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Supplementary Information S1: representative videos showing S. ratti L3 with indicated motility Scores, Peer reviewed

(A) Left panel, surface representation of the AliD:permease complex predicted by AF. Each protein is color-coded and labeled accordingly. CH: coupling helix. (B) Different configurations of the NBDs in the inward (closed, upper panel) and outward (open, lower panel) conformations. AmiF and AmiD are represented in cartoon form, and the ATP molecules are shown as spheres. (C) View corresponding to a slice located at the blue plane depicted in A, right panel. The upper panel highlights the identified hydrophobic residues that obstruct the channel in the inward, closed conformation. In contrast, the lower panel shows the location of the same residues in the outward, open conformation. The generated cavity, presumably allowing oligopeptide transport, is marked in blue and outlined with a dashed black line. Relevant residues are labeled and represented as spheres. (D) Different proteins of the Ami system and conceptual schematic model for the mechanism of oligopeptide transport., Peer reviewed

(A) Right panel, molecular electrostatic potential surface (MEPS) of AliB’s lobe 2 is shown. The three peptides complexed with AliB are depicted as capped sticks with colors orange (peptide 2), green (peptide 3) and cyan (peptide 4). The oligopeptide positions 1 to 4 and the pockets P1 to P4 are labeled. Left panel, cartoon representation corresponding to the boxed area displayed in the right panel. Conserved Aspartate (D) and Tryptophan (W) residues are depicted in capped gray sticks. The residue F521, crucial for stabilizing the lateral chain in oligopeptide position 2, is also shown as capped gray sticks. (B) Right panel, MEPS of AmiA’s lobe 2 is shown. The peptide corresponding to the AmiA:peptide 5 complex is depicted in capped sticks (colored green). The oligopeptide positions 1 to 4 and the pockets P1 to P4 are labeled. Left panel, cartoon representation corresponding to the boxed area is in the right panel is displayed. Conserved Asp (D) and Trp (W) residues are depicted as capped deep olive sticks. The residue E524, critical for stabilizing the lateral chain in oligopeptide position 2, is also shown as capped gray sticks. Polar interactions are represented with dashed black lines. R1 and R2 represent variable regions 1 and 2, respectively. The small panel between panels A and B provides a view of a typical Ami OBP with both lobes indicated. The black dashed line between the two lobes indicates the position of the slice required to obtain the view of the MEPS of lobe 2 for both panels A and B. (C) Structural superposition of lobe 2 for the five Ami OBPs is presented, with a focus on strand β20. Regions R1 and R2 are indicated. (D) Schematic cartoon representing the relevant OBP structural features involved in oligopeptide recognition (colored in pink)., Peer reviewed

(A) Schematic representation illustrating the different elements composing the lipoproteins of the Ami system. Distinctive coloring is applied to each domain. (B) Alignment of sequences encompassing the signal peptide (in yellow), the lipobox (in red) and the linker region (in gray) in the five lipoproteins of the Ami system. The cysteine residue designated for lipidation is denoted by a black arrow., Peer reviewed

The structure of AliD is presented in a cartoon oval representation. Domain I, II and III are color-coded as green, pale brown and blue, respectively. Peptide 1 is represented as yellow capped sticks. Hydrophobic residues, forming a hydrophobic patch that encloses the ligand when the protein adopts a closed conformation, are also visualized in capped sticks. (MPG), Peer reviewed

AliD residues interacting with the two permease subunits (AmiC and AmiD) were identified with LigPlot+ v.2.2 [35]. (A) Upper panel, cartoon depiction of AliD (colored orange) is presented in a manner akin to that in Fig 2B (upper panel). AliD residues involved in the interaction with the permease are labeled and depicted in yellow capped sticks, while the semi-transparent representation illustrates the AliD surface. Peptide 1 is visualized as blue spheres. The boxed inset exhibits a surface representation of AliD (orange), highlighting the positions of residues engaging with AmiC (pink) and AmiD (purple). The orientation of AliD corresponds to that in A, upper panel. The middle panel mirrors the representation in A, upper panel, but with a 60° rotation through the indicated axes. The lower panel replicates the view from A, middle panel, incorporating the two permease subunits, AmiC (pink cartoon), and AmiD (soft purple cartoon). ECD, extra-cellular domain. (B) In the upper panel, a table is provided summarizing AliD residues involved in interactions with AmiD. The lower panel presents a table summarizing AliD residues engaged with AmiC. The symbol ’*’ denotes residues that are absolutely conserved across all five OBPs of the Ami system. The symbol ’#’, on the other hand, designates residues with physicochemical properties conserved throughout all five OBPs of the Ami system., Peer reviewed

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(A) Left, Structural superimposition of Lobe 2 from AliB: peptide 2, AliB:peptide 3 and AliB:peptide 4 complexes. Right, Electrostatic potential surface of Lobe 2 AliB:peptide 2 complex showing peptide recognition and binding pockets (see main text). (B) Electrostatic potential surface of Lobe 2 in AliD:peptide1 complex, AliC, AmiA:peptide 5 complex and in AliA as predicted by AlphaFold (AF). P1 to P4, pocket 1 to pocket 4., Peer reviewed

Data for this article, including PXRD, Le Bail fitting, multinuclear liquid and MAS NMR, calculated bond lengths and angles and infrared data, are available at DIGITAL.CSIC at https://hdl.handle.net/10261/374491., Financial support from the Spanish Ministry of Science Innovation (PID2022-137889OB-I00, TED2021-131223B-I00, MCIN/AEI/10.13039/501100011033) is gratefully acknowledged. Synchrotron powder X-ray diffraction data experiments were performed at the Materials Science Powder Diffraction beamline bl04 at the ALBA Spanish Synchrotron with the collaboration of the ALBA staff. H. Y. is grateful to the China Scholarship Council for a PhD grant. S. R. G. B. acknowledges grant RYC2022-036070-I funded by MICIU/AEI/10.13039/501100011033 and by “ESF+”. Authors would like to thank the computing time provided by the Servicio de Supercomputación de la Universidad de Granada, Data for this article, including PXRD, Le Bail fitting, multinuclear liquid and MAS NMR, calculated bond lengths and angles and infrared data, are available at DIGITAL.CSIC at https://hdl.handle.net/10261/374491., Peer reviewed

(A) The bound peptides visualized inside the binding cavity of OBPs belonging to the Ami permease reveal the extent of the unoccupied space. The cavity is shown as a gray semi-transparent surface, the peptide is shown in yellow capped stick, and the protein is not shown. AmiA:5, AmiA in complex with peptide 5; AliD:1, AliD in complex with peptide 1; AliB:2, AliB in complex with peptide 2; AmiB:3, AmiB in complex with peptide 3 and AmiB:4, AmiB in complex with peptide 4. (B) Comparison of the binding cavity volumes found on SBPs of the Ami permease, including the cavities of OppA from L. lactis [PDB 3DRF [30]], AppA from B. subtillis (PDB 1XOC [31]), OppA from S. typhimurium [PDB 1B4Z [26]], DppA [PDB 1DPE] and ProX from E. coli [PDB 1SW2 [33]], for comparative purposes. The volumes were calculated with the program POCASA v1.1 [34] using default parameters and a Probe Radius of 1 Å., Peer reviewed