Untitled

Abstract

The nicotinamide nucleotide transhydrogenases (TH) of mitochondria and bacteria are membrane-intercalated proton pumps that transduce substrate binding energy and protonmotive force via protein conformational changes. In mitochondria, TH utilizes protonmotive force to promote direct hydride ion transfer from NADH to NADP, which are bound at the distinct extramembranous domains I and III, respectively. Domain II is the membrane-intercalated domain and contains the enzyme's proton channel. This paper describes the crystal structure of the NADP(H) binding domain III of bovine TH at 1.2 A resolution. The structure reveals that NADP is bound in a manner inverted from that previously observed for nucleotide binding folds. The non-classical binding mode exposes the NADP(H) nicotinamide ring for direct contact with NAD(H) in domain I, in accord with biochemical data. The surface of domain III surrounding the exposed nicotinamide is comprised of conserved residues presumed to form the interface with domain I during hydride ion transfer. Further, an adjacent region contains a number of acidic residues, forming a surface with negative electrostatic potential which may interact with extramembranous loops of domain II. Together, the distinctive surface features allow mechanistic considerations regarding the NADP(H)-promoted conformation changes that are involved in the interactions of domain III with domains I and II for hydride ion transfer and proton translocation.

Critical Role of Water Molecules in Proton Translocation by the Membrane-Bound Transhydrogenase

“…For example, the human mitochondrial TH has all 3 domains as parts of a single polypeptide subunit, whereas the enzyme from Thermus thermophilus has 3 different polypeptide subunits (α 1 , α 2 and β) (Figure S1) (Leung et al, 2015). X-ray structures have been determined for the isolated nucleotide-binding domains of dI and dIII from several species, both with and without bound nucleotide (Bhakta et al, 2007; Johansson et al, 2005; Prasad et al, 1999; Prasad et al, 2002; Sundaresan et al, 2005; Sundaresan et al, 2003). Domain dI has always been a dimer in these structures and the soluble part of domain dIII crystallizes as a monomer (Prasad et al, 1999; Prasad et al, 2002).…”

“…X-ray structures have been determined for the isolated nucleotide-binding domains of dI and dIII from several species, both with and without bound nucleotide (Bhakta et al, 2007; Johansson et al, 2005; Prasad et al, 1999; Prasad et al, 2002; Sundaresan et al, 2005; Sundaresan et al, 2003). Domain dI has always been a dimer in these structures and the soluble part of domain dIII crystallizes as a monomer (Prasad et al, 1999; Prasad et al, 2002). When these hydrophilic domains are combined in solution, they form a stable heterotrimer consisting of a dimer of domain dI and one copy of domain dIII (PDB ID 4J1T) (Bhakta et al, 2007).…”

Division of labor in transhydrogenase by alternating proton translocation and hydride transfer

“…Structures of hydrophilic dI and dIII were previously solved for TH isolated from bovine (8) and human (9) mitochondria as well as from Rhodospirillum rubrum (10) and Escherichia coli (11). Co-crystallization of dI with dIII results in an asymmetric heterotrimer (dI) 2 :dIII (12, 13).…”

Proton-Translocating Nicotinamide Nucleotide Transhydrogenase: A Structural Perspective

“…Individual crystal structures of the two soluble domains of TH showed overall similarity across multiple species including human, bovine, E. coli , and R. rubrum (Prasad et al, 1999 , 2002 ; White et al, 2000 ; Sundaresan et al, 2003 ; Johansson et al, 2005 ). Domain I is present as a dimer in solution and always crystallized as such, with the dimeric interface stabilized by a swapping beta-hairpin structure and two helices.…”

“…In E. coli TH holoenzyme, the dissociation constant (Kd) for NAD + is 100–500 μM and that for NADH is 50 μM, comparable to values for the isolated domain I (Bizouarn et al, 2005 ). Domain III has hitherto only been crystallized in the presence of NADP(H), with loop D either in open or closed conformation (Prasad et al, 1999 ; White et al, 2000 ; Sundaresan et al, 2003 ). Notably, NADP(H) has extremely high binding affinity with the isolated domain III (apparent Kd value < nM) (Diggle et al, 1995 ; Fjellstrom et al, 1997 ; Peake et al, 1999 ), which is partly attributed to adjacent loop E folding over NADP(H) like a lid.…”