Volume 25, Issue 7, Pages 1111-1119.e3 (July 2017) Critical Role of Water Molecules in Proton Translocation by the Membrane-Bound Transhydrogenase  Pius S. Padayatti, Josephine H. Leung, Paween Mahinthichaichan, Emad Tajkhorshid, Andrii Ishchenko, Vadim Cherezov, S. Michael Soltis, J. Baz Jackson, C. David Stout, Robert B. Gennis, Qinghai Zhang  Structure  Volume 25, Issue 7, Pages 1111-1119.e3 (July 2017) DOI: 10.1016/j.str.2017.05.022 Copyright © 2017 Elsevier Ltd Terms and Conditions

Structure 2017 25, 1111-1119.e3DOI: (10.1016/j.str.2017.05.022) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 1 Overall Structural Model of Holo-TH and Alignment of the Dimeric dII Structures at pH 6.5 and pH 8.5 (A) A cartoon representation of holo-TH architecture (based on PDB: 409U). The holoenzyme functions as a dimer with each protomer containing two cytosolic domains designated as dI (binds NAD(H)) and dIII (binds NADP(H)), and a transmembrane (TM) domain dII (proton channel). (B) Comparison of dimeric dII structures determined at pH 6.5 (this study, shown in color) and pH 8.5 (PDB: 4O93, in gray) shows a slight change in the TM positions. The TM helices in each protomer are slightly tilted about a vertical axis near the middle of the TM bundles (indicated by a red star). Structures shown is a periplasmic view of the dII dimer from T. thermophilus which contain two polypeptide chains α2 (three TM helices numbered from 2 to 4 shown in purple and green) and β (nine TM helices numbered from 6 to 14 in orange and cyan). Structure 2017 25, 1111-1119.e3DOI: (10.1016/j.str.2017.05.022) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 2 Cartoon Representation of Overall Structure of dII Domain at pH 6.5 and Electrostatic Potential Displayed on Solvent-Accessible Surface (A) TM helices from the α2 subunit are shown in green. TM helices from the β (truncated) subunit are shown in cyan. The positions of all solvent molecules observed crystallographically are represented as balls. Other small molecules modeled into the density around the protein are in stick representation: MAG 8.7 (pink), benzamidines (gray), and polyethylene glycol 400 (yellow). I, II, and III are views of dII domain from periplasm, side profile, and cytosol, respectively. (B) The electrostatic potential displayed on the solvent-accessible surface of membrane domain of TH. The scale is shown as kT/e, at pH 7.0 and 25°C. The potential gradient is shown with electropositive (blue) to electronegative (red) regions. The calculations are performed using the APBS tool in PyMOL. Putative channel entry points are indicated on both the periplasmic side and the cytosolic side. Structure 2017 25, 1111-1119.e3DOI: (10.1016/j.str.2017.05.022) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 3 pH 6.5 Structure of dII Showing the Nine Bound Internal Water Molecules W1 to W3 are shown as red balls. W4 to W6 are shown as purple balls. W7 to W9 are shown as orange balls. TM helices from the α2 subunit are shown in green. TM helices from the β (truncated) subunit are shown in cyan. I, II, and III are views of dII domain from periplasm, side profile and cytosol, respectively. Putative channel entry positions are indicated by red arrows in the middle structure. Structure 2017 25, 1111-1119.e3DOI: (10.1016/j.str.2017.05.022) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 4 Cytosolic Chamber of TH Proton Translocation Machinery (A) A view from the top of the dII domain from its cytosolic end. Three water molecules (W1, W2, and W3, purple balls) together with a cluster of hydrogen-bonded polar residues are shown to provide a backbone for the proton translocation. The Asp202β on the cytosolic end of TM13 is the last link from the TM domain dII, and its connection with Arg254β on a cytosolic loop (CL8) brings the communication link between dII and the cytosolic domain dIII (Leung et al., 2015). (B) The representation of the microenvironment of His42α2, and associated hydrogen-bonded residues and water molecules (W2 and W3). Right and left panels are same views with a rotation of viewpoint as indicated by the arrow. The yellow dashed lines represent the shortest distances. The two red balls represent waters W2 and W3 that are bound to Asn211β and Asn39α2, respectively. The green mesh represents the |Fo −Fc| omit map contoured at 3.5σ. Structure 2017 25, 1111-1119.e3DOI: (10.1016/j.str.2017.05.022) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 5 Proton Transport Machinery in the Periplasmic Chamber Water molecules W4, W5, and W6 are clustered at the periplasmic entrance and hydrogen bonded to Asn227β and Glu103β. Water molecules W8 and W9 are hydrogen bonded to W4 through main-chain interactions involving residues Pro228β and Ala229β. The other major polar residue in the vicinity that may participate in the proton conduction from periplasmic chamber is Glu221β, whose side chain in this structure is turned away from the channel. Structure 2017 25, 1111-1119.e3DOI: (10.1016/j.str.2017.05.022) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 6 MD Simulations of Water Flows in the dII Domain (A) Water positions determined in 300-ns MD simulations with His42α2 being fully protonated or cationic. The blue-transparent surface is a map highlighting regions within the protein that were highly occupied by water molecules and corresponds to the time-average probability of at least 20% for water insertion. A notable feature is the clear interruption of the proton channel by a “dry region” in the middle of the channel between Glu221β and Thr214β (shown within a vertical bracket), indicated by the discontinuity of the map, which became hydrated only transiently during the simulations as illustrated in Figure 7A. Blue arrow lines highlight the putative water entrance(s) from the periplasm. Thr214β is at −60° with respect to its N-Cα-Cβ-Cγ dihedral angle. (B) The time-average water content along each section of the proton channel. The solid blue line represents the “water wire,” a region where connections form between polar residues within the cytosolic chamber at a certain point during the simulation. The gray line represents His42α2 and associated hydrogen-bonded residues that form the gating region separating the periplasmic chamber and the cytoplasmic chamber. The periplasmic chamber is connected to this gated barrier by the “dry region” (indicated by the orange line) composed of hydrophobic aliphatic amino acids. (C) The average pore radii along the proton channel are calculated from the MD trajectory using the HOLE program (Smart, 1996). Structure 2017 25, 1111-1119.e3DOI: (10.1016/j.str.2017.05.022) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 7 MD Simulations Revealed Transient Formation of Water Wires between Glu221β and Thr214β (A) During the simulation, the “dry region” becomes transiently hydrated, resulting in the formation of a water wire establishing communication between the periplasmic chamber and His42α2. This is the result of a movement of the N-Cα-Cβ-Cγ dihedral angle of Thr214β from −60° to +60°. (B) Conformational dynamics of Thr214β side chain during the simulation, quantified by its N-Cα-Cβ-Cγ dihedral angle. (C) Number of water molecules localized at the “dry region” of the proton channel during the simulation. The formation of water wires occurs between 150 and 170 ns when Thr214β is at +60°. Structure 2017 25, 1111-1119.e3DOI: (10.1016/j.str.2017.05.022) Copyright © 2017 Elsevier Ltd Terms and Conditions