Limits...
Architecture and dynamics of the autophagic phosphatidylinositol 3-kinase complex.

Baskaran S, Carlson LA, Stjepanovic G, Young LN, Kim do J, Grob P, Stanley RE, Nogales E, Hurley JH - Elife (2014)

Bottom Line: Dynamic transitions occur in which the lipid kinase domain is ejected from the complex and VPS15 pivots at the base of the V.The N-terminus of BECN1, the target for signaling inputs, resides near the pivot point.These observations provide a framework for understanding the allosteric regulation of lipid kinase activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States.

ABSTRACT
The class III phosphatidylinositol 3-kinase complex I (PI3KC3-C1) that functions in early autophagy consists of the lipid kinase VPS34, the scaffolding protein VPS15, the tumor suppressor BECN1, and the autophagy-specific subunit ATG14. The structure of the ATG14-containing PI3KC3-C1 was determined by single-particle EM, revealing a V-shaped architecture. All of the ordered domains of VPS34, VPS15, and BECN1 were mapped by MBP tagging. The dynamics of the complex were defined using hydrogen-deuterium exchange, revealing a novel 20-residue ordered region C-terminal to the VPS34 C2 domain. VPS15 organizes the complex and serves as a bridge between VPS34 and the ATG14:BECN1 subcomplex. Dynamic transitions occur in which the lipid kinase domain is ejected from the complex and VPS15 pivots at the base of the V. The N-terminus of BECN1, the target for signaling inputs, resides near the pivot point. These observations provide a framework for understanding the allosteric regulation of lipid kinase activity.

Show MeSH

Related in: MedlinePlus

Time course of deuteration of CHIL motif peptides.Hydrogen–deuterium incorporation as a function of time (10 s to 1 hr) for selected peptides in the CHIL motif of VPS34. The deuteration level of an individual peptide was determined via HDexaminer, and it was assumed that the first residue in a peptide is non-deuterated.DOI:http://dx.doi.org/10.7554/eLife.05115.011
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4281882&req=5

fig8: Time course of deuteration of CHIL motif peptides.Hydrogen–deuterium incorporation as a function of time (10 s to 1 hr) for selected peptides in the CHIL motif of VPS34. The deuteration level of an individual peptide was determined via HDexaminer, and it was assumed that the first residue in a peptide is non-deuterated.DOI:http://dx.doi.org/10.7554/eLife.05115.011

Mentions: One significant and unexpected zone of protection was discovered within the linker regions, a ‘cold spot’ comprising residues 218–237 of VPS34 (Figure 1A, yellow box between C2 and helical domains; Figure 7A,C). This region is part of the C2-helical domain linker. It is highly conserved from yeast to humans (Figure 7D), so we christened it the C2-Helical Internal Linker (CHIL) motif. Peptides covering this region show very little increase in deuteration over the interval from 10 s to 1 hr (Figure 8). Because this 20-residue motif is too small to form a folded entity on its own, we presume that it assembles with a nearby unit, likely the VPS15 HEAT or the VPS34 helical domain.10.7554/eLife.05115.011Figure 8.Time course of deuteration of CHIL motif peptides.


Architecture and dynamics of the autophagic phosphatidylinositol 3-kinase complex.

Baskaran S, Carlson LA, Stjepanovic G, Young LN, Kim do J, Grob P, Stanley RE, Nogales E, Hurley JH - Elife (2014)

Time course of deuteration of CHIL motif peptides.Hydrogen–deuterium incorporation as a function of time (10 s to 1 hr) for selected peptides in the CHIL motif of VPS34. The deuteration level of an individual peptide was determined via HDexaminer, and it was assumed that the first residue in a peptide is non-deuterated.DOI:http://dx.doi.org/10.7554/eLife.05115.011
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4281882&req=5

fig8: Time course of deuteration of CHIL motif peptides.Hydrogen–deuterium incorporation as a function of time (10 s to 1 hr) for selected peptides in the CHIL motif of VPS34. The deuteration level of an individual peptide was determined via HDexaminer, and it was assumed that the first residue in a peptide is non-deuterated.DOI:http://dx.doi.org/10.7554/eLife.05115.011
Mentions: One significant and unexpected zone of protection was discovered within the linker regions, a ‘cold spot’ comprising residues 218–237 of VPS34 (Figure 1A, yellow box between C2 and helical domains; Figure 7A,C). This region is part of the C2-helical domain linker. It is highly conserved from yeast to humans (Figure 7D), so we christened it the C2-Helical Internal Linker (CHIL) motif. Peptides covering this region show very little increase in deuteration over the interval from 10 s to 1 hr (Figure 8). Because this 20-residue motif is too small to form a folded entity on its own, we presume that it assembles with a nearby unit, likely the VPS15 HEAT or the VPS34 helical domain.10.7554/eLife.05115.011Figure 8.Time course of deuteration of CHIL motif peptides.

Bottom Line: Dynamic transitions occur in which the lipid kinase domain is ejected from the complex and VPS15 pivots at the base of the V.The N-terminus of BECN1, the target for signaling inputs, resides near the pivot point.These observations provide a framework for understanding the allosteric regulation of lipid kinase activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States.

ABSTRACT
The class III phosphatidylinositol 3-kinase complex I (PI3KC3-C1) that functions in early autophagy consists of the lipid kinase VPS34, the scaffolding protein VPS15, the tumor suppressor BECN1, and the autophagy-specific subunit ATG14. The structure of the ATG14-containing PI3KC3-C1 was determined by single-particle EM, revealing a V-shaped architecture. All of the ordered domains of VPS34, VPS15, and BECN1 were mapped by MBP tagging. The dynamics of the complex were defined using hydrogen-deuterium exchange, revealing a novel 20-residue ordered region C-terminal to the VPS34 C2 domain. VPS15 organizes the complex and serves as a bridge between VPS34 and the ATG14:BECN1 subcomplex. Dynamic transitions occur in which the lipid kinase domain is ejected from the complex and VPS15 pivots at the base of the V. The N-terminus of BECN1, the target for signaling inputs, resides near the pivot point. These observations provide a framework for understanding the allosteric regulation of lipid kinase activity.

Show MeSH
Related in: MedlinePlus