High-resolution structure of the amino acid transporter AdiC reveals insights into the role of water molecules and networks in oligomerization and substrate binding

Abstract

Background The L-arginine/agmatine transporter AdiC is part of the arginine-dependent extreme acid resistance system of the bacterium Escherichia coli and its pathogenic varieties such as strain E. coli O157:H7. At the present time, there is a lack of knowledge concerning the role of water molecules and networks for the structure and function of AdiC, and solute transporters in general. Results The structure of the L-arginine/agmatine transporter AdiC was determined at 1.7 Å resolution by X-ray crystallography. This high resolution allowed for the identification of numerous water molecules buried in the structure. In combination with molecular dynamics (MD) simulations, we demonstrate that water molecules play an important role for stabilizing the protein and key residues, and act as placeholders for atoms of the AdiC substrates L-arginine and agmatine. MD simulations unveiled flexibility and restrained mobility of gating residues W202 and W293, respectively. Furthermore, a water-filled cavity was identified at the dimer interface of AdiC. The two monomers formed bridging interactions through water-mediated hydrogen bonds. The accessibility and presence of water molecules in this cavity was confirmed with MD simulations. Point mutations disrupting the interfacial water network validated the importance of water molecules for dimer stabilization. Conclusions This work gives new insights into the role and importance of water molecules in the L-arginine/agmatine transporter AdiC for protein stabilization and substrate-binding site shaping and as placeholders of substrate atoms. Furthermore, and based on the observed flexibility and restrained mobility of gating residues, a mechanistic role of the gate flexibility in the transport cycle was proposed. Finally, we identified a water-filled cavity at the dimeric interface that contributes to the stability of the amino acid transporter oligomer.

Tracking multiple conformations occurring on angstrom-and-millisecond scales in single amino-acid-transporter molecules

“…Fig. 6 illustrates four superimposed partial structures of AdiC and related transporters (Shaffer et al, 2009, Gao et al, 2010, Errasti-Murugarren et al, 2019, Ilgu et al, 2021), in which the orientation of helix 6 or its counterpart shown in Fig. 6A,B is represented by a vector that is color-coded for a specific structural state (Fig.…”

“…To examine conformational changes of AdiC, we chose to monitor helix 6a in one of its subunits (Fig. 1D,E) (Gao et al, 2009, Fang et al, 2009, Gao et al, 2010, Kowalczyk et al, 2011, Ilgu et al, 2016, Ilgu et al, 2021). This helix adopts differing spatial orientations among different known structural states in AdiC (Fig.…”

“…The molecule in the E x or I x state is inaccessible to ligands from either side. Thus far, the crystal structures of AdiC in the E o and E x state have been solved (Gao et al, 2009, Fang et al, 2009, Gao et al, 2010, Kowalczyk et al, 2011, Ilgu et al, 2016, Ilgu et al, 2021). Additionally, the structures of BasC and ApcT (Shaffer et al, 2009, Errasti-Murugarren et al, 2019), which have the same folds as AdiC, were solved in I o and I x states, respectively.…”

Integrative 4D-conformational mechanisms of single AdiC transporter molecules

“…1A shows a set of intensity traces recorded at a rate of 10 ms per frame from a bifunctional rhodamine attached to helix-6A of AdiC, which was inserted into nanodiscs, attached to a coverslip, and exposed to 50 μM of the ligand Agm 2+ . Judging from crystal structures, helix-6A adopts different orientations in different states (Gao et al, 2009, Fang et al, 2009, Gao et al, 2010, Kowalczyk et al, 2011, Ilgu et al, 2016, Ilgu et al, 2021). Unless specified otherwise, all references to AdiC, a structural dimer, are in the context of an independent functional subunit; Agm 2+ or Arg + are referred to as substrate and ligand in the context of their transport and binding, respectively.…”

“…1B), under which is an illustrative color-coded state profile. The conformational states are shown in the form of electron density maps (PDB: 7O82, 3L1L, 3GIA, 6F2G) (Shaffer et al, 2009, Gao et al, 2010, Errasti-Murugarren et al, 2019, Ilgu et al, 2021), colored for states as for the state profile trace. Two segments of video frames from the movie are shown in Fig.…”