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

Abstract

Summary The nicotinamide nucleotide transhydrogenase (TH) is an integral membrane enzyme that uses the proton motive force to drive hydride transfer from NADH to NADP+ in bacteria and eukaryotes. Here we solved a 2.2 Å crystal structure of the TH transmembrane domain (Thermus thermophilus) at pH 6.5. This structure exhibits conformational changes of helix positions from a previous structure solved at pH 8.5, and reveals internal water molecules interacting with residues implicated in proton translocation. Together with molecular-dynamic simulations, we show transient water flows across a narrow pore and a hydrophobic “dry” region in the middle of the membrane channel with key residues His42α2 (chain A) being protonated and Thr214β (chain B) displaying a conformational change, respectively, to gate the channel access to both cytoplasmic and periplasmic chambers. Mutation of Thr214β to Ala deactivated the enzyme. These data provide new insights into the gating mechanism of proton translocation in TH.

Proton-Translocating Nicotinamide Nucleotide Transhydrogenase: A Structural Perspective

“…During MD simulations, a transient water wire is formed connecting His42 α2 and Glu221 β through the hydrophobic barrier, and the presence of the water wire coincides with a conformational change of Thr214 β side chain located slightly below His42 α2 . Mutation of Thr214 β to Ala causes >90% loss of channel-coupled enzymatic activity, biochemically supporting the proposed role of Thr214 β in proton translocation (Padayatti et al, 2017 ).…”

“…Structural determination of the transmembrane domain of TH has recently been achieved by the use of lipidic cubic phase (LCP) crystallization (Leung et al, 2015 ; Padayatti et al, 2017 ). The LCP is a viscous bilayer matrix that is thought to better stabilize membrane proteins than detergents and more frequently results in a tighter and layered crystal packing (Caffrey and Cherezov, 2009 ).…”

“…The pH 6.5 crystal structure solved at a resolution of 2.1 Å revealed water molecules inside the TM helix bundles, which, in combination with molecular dynamics (MD) simulations, allows a clearer interpretation of the proton translocation channel and its key functional residues (Figure 2B ) (Padayatti et al, 2017 ). A cluster of polar residues including a central histidine (His42 α2 ) form an extended H-bonded network together with two bound water molecules in the cytoplasmic half of the channel, and this connection likely provides a proton conduit to the cytoplasmic side.…”

“…Mechanistic understanding of TH has been advanced significantly by the structure determinations of isolated soluble domains and more recently of the transmembrane domain and the holo-enzyme from T. thermophilus (Leung et al, 2015 ; Padayatti et al, 2017 ). The architecture of the three domains of TH is explicitly established with the NADP(H)-binding domain III sandwiched between domains I and II and showing significant conformational mobility.…”

Deep learning model can predict water binding sites on the surface of proteins using limited-resolution data

“…With regard to proteins, not only does it provide the necessary energetic conditions required for faithful folding [2][3][4] but also mediates enzymatic activity and facilitates substrate specificity [5,6]. In addition, there are instances of specific water molecules that fulfill critical biological roles contributing to a protein's conformational stability [7], enabling proton translocation in the membrane-bound transhydrogenase [8], mediating an antigenantibody association [9], or manifestly contributing to ligand binding [10][11][12]. Recently, we demonstrated that considering the particularly strongly interacting water molecules when designing drugs allows the identification of ligands that are specific to a particular protein ortholog [13].Although the surface of most proteins is generally hydrophilic, interactions between water molecules and atoms comprising the protein are transient.…”