TY - CHAP

T1 - Structural model of a2-subunit N-terminus and its binding interface for Arf-GEF CTH2

T2 - Implication for regulation of V-ATPase, CTH2 function and rational drug design

AU - Marshansky, Vladimir

AU - Hosokawa, Hiroyuki

AU - Merkulova, Maria

AU - Bakulina, Anastasia

AU - Dip, Phat Vinh

AU - Thaker, Youg Raj

AU - Bjargava, Ajay

AU - Tonra, James R.

AU - Ausiello, Dennis A.

AU - Grüber, Gerhard

N1 - Publisher Copyright: © 2019 Elsevier Inc.

PY - 2019/1/1

Y1 - 2019/1/1

N2 - We have previously identified the interaction between mammalian V-ATPase a2-subunit isoform and cytohesin-2 (CTH2) and studied molecular details of binding between these proteins. In particular, we found that six peptides derived from the N-terminal cytosolic domain of a2 subunit (a2N 1–402 ) are involved in interaction with CTH2 (Merkulova, Bakulina, Thaker, Grüber, & Marshansky, 2010). However, the actual 3D binding interface was not determined in that study due to the lack of high-resolution structural information about a-subunits of V-ATPase. Here, using a combination of homology modeling and NMR analysis, we generated the structural model of complete a2N 1–402 and uncovered the CTH2-binding interface. First, using the crystal-structure of the bacterial M. rubber I cyt -subunit of A-ATPase as a template (Srinivasan, Vyas, Baker, & Quiocho, 2011), we built a homology model of mammalian a2N 1–352 fragment. Next, we combined it with the determined NMR structures of peptides a2N 368–395 and a2N 386–402 of the C-terminal section of a2N 1–402 . The complete molecular model of a2N 1–402 revealed that six CTH2 interacting peptides are clustered in the distal and proximal lobe sub-domains of a2N 1–402 . Our data indicate that the proximal lobe sub-domain is the major interacting site with the Sec7 domain of first CTH2 protein, while the distal lobe sub-domain of a2N 1–402 interacts with the PH-domain of second CTH2. Indeed, using Sec7/Arf-GEF activity assay we experimentally confirmed our model. The interface formed by peptides a2N 1–17 and a2N 35–49 is involved in specific interaction with Sec7 domain and regulation of GEF activity. These data are critical for understanding of the cross-talk between V-ATPase and CTH2 as well as for the rational drug design to regulate their function.

AB - We have previously identified the interaction between mammalian V-ATPase a2-subunit isoform and cytohesin-2 (CTH2) and studied molecular details of binding between these proteins. In particular, we found that six peptides derived from the N-terminal cytosolic domain of a2 subunit (a2N 1–402 ) are involved in interaction with CTH2 (Merkulova, Bakulina, Thaker, Grüber, & Marshansky, 2010). However, the actual 3D binding interface was not determined in that study due to the lack of high-resolution structural information about a-subunits of V-ATPase. Here, using a combination of homology modeling and NMR analysis, we generated the structural model of complete a2N 1–402 and uncovered the CTH2-binding interface. First, using the crystal-structure of the bacterial M. rubber I cyt -subunit of A-ATPase as a template (Srinivasan, Vyas, Baker, & Quiocho, 2011), we built a homology model of mammalian a2N 1–352 fragment. Next, we combined it with the determined NMR structures of peptides a2N 368–395 and a2N 386–402 of the C-terminal section of a2N 1–402 . The complete molecular model of a2N 1–402 revealed that six CTH2 interacting peptides are clustered in the distal and proximal lobe sub-domains of a2N 1–402 . Our data indicate that the proximal lobe sub-domain is the major interacting site with the Sec7 domain of first CTH2 protein, while the distal lobe sub-domain of a2N 1–402 interacts with the PH-domain of second CTH2. Indeed, using Sec7/Arf-GEF activity assay we experimentally confirmed our model. The interface formed by peptides a2N 1–17 and a2N 35–49 is involved in specific interaction with Sec7 domain and regulation of GEF activity. These data are critical for understanding of the cross-talk between V-ATPase and CTH2 as well as for the rational drug design to regulate their function.

KW - Arf GEF cytohesin-2

KW - Cross-talk between V-ATPase and CTH2

KW - Homology modeling

KW - NMR analysis

KW - Rational drug design

KW - Sec7 domain enzymatic GEF activity

KW - V-ATPase a2-subunit isoform N-terminus

KW - DOMAIN

KW - PROTEIN

KW - RENIN

KW - TORSION ANGLE DYNAMICS

KW - ARNO

KW - INTERACTS

KW - H+-ATPASE

KW - (PRO)RENIN RECEPTOR

KW - SUBUNIT

UR - http://www.scopus.com/inward/record.url?scp=85063115359&partnerID=8YFLogxK

U2 - 10.1016/bs.ctm.2019.01.008

DO - 10.1016/bs.ctm.2019.01.008

M3 - Chapter

C2 - 31196611

AN - SCOPUS:85063115359

SN - 9780128177648

VL - 83

T3 - Current Topics in Membranes

SP - 77

EP - 106

BT - Membrane Transporters in the Pathogenesis of Cardiovascular and Lung Disorders

A2 - Orlov, Sergei N.

PB - Academic Press Inc.

ER -