2LX4

NMR solution structure of peptide a2N(1-17) from Mus musculus V-ATPase

Summary for 2LX4

• Entry DOI: 10.2210/pdb2lx4/pdb
• NMR Information: BMRB: 18658
• Descriptor: V-type proton ATPase 116 kDa subunit a isoform 2 (1 entity in total)
• Functional Keywords: v-atpase, subunit a, alpha helix, ph sensor, sec7 binding motif, proton transport
• Biological source: Mus musculus (mouse)
• Cellular location: Cell membrane; Multi-pass membrane protein: P15920
• Total number of polymer chains: 1
• Total formula weight: 1934.33

Authors

Dip, P.,Gruber, G.,Marshansky, V. (deposition date: 2012-08-14, release date: 2013-01-09, Last modification date: 2024-05-01)

Primary citation

Hosokawa, H.,Dip, P.V.,Merkulova, M.,Bakulina, A.,Zhuang, Z.,Khatri, A.,Jian, X.,Keating, S.M.,Bueler, S.A.,Rubinstein, J.L.,Randazzo, P.A.,Ausiello, D.A.,Gruber, G.,Marshansky, V.
The N Termini of a-Subunit Isoforms Are Involved in Signaling between Vacuolar H+-ATPase (V-ATPase) and Cytohesin-2.
J.Biol.Chem., 288:5896-5913, 2013

PubMed Abstract: Previously, we reported an acidification-dependent interaction of the endosomal vacuolar H(+)-ATPase (V-ATPase) with cytohesin-2, a GDP/GTP exchange factor (GEF), suggesting that it functions as a pH-sensing receptor. Here, we have studied the molecular mechanism of signaling between the V-ATPase, cytohesin-2, and Arf GTP-binding proteins. We found that part of the N-terminal cytosolic tail of the V-ATPase a2-subunit (a2N), corresponding to its first 17 amino acids (a2N(1-17)), potently modulates the enzymatic GDP/GTP exchange activity of cytohesin-2. Moreover, this peptide strongly inhibits GEF activity via direct interaction with the Sec7 domain of cytohesin-2. The structure of a2N(1-17) and its amino acids Phe(5), Met(10), and Gln(14) involved in interaction with Sec7 domain were determined by NMR spectroscopy analysis. In silico docking experiments revealed that part of the V-ATPase formed by its a2N(1-17) epitope competes with the switch 2 region of Arf1 and Arf6 for binding to the Sec7 domain of cytohesin-2. The amino acid sequence alignment and GEF activity studies also uncovered the conserved character of signaling between all four (a1-a4) a-subunit isoforms of mammalian V-ATPase and cytohesin-2. Moreover, the conserved character of this phenomenon was also confirmed in experiments showing binding of mammalian cytohesin-2 to the intact yeast V-ATPase holo-complex. Thus, here we have uncovered an evolutionarily conserved function of the V-ATPase as a novel cytohesin-signaling receptor.

PubMed: 23288846
DOI: 10.1074/jbc.M112.409169

Experimental method

SOLUTION NMR