Limits...
Germ plasm localisation of the HELICc of Vasa in Drosophila: analysis of domain sufficiency and amino acids critical for localisation.

Wang SC, Hsu HJ, Lin GW, Wang TF, Chang CC, Lin MD - Sci Rep (2015)

Bottom Line: We found that HELICc itself, through the interaction with Oskar (Osk), was sufficient for germ-plasm localisation.We further identified that glutamine (Gln) 527 within HELICc of DmVas was critical for localisation, and its corresponding residue could also be detected in grasshopper Vas yet missing in the other three species.This suggests that Gln527 is a direct target of Osk or critical to the maintenance of HELICc conformation.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Human Genetics, Tzu-Chi University, Hualien, Taiwan.

ABSTRACT
Formation of the germ plasm drives germline specification in Drosophila and some other insects such as aphids. Identification of the DEAD-box protein Vasa (Vas) as a conserved germline marker in flies and aphids suggests that they share common components for assembling the germ plasm. However, to which extent the assembly order is conserved and the correlation between functions and sequences of Vas remain unclear. Ectopic expression of the pea aphid Vas (ApVas1) in Drosophila did not drive its localisation to the germ plasm, but ApVas1 with a replaced C-terminal domain (HELICc) of Drosophila Vas (DmVas) became germ-plasm restricted. We found that HELICc itself, through the interaction with Oskar (Osk), was sufficient for germ-plasm localisation. Similarly, HELICc of the grasshopper Vas could be recruited to the germ plasm in Drosophila. Nonetheless, germ-plasm localisation was not seen in the Drosophila oocytes expressing HELICcs of Vas orthologues from aphids, crickets, and mice. We further identified that glutamine (Gln) 527 within HELICc of DmVas was critical for localisation, and its corresponding residue could also be detected in grasshopper Vas yet missing in the other three species. This suggests that Gln527 is a direct target of Osk or critical to the maintenance of HELICc conformation.

No MeSH data available.


Related in: MedlinePlus

Structures and predicted protein interaction sites in DmVas460–621, DmVas460–621/Q527A, and DmVas470–621.(A–C) Molecular dynamics (MD) simulated structures of DmVas460–621, DmVas460–621/Q527A, and DmVas470–621 after 100-ns simulations (Supplementary Methods). Protein–protein interaction sites were predicted using the Site Finder of the Molecular Operating Environment (MOE) software package, and dummy atoms were placed within the MD-simulated structures. Red and grey dummy atoms represent potential hydrophilic and hydrophobic interactions, respectively. Residues located in the individual interaction sites are listed in the supplementary material Table S2. Red: exposed surface; pink: hydrophilic region; green: hydrophobic region; blue: the back bone of residues 460–469. Panels (A’–C’) are horizontal rotations of (A–C), respectively. (A,A’) DmVas460–621: Gln527 is located within Site 1; 6 residues (Ser463, Asp464, Val465, Lys466, Gln467, and Thr468) from amino acids 460–469 are located within Site 2. (B,B’) DmVas460–621/Q527A: a conformational change excluded the residues around Q527A from Site 1 and residues 460–469 from Site 2. (C,C’) DmVas470–621: Site 1 was greatly reduced in size; Site 2 vanished. (D) Crystal structure of DmVas bound with the poly(U) RNA (PDB code: 2DB3; residues 200-621). Residues Gln525 and Arg528 interacted with the RNA, whereas Gln527 did not. Yellow: DEXDc; white: HELICc; pink: poly(U) RNA; blue: residues 460–469; green: QxxR RNA-binding motif. (E) A presumptive model for the germ plasm localisation of DmVas. After the Oskar (Osk) protein accumulated in the posterior pole of the oocyte at Stage 9 of oogenesis, it interacted with DmVas through residues Gln527 (Q527) and 460–469 in HELICc. The OIM (residues 163–319) might promote or stabilise the interaction between HELICc and Osk. Moreover, we proposed that some germ plasm components (I) could pre-associate with the DmVas during its transportation from nurse cells to the oocyte. In the germ plasm, DmVas could localise additional germ plasm components (II) with the aid of Osk and/or other Osk-bound molecules. Pole cell formation follows germ plasm assembly in the early embryogenesis.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4588571&req=5

f8: Structures and predicted protein interaction sites in DmVas460–621, DmVas460–621/Q527A, and DmVas470–621.(A–C) Molecular dynamics (MD) simulated structures of DmVas460–621, DmVas460–621/Q527A, and DmVas470–621 after 100-ns simulations (Supplementary Methods). Protein–protein interaction sites were predicted using the Site Finder of the Molecular Operating Environment (MOE) software package, and dummy atoms were placed within the MD-simulated structures. Red and grey dummy atoms represent potential hydrophilic and hydrophobic interactions, respectively. Residues located in the individual interaction sites are listed in the supplementary material Table S2. Red: exposed surface; pink: hydrophilic region; green: hydrophobic region; blue: the back bone of residues 460–469. Panels (A’–C’) are horizontal rotations of (A–C), respectively. (A,A’) DmVas460–621: Gln527 is located within Site 1; 6 residues (Ser463, Asp464, Val465, Lys466, Gln467, and Thr468) from amino acids 460–469 are located within Site 2. (B,B’) DmVas460–621/Q527A: a conformational change excluded the residues around Q527A from Site 1 and residues 460–469 from Site 2. (C,C’) DmVas470–621: Site 1 was greatly reduced in size; Site 2 vanished. (D) Crystal structure of DmVas bound with the poly(U) RNA (PDB code: 2DB3; residues 200-621). Residues Gln525 and Arg528 interacted with the RNA, whereas Gln527 did not. Yellow: DEXDc; white: HELICc; pink: poly(U) RNA; blue: residues 460–469; green: QxxR RNA-binding motif. (E) A presumptive model for the germ plasm localisation of DmVas. After the Oskar (Osk) protein accumulated in the posterior pole of the oocyte at Stage 9 of oogenesis, it interacted with DmVas through residues Gln527 (Q527) and 460–469 in HELICc. The OIM (residues 163–319) might promote or stabilise the interaction between HELICc and Osk. Moreover, we proposed that some germ plasm components (I) could pre-associate with the DmVas during its transportation from nurse cells to the oocyte. In the germ plasm, DmVas could localise additional germ plasm components (II) with the aid of Osk and/or other Osk-bound molecules. Pole cell formation follows germ plasm assembly in the early embryogenesis.

Mentions: To understand how Gln527 participated in the Osk–Vas interaction in Drosophila, we performed MD simulations and protein-protein interaction site prediction to analyse the possible conformational change in HELICc caused by the Q527A substitution. In the MD simulated structure of HELICc, two protein–protein interaction pockets containing the residues 460–469 and Gln527 were predicted (Fig. 8A,A’). Gln527 was identified within a predicted interaction pocket entitled as ‘site 1’ whereas residues 463–468 were included in ‘site 2’ (DmVas460–621 in Fig. 8A; Supplementary Table S2). The Q527A substitution excludes Arg523, Lys524 and Arg528, all of which are amino acids surrounding Gln527, from site 1. Meanwhile it expels residues 463–470 from site 2 (Fig. 8B; Supplementary Table S2). Consequently, conformational distortion of sites 1 and 2 in HELICc may explain why DmVas460–661/Q527A cannot be localised to the germ plasm. According to the published crystal structure of DmVas, the residue Gln527 is located within the RNA-binding motif QxxR22. However, because the side chain of Gln527 flips outside the RNA-binding pocket, we believe that it does not directly interact with the target RNA (Fig. 8D). Further evidence is required for understanding whether the protruding side chain of Gln527 is a direct target of Osk. We also simulated the structure of DmVas470–621 and found that truncation of the HELICc N-terminal sequence, namely the residues 460–469 of DmVas, led to distortion and shrinkage of site 1 and a deletion of site 2 (Fig. 8C,C’). The fact that DmVas470–661 could not be detected in the posterior germ plasm (Fig. 3D–D”’) suggests that the residues 460–469, similar to Gln527, contribute to the Osk–Vas interaction, and that the predicted sites 1 and 2 could play a role. Liang et al. (1994) identified four EMS-induced mutations in HELICc that could disrupt germ plasm localisation of Vas23. Likewise, a recent study carried out by Dehghani and Lasko (2015) shows that substitution of Thr546 with Ala (T546A) in HELICc results in the same outcome45. MD simulation shows that they are respectively located within site 1 (Ser518, His520, Thr546) and site 2 (Val465, Gly587). MD simulation predicts that: (1) these 5 amino acids are respectively located within site 1 (Ser518, His520, Thr546) and site 2 (Val465, Gly587); (2) the Q527A substitution expels Thr546 from site 1 and Val465 from site 2; and (3) the deletion of residues 460–469 excludes His520/Thr546 from site 1 (Supplementary Table S2). We therefore infer that Gln527 and residues 460–469 stabilize the conformation of HELICc.


Germ plasm localisation of the HELICc of Vasa in Drosophila: analysis of domain sufficiency and amino acids critical for localisation.

Wang SC, Hsu HJ, Lin GW, Wang TF, Chang CC, Lin MD - Sci Rep (2015)

Structures and predicted protein interaction sites in DmVas460–621, DmVas460–621/Q527A, and DmVas470–621.(A–C) Molecular dynamics (MD) simulated structures of DmVas460–621, DmVas460–621/Q527A, and DmVas470–621 after 100-ns simulations (Supplementary Methods). Protein–protein interaction sites were predicted using the Site Finder of the Molecular Operating Environment (MOE) software package, and dummy atoms were placed within the MD-simulated structures. Red and grey dummy atoms represent potential hydrophilic and hydrophobic interactions, respectively. Residues located in the individual interaction sites are listed in the supplementary material Table S2. Red: exposed surface; pink: hydrophilic region; green: hydrophobic region; blue: the back bone of residues 460–469. Panels (A’–C’) are horizontal rotations of (A–C), respectively. (A,A’) DmVas460–621: Gln527 is located within Site 1; 6 residues (Ser463, Asp464, Val465, Lys466, Gln467, and Thr468) from amino acids 460–469 are located within Site 2. (B,B’) DmVas460–621/Q527A: a conformational change excluded the residues around Q527A from Site 1 and residues 460–469 from Site 2. (C,C’) DmVas470–621: Site 1 was greatly reduced in size; Site 2 vanished. (D) Crystal structure of DmVas bound with the poly(U) RNA (PDB code: 2DB3; residues 200-621). Residues Gln525 and Arg528 interacted with the RNA, whereas Gln527 did not. Yellow: DEXDc; white: HELICc; pink: poly(U) RNA; blue: residues 460–469; green: QxxR RNA-binding motif. (E) A presumptive model for the germ plasm localisation of DmVas. After the Oskar (Osk) protein accumulated in the posterior pole of the oocyte at Stage 9 of oogenesis, it interacted with DmVas through residues Gln527 (Q527) and 460–469 in HELICc. The OIM (residues 163–319) might promote or stabilise the interaction between HELICc and Osk. Moreover, we proposed that some germ plasm components (I) could pre-associate with the DmVas during its transportation from nurse cells to the oocyte. In the germ plasm, DmVas could localise additional germ plasm components (II) with the aid of Osk and/or other Osk-bound molecules. Pole cell formation follows germ plasm assembly in the early embryogenesis.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4588571&req=5

f8: Structures and predicted protein interaction sites in DmVas460–621, DmVas460–621/Q527A, and DmVas470–621.(A–C) Molecular dynamics (MD) simulated structures of DmVas460–621, DmVas460–621/Q527A, and DmVas470–621 after 100-ns simulations (Supplementary Methods). Protein–protein interaction sites were predicted using the Site Finder of the Molecular Operating Environment (MOE) software package, and dummy atoms were placed within the MD-simulated structures. Red and grey dummy atoms represent potential hydrophilic and hydrophobic interactions, respectively. Residues located in the individual interaction sites are listed in the supplementary material Table S2. Red: exposed surface; pink: hydrophilic region; green: hydrophobic region; blue: the back bone of residues 460–469. Panels (A’–C’) are horizontal rotations of (A–C), respectively. (A,A’) DmVas460–621: Gln527 is located within Site 1; 6 residues (Ser463, Asp464, Val465, Lys466, Gln467, and Thr468) from amino acids 460–469 are located within Site 2. (B,B’) DmVas460–621/Q527A: a conformational change excluded the residues around Q527A from Site 1 and residues 460–469 from Site 2. (C,C’) DmVas470–621: Site 1 was greatly reduced in size; Site 2 vanished. (D) Crystal structure of DmVas bound with the poly(U) RNA (PDB code: 2DB3; residues 200-621). Residues Gln525 and Arg528 interacted with the RNA, whereas Gln527 did not. Yellow: DEXDc; white: HELICc; pink: poly(U) RNA; blue: residues 460–469; green: QxxR RNA-binding motif. (E) A presumptive model for the germ plasm localisation of DmVas. After the Oskar (Osk) protein accumulated in the posterior pole of the oocyte at Stage 9 of oogenesis, it interacted with DmVas through residues Gln527 (Q527) and 460–469 in HELICc. The OIM (residues 163–319) might promote or stabilise the interaction between HELICc and Osk. Moreover, we proposed that some germ plasm components (I) could pre-associate with the DmVas during its transportation from nurse cells to the oocyte. In the germ plasm, DmVas could localise additional germ plasm components (II) with the aid of Osk and/or other Osk-bound molecules. Pole cell formation follows germ plasm assembly in the early embryogenesis.
Mentions: To understand how Gln527 participated in the Osk–Vas interaction in Drosophila, we performed MD simulations and protein-protein interaction site prediction to analyse the possible conformational change in HELICc caused by the Q527A substitution. In the MD simulated structure of HELICc, two protein–protein interaction pockets containing the residues 460–469 and Gln527 were predicted (Fig. 8A,A’). Gln527 was identified within a predicted interaction pocket entitled as ‘site 1’ whereas residues 463–468 were included in ‘site 2’ (DmVas460–621 in Fig. 8A; Supplementary Table S2). The Q527A substitution excludes Arg523, Lys524 and Arg528, all of which are amino acids surrounding Gln527, from site 1. Meanwhile it expels residues 463–470 from site 2 (Fig. 8B; Supplementary Table S2). Consequently, conformational distortion of sites 1 and 2 in HELICc may explain why DmVas460–661/Q527A cannot be localised to the germ plasm. According to the published crystal structure of DmVas, the residue Gln527 is located within the RNA-binding motif QxxR22. However, because the side chain of Gln527 flips outside the RNA-binding pocket, we believe that it does not directly interact with the target RNA (Fig. 8D). Further evidence is required for understanding whether the protruding side chain of Gln527 is a direct target of Osk. We also simulated the structure of DmVas470–621 and found that truncation of the HELICc N-terminal sequence, namely the residues 460–469 of DmVas, led to distortion and shrinkage of site 1 and a deletion of site 2 (Fig. 8C,C’). The fact that DmVas470–661 could not be detected in the posterior germ plasm (Fig. 3D–D”’) suggests that the residues 460–469, similar to Gln527, contribute to the Osk–Vas interaction, and that the predicted sites 1 and 2 could play a role. Liang et al. (1994) identified four EMS-induced mutations in HELICc that could disrupt germ plasm localisation of Vas23. Likewise, a recent study carried out by Dehghani and Lasko (2015) shows that substitution of Thr546 with Ala (T546A) in HELICc results in the same outcome45. MD simulation shows that they are respectively located within site 1 (Ser518, His520, Thr546) and site 2 (Val465, Gly587). MD simulation predicts that: (1) these 5 amino acids are respectively located within site 1 (Ser518, His520, Thr546) and site 2 (Val465, Gly587); (2) the Q527A substitution expels Thr546 from site 1 and Val465 from site 2; and (3) the deletion of residues 460–469 excludes His520/Thr546 from site 1 (Supplementary Table S2). We therefore infer that Gln527 and residues 460–469 stabilize the conformation of HELICc.

Bottom Line: We found that HELICc itself, through the interaction with Oskar (Osk), was sufficient for germ-plasm localisation.We further identified that glutamine (Gln) 527 within HELICc of DmVas was critical for localisation, and its corresponding residue could also be detected in grasshopper Vas yet missing in the other three species.This suggests that Gln527 is a direct target of Osk or critical to the maintenance of HELICc conformation.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Human Genetics, Tzu-Chi University, Hualien, Taiwan.

ABSTRACT
Formation of the germ plasm drives germline specification in Drosophila and some other insects such as aphids. Identification of the DEAD-box protein Vasa (Vas) as a conserved germline marker in flies and aphids suggests that they share common components for assembling the germ plasm. However, to which extent the assembly order is conserved and the correlation between functions and sequences of Vas remain unclear. Ectopic expression of the pea aphid Vas (ApVas1) in Drosophila did not drive its localisation to the germ plasm, but ApVas1 with a replaced C-terminal domain (HELICc) of Drosophila Vas (DmVas) became germ-plasm restricted. We found that HELICc itself, through the interaction with Oskar (Osk), was sufficient for germ-plasm localisation. Similarly, HELICc of the grasshopper Vas could be recruited to the germ plasm in Drosophila. Nonetheless, germ-plasm localisation was not seen in the Drosophila oocytes expressing HELICcs of Vas orthologues from aphids, crickets, and mice. We further identified that glutamine (Gln) 527 within HELICc of DmVas was critical for localisation, and its corresponding residue could also be detected in grasshopper Vas yet missing in the other three species. This suggests that Gln527 is a direct target of Osk or critical to the maintenance of HELICc conformation.

No MeSH data available.


Related in: MedlinePlus