Exploring the Ligand Binding and Conformational Dynamics of the Substrate-Binding Domain 1 of the ABC Transporter GlnPQ

The adenosine triphosphate (ATP)-binding cassette (ABC) importer GlnPQ from Lactococcus lactis has two sequential covalently linked substrate-binding domains (SBDs), which capture the substrates and deliver them to the translocon. The two SBDs differ in their ligand specificities, binding affinities and the distance to the transmembrane domain; interestingly, both SBDs can bind their ligands simultaneously without affecting each other. In this work, we studied the binding of ligands to both SBDs using X-ray crystallography and molecular dynamics simulations. We report three high-resolution structures of SBD1, namely, the wild-type SBD1 with bound asparagine or arginine, and E184D SBD1 with glutamine bound. Molecular dynamics (MD) simulations provide a detailed insight into the dynamics associated with open-closed transitions of the SBDs.


SUPPLEMENTARY NOTE 1:
Table S1.Binding characteristics of SBDs determined by ITC.

Figure S1 .
Figure S1.Snapshots of the conformations of the SBDs domains of the tandem relative to each other in the closure of one of the domains (left, middle) and the opening of both domains (right) obtained after CG MD simulations.

FigureFigure S3 .
Figure S2.(A) Binding sites of SBDs after CG MD simulations.Binding residues labelled with numbers have interactions with the substrate (in stick representation).The ligands are shown in magenta.Oriented water molecules are shown in stick representation and these have interactions with residues important for ligand binding.The dashed lines show polar contacts in the binding sites, which range from 1.8 Å to 3.6 Å. (B) Ligand densities around SBD1, SBD1_E184D mutant and SBD2 of the GlnPQ transporter obtained from averaging unbiased CG MD simulations.The blue isosurfaces correspond to a 1,000 fold higher ligand density than in solution.(C) Overlay of the crystallographic structure (PDB ID: 6FXG, in green) with the one simulated bound state of SBD1-Asn (in yellow).Aligning the structures gives a RMSD of ~0.6 Å. Binding residues labelled with numbers have interactions with the substrate (in stick representation).Residues interacting with Asn are labeled by number.

Figure S4 .
Figure S4.Probability distributions of obtained distances in Fig.2 in the main text between subdomains in CG MD simulations of SBD1 wild type, SBD1 E184D mutant and SBD2 in the presence of Gln and / or Asn ligands.

Figure S5 .
Figure S5.Opening-closing transitions of SBD1_E184D mutant observed with CG MD simulations.Distance profiles were calculated between the centers of geometry of the D1 and D2 subdomains in the absence of the ligands and probability distributions of obtained distance.For the CG MD simulation in the presence of the ligand, the protein model was used after 1 μs of CG MD simulation in the absence of the ligand, which was carried out after 500 μs of preliminary MD CG simulation.

Figure S6 .
Figure S6.Superposition of unliganded and Arg-bound structures of SBD1.Two structures (PDB ID 4LA9 (green) and 8B5E (cyan), respectively) were used.Overlay of the residues in the active site was done by aligning the hinge region as an anchor point.Aligning the structures gives a RMSD of ~0.23 Å.Residues interacting with arginine are labeled by number.The dashed lines show polar contacts in the binding sites, which range from 1.8 to 3.6 Å.

Figure S7 .
Figure S7.Superposition of liganded and unliganded structures to show the venus-fly trap movement of the SBDs upon binding of a substrate.Sequences were aligned using the hinge region as anchor point (shown in red).(A) shows the subdomain motions upon binding of asparagine to unliganded SBD1 (green, open unliganded; purple, closed-liganded).(B) shows the subdomain motions of SBD2 upon binding of glutamine (blue, open-unliganded; orange, closed-liganded.Binding of substrate results in movement of the domains relative to each other as shown in Figure1B.For SBD2 this movement displaces the lobes relative to the other more than for SBD1, where this relative movement is smaller and best described by a twisting/rotational movement.This difference might relate to a different structure of the linker between the lobes; for SBD2 this linker is a clear double eta-sheet, for SBD1 the linker has more flexibility.Polar interactions between SBD1 D35 (SBD2 D267) from the large lobe, the substrate and SBD1 K140 (SBD2 K373) of the small lobe probably play a role in the closure of the SBDs (Fig.S8).

Figure S8 .
Figure S8.Binding sites of SBD1 and SBD2.(A) Asparagine (yellow) bound to SBD1.In pink residues from the large subdomain are shown; in green the residues and chains from the small subdomain.Residues labelled with numbers have interactions with the substrate.(B) SBD2 with bound glutamine (yellow).In light blue residues from the large subdomain are shown; in orange the residues and chains from the small domain.Residues interacting with glutamine are labelled by number.The dashed lines show polar contacts in the binding sites, which range from 1.8 to 3.6Å.Oriented water molecules are shown by grey balls and these have interactions with residues important for ligand binding.(C) Overlay of the residues in the active site was done by aligning the hinge region, which is the distinguishing features of SBDs.Asparagine is shown in red; glutamine in yellow.Black labels for SBD1 residues; blue labels for SBD2 residues.