NMR studies on the interactions between yeast Vta1 and Did2 during the multivesicular bodies sorting pathway.

Jie Shen,Cody J Wild,Maili Liu,Chunxi Wang,Wenxian Lan,Zhaohui Xu,Bin Zhao,Xu Zhang,Jiaolong Wang,Chunyang Cao,Zhongzheng Yang

doi:10.1038/srep38710

Abstract

As an AAA-ATPase, Vps4 is important for function of multivesicular bodies (MVB) sorting pathway, which involves in cellular phenomena ranging from receptor down-regulation to viral budding to cytokinesis. The activity of Vps4 is stimulated by the interactions between Vta1 N-terminus (named as Vta1NTD) and Did2 fragment (176–204 aa) (termed as Did2176–204) or Vps60 (128–186 aa) (termed as Vps60128–186). The structural basis of how Vta1NTD binds to Did2176–204 is still unclear. To address this, in this report, the structure of Did2176–204 in complex with Vta1NTD was determined by NMR techniques, demonstrating that Did2176–204 interacts with Vta1NTD through its helix α6′ extending over the 2nd and the 3rd α-helices of Vta1NTD microtubule interacting and transport 1 (MIT1) domain. The residues within Did2176–204 helix α6′ in the interface make up of an amino acid sequence as E192′xxL195′xxR198′L199′xxL202′R203′, identical to type 1 MIT-interacting motif (MIM1) (D/E)xxLxxRLxxL(K/R) of CHMP1A180–196 observed in Vps4-CHMP1A complex structure, indicating that Did2 binds to Vta1NTD through canonical MIM1 interactions. Moreover, the Did2 binding does not result in Vta1NTD significant conformational changes, revealing that Did2, similar to Vps60, enhances Vta1 stimulation of Vps4 ATPase activity in an indirect manner.

Highlights

The biogenesis of lysosomes involves the maturation of early endosomes into MVBs
To investigate how Did[2] interacted with Vta1NTD, we first measured the binding affinity of Vta1NTD to Did2176–204 by isothermal titration calorimetry (ITC) assay (KD = 12.8 ± 1.0 μM, the number N = 1.16 ± 0.0266) (Fig. 1B), and determined the solution structure of Vta1NTD in complex with Did2176–204. This structure revealed that Did2176–204 bound to Vta1NTD through canonical type 1 microtubule-interacting and transport (MIT)-interacting motif (MIM1) interactions
Secondary structure prediction and perceived structural homology to ESCRT-III protein Vps24/CHMP3 suggest that Did2176–204 corresponds to the 6th helix within the Did[2] structure[37]

Summary

Results and Discussion

The suggested MIM region of Did2176–204 forms an α-helix conformation. Secondary structure prediction and perceived structural homology to ESCRT-III protein Vps24/CHMP3 suggest that Did2176–204 corresponds to the 6th helix within the Did[2] structure[37]. In Vta1NTD-Did2176–204 complex structure, the MIM region utilizes conserved hydrophobic residues L195′,L199′, and L202′and hydrophilic residues R198′, R203′to interact with MIT These residues within Did2176–204 helix α6′in the interface make up of an amino acid sequence as E192′xxL195′xxR198′L199′xxL202′R203′, nearly identical to CHMP1A180–196 MIM1 motif (D/E)xxLxxRLxxL(K/R)[19,20]. The NMR structures of complex Vta1NTD-Vps60128–18633–35 and Vta1NTD-Did2176–204 provided evidences that the Vps[60] or Did2-binding did not induce overall conformational changes in the N-terminus of Vta[1] These observations suggested that either Did[2] or Vps[60] did not allosterically regulate Vta1NTD and could not potentiate its ability to directly activate Vps[4]. Both Vps60128–186 and Did2176–204 stimulate Vps[4] activities by releasing VSE through interaction with Vta1NTD

Methods

Author Contributions

Additional Information