were purchased from Intavis Celluspots.

Techniques: Microscale Thermophoresis, Binding Assay, Concentration Assay, Standard Deviation, Sedimentation, Liposomes, Pyrene Actin Assay, Inhibition

Journal: Communications Biology

Article Title: Competitive binding of actin and SH3 domains at proline-rich regions of Las17/WASP regulates actin polymerisation

doi: 10.1038/s42003-025-08188-4

Figure Lengend Snippet: A Purified individual GST-tagged Sla1 SH3 domains were used to probe a Celluspot array consisting of a series of 12 amino acid peptides starting with residue 181, and subsequent peptides starting at two amino acid intervals, up to amino acid 540. Binding of GST-Sla1 SH3 to the array was identified by further probing of HRP-tagged anti-GST and subsequent visualisation using chemiluminescence. Peptide spots corresponding to actin binding sites on Las17 (pink), additional spots including P387A (red) and P388A (green) and their corresponding wild type peptide (blue) are also shown. Binding to the peptide containing P387A is strongly reduced or abrogated for each Sla1 SH3 domain. B Microscale Thermophoresis of Las17 300–422 wild type (blue), P387A (red) and P388A (green) showing that neither of these mutations affects Las17 binding to actin. Error bars are standard error of mean.

Article Snippet: Membrane dot blots carrying overlapping 12-mer peptides of Las17 over the region 300-536 were purchased from Intavis Celluspots.

Techniques: Purification, Residue, Binding Assay, Microscale Thermophoresis

Journal: Communications Biology

Article Title: Competitive binding of actin and SH3 domains at proline-rich regions of Las17/WASP regulates actin polymerisation

doi: 10.1038/s42003-025-08188-4

Figure Lengend Snippet: A – C Lifetime of patches from wild type (red) or las17 P387A (blue) expressing cells tagged with fluorescent markers, alongside representative kymographs. A Las17-GFP, ( B ) GFP-Abp1, ( C ) Arc-15 mCherry (describing Arp2/3 complex lifetime). Statistical test Unpaired Mann Whitney test, **** indicates p value < 0.0001. A size bar for each set of kymographs is shown on the upper right of each set. For ( A ) and ( C ) size bar is 100 nm and for B is 200 nm. Error bars are standard deviation. D Time lapse images showing co-localisation of Las17-GFP (green dots) and Arc15-mCherry (red dots) in wild type cells and las17 P387A cells. The initial las17 P387A images contain two adjacent endocytic sites. The site of interest is labelled in the first two time panels with a white arrow. Exposure 0.5 sec with 1 second time lapse. 120 s recorded. E Quantification of lifetime of Las17-GFP alone (green), Arc15-mCherry alone (red) and overlap between Las17-GFP and Arc15-mCherry (yellow) from multiple endocytic patches is shown ( n = 13). Error bars are standard deviation.

Article Snippet: Membrane dot blots carrying overlapping 12-mer peptides of Las17 over the region 300-536 were purchased from Intavis Celluspots.

Techniques: Expressing, MANN-WHITNEY, Standard Deviation