Ksenia Rostislavleva1, Nicolas Soler1, Yohei Ohashi1, Lufei Zhang1, Els Pardon2,3, John E. Burke1, Glenn R. Masson1, Chris Johnson1, Jan Steyaert2,3, Nicholas T. Ktistakis4, and Roger L. Williams1
- Medical Research Council (MRC) Laboratory of Molecular Biology, Cambridge CB2 0QH, UK.
- Structural Biology Research Center, VIB, B-1050 Brussels, Belgium.
- Structural Biology Brussels, Vrije Universiteit Brussel, B-1050 Brussels, Belgium.
- The Babraham Institute, Cambridge, UK.
Phosphatidylinositol 3-kinase Vps34 complexes regulate intracellular membrane trafficking in endocytic sorting, cytokinesis, and autophagy. We present the 4.4 angstrom crystal structure of the 385-kilodalton endosomal complex II (PIK3C3-CII), consisting of Vps34, Vps15 (p150), Vps30/Atg6 (Beclin 1), and Vps38 (UVRAG). The subunits form a Y-shaped complex, centered on the Vps34 C2 domain. Vps34 and Vps15 intertwine in one arm, where the Vps15 kinase domain engages the Vps34 activation loop to regulate its activity. Vps30 and Vps38 form the other arm that brackets the Vps15/Vps34 heterodimer, suggesting a path for complex assembly. We used hydrogen-deuterium exchange mass spectrometry (HDX-MS) to reveal conformational changes accompanying membrane binding and identify a Vps30 loop that is critical for the ability of complex II to phosphorylate giant liposomes on which complex I is inactive.
CovalX Technology Used (Click each option to learn more)
Protein from 10 liters of yeast cell culture was used to generate nanobodies against cross-linked complex II (S. cerevisiae P13K subunits). Complex II was purified and proteins on IgG beads were incubated with TEV protease in 10 ml of wash 2 buffer (50 mM HEPES (pH 7.4), 300 mM NaCl, 1 mM TCEP, 0.5 mM PMSF) overnight at 4 °C. Following the first elution, 5 ml of wash 2 buffer without TEV protease was added and a second extraction was performed. CHAPS was added to a concentration of 0.1% to a combination of the two TEV elution fractions. The protein was then concentrated and gel filtration was performed. Peak fractions were combined with one another and then concentrated to 400 μl at 6.3 mg/ml (16 μM) and the entire amount was used for crosslinking. 10 μM of Complex II was crosslinked using the CovalX K200 Stabilization Kit and precipitates were removed by centrifugation before the material was frozen in liquid nitrogen for shipping. Llamas were then immunized using this crosslinked Complex II.