Table 1.
List of peptides selected using SH3 HUNTER software.
Sequences shown in bold are the ones similar to those recognized by yeast myosin I (Myo3 and Myo5). Underlined sequences were used for binding studies using Surface Plasmon Resonance and co-crystallization experiments.
Table 2.
Details of peptides used for for SPR binding studies and co-crystallization experiments.
Selected peptides belonged to all ligand types: Class I [(K/R)xxPxxP], Class II [xPxxPx(K/R)] and PxxP. Binding affinity denotes KD (equilibrium dissociation constant).
Fig 1.
Interaction studies of EhMySH3 with peptides from EhFP10 and other proteins (Table 2) using Surface Plasmon Resonance.
(A) SPR sensorgrams depicting binding responses when 0.8 mM of each peptide was passed over immobilized EhMySH3 protein using Autolab SPR. (B) SPR kinetic analysis with each of the selected five peptides (P1, P2, P3, P4, and P5) over immobilized EhMySH3 protein as a ligand.
Fig 2.
Crystal structure of EhFP10-P2 complex.
(A) Crystal structure of the EhMySH3-P2 peptide complex, showing the asymmetric unit to be composed of two protein molecules, and one molecule of P2 near chain B. (B) 2Fo-Fc electron density map, of the P2 peptide of the EhMySH3-P2 complex at 1.5σ cut-off. (C) The structure of two asymmetric units of the crystal, showing one P2 peptide bound to two EhMySH3 molecules. (D) Surface charge representation of the P2 peptide-binding site of chain B and chain A. The c-terminal Arg residue of P2 is bound in a class II orientation in the specificity pocket of chain B.
Fig 3.
EhMySH3 interacts with c-terminal domain of EhFP10.
(A) Schematic representation of EhFP10 protein showing RhoGEF, PH, FYVE, and c-terminal domains. The green line in the c-terminal domain marks the position of the peptide P2. (B) 12% SDS PAGE showing results of an in-vitro GST-pull down assay. ‘FT’ stands for flow through after binding, ‘Wash’ is the sample after using wash buffer and ‘Elution’ is the sample collected after using elution buffer, buffer composition is mentioned in materials and methods. cterEhFP10 was seen only in the GSTSH3+cterEhFP10 elution fraction, i.e., not in that of GST+cterEhFP10 elution. (C) SPR sensorgrams depicting results after cterEhFP10 protein samples of various concentrations were passed over immobilized EhMySH3 protein. The KD was calculated to be 200 nM.
Fig 4.
Localization of EhFP10 in E. histolytica cells showing its involvement in the phagocytic process.
(A) Montage representing time series of images of GFP-EhFP10-expressing trophozoites under normal conditions. Several pseudopods leading to endocytic cup formation (marked with arrow), closing cups (marked with asterisks) and mature endosomes (marked with dots) were observed. (B) Montage representing time series of images of GFP-EhFP10-expressing trophozoites undergoing erythrophagocytosis. Enrichment of EhFP10 (bright green) in progressing pseudopods and in cups encircling RBCs (shown in red) leading to engulfment of these cells are marked with arrows. Closing phagocytic cups are marked with asterisks. Scale bar represents 10 μm.
Fig 5.
Localization of EhFP10 in E. histolytica cells showing its involvement in the macropinocytic process.
(A) A montage representing a time series (0 to 9.5 seconds) of images of GFP-EhFP10-expressing trophozoites undergoing macropinocytosis from live cell microscopy. Enrichment of EhFP10 in progressing pseudopods (marked with arrows) and engulfment of labeled media (red colour) within the vesicle (marked with asterisks) were observed. Scale bar represents 10 μm. (B) The intensity profile (ROI) of red (i.e. labelled media) and green fluorescence (i.e. GFP tagged EhFP10 protein) is shown as a function of time. Image at center depicts the snapshot of an E. histolytica trophozoite undergoing macropinocytosis. The rectangle marks the selected region that encloses a macropinocytic cup. An increase in the intensity of red colour from 9 s to 14 s was observed within the green region (i.e., amoeba cell), indicating the formation of a macropinocytic cup and presence of red fluorescence of media within the E. histolytica cell. Bottom image demonstrates overlapped red and green intensity profile in the marked region and top image displays individual profiles of each colour in the marked region.
Fig 6.
Colocalization of myosin IB with EhFP10 during phagocytosis and pinocytosis.
(A) Confocal images of immunostained fixed E. histolytica cells depicting colocalization of myosin IB with EhFP10 at different stages of phagocytosis. (B) Immunostained fixed confocal images showing myosin IB to have also colocalized with EhFP10 during pinocytosis from initiation to cup closure as well as in internalized pinosomes. Scale bar represents 5 μm. (C, D) Quantitative depictions of the colocalization of EhFP10 with (C) EhMyosin IB and with (D) actin, each at various stages of phagocytosis and pinocytosis. (E) Quantitative depiction of the colocalization of EhFP10 with myosin IB and actin in the base and tip region of closing vesicles during phagocytosis and pinocytosis. Pearson’s correlation coefficient (PCC) was used to quantify colocalization. PCC values were calculated using Olympus Fluoview FV1000 software by selecting area enclosing the phagocytic and pinocytic cups, and also the area at the base and the tip of the endocytic cups from different trophozoites (n) and the mean values were plotted. Statistical analysis was performed using the unpaired two-tailed Student’s t-test (*P < 0.05, **P<0.01, ***P< 0.001, ****P< 0.0001 was considered significant). Error bar = SEM. EhFP10-Myosin [Phagocytosis/ Pinocytosis (initiation, n = 19/ n = 7; Extension, n = 26/ n = 13; Phagosome, n = 5/ n = 13; Base, n = 9/ n = 12; Tip, n = 9/n = 12)]. EhFP10-Actin [Phagocytosis/ Pinocytosis (initiation, n = 19/ n = 6; Extension, n = 26/ n = 15; Phagosome, n = 5/ n = 19; Base, n = 9/ n = 12; Tip, n = 9/n = 12)].
Fig 7.
EhFP10 binds and bundles actin filament.
(A) 10% SDS-PAGE gel of the supernatant (S) and pellet (P) fractions obtained after co-sedimentation assay of purified filamentous rabbit muscle actin with purified cterEhFP10. Purified cterEhFP10 protein was seen to co-sediment with actin and was found in the pellet fraction. Purified Alpha-actinin (cytoskeleton Inc.) was used as positive control and bovine serum albumin (SRL) was used as a negative control in the experiment. (B) SPR sensorgrams obtained after cterEhFP10 proteins of various concentrations were passed over immobilized F-actin. The coresponding KD value was determined to be 87 nM. (C) 10% SDS-PAGE gel of the supernatant (S) and pellet (P) fractions obtained after actin bundling assay of purified filamentous rabbit muscle actin with purified cterEhFP10. cterEhFP10 was found to bundle F-actin as only F-actin stayed in supernatant while heavier bundled actin went into the pellet fraction along with cterEhFP10 upon low-speed centrifugation. Purified Alpha-actinin (cytoskeleton Inc.) was used as positive control and bovine serum albumin (SRL) was used as a negative control in the experiment. (D) TEM images showing changes in the thickness of F-actin as the cterEhFP10 concentration was changed. Scale bar represents 100 nm.
Fig 8.
SH3 domain of EhMyosin IB interacts with c-terminal domain of EhFP10 and inhibits its actin bundling activity.
(A) 15% SDS PAGE showing actin co-sedimentation assay with EhMySH3 in the presence of cterEhFP10. Actin filament was observed in the pellet fraction in all lanes. cterEhFP10 was seen in the supernatant as well as in the pellet fraction with actin at all concentrations. EhMySH3 was present in the supernatant fraction in the absence of cterEhFP10. (B) 15% SDS PAGE gel showing the presence of EhMySH3 in the supernatant fraction during actin co-sedimentation assay. This result indicated that EhMySH3 does not bind to filamentous actin. (C) 12% SDS PAGE showing competitive inhibition of cterEhFP10 mediated actin bundling activity by EhMySh3. In the presence of the EhMySH3 domain, a fraction of the actin filament remained in the supernatant, i.e., could not form bundles. As the concentration of cterEhFP10 was increased, the amount of actin in the supernatant fraction decreased. (D) A bar diagram of the percentages of actin that remained in the supernatant samples, i.e., that could not form actin bundles, in the presence and absence of the EhMySH3. Densitometry analysis was done by image J software. Statistical analysis was performed using unpaired two tailed Student’s t-test (n = 3, *Pvalue < 0.05). Error bars = SEM.
Table 3.
Data collection and refinement statistics for the EhMySH3-P2 complex.
Values given in brackets are for the higher-resolution shell.