BUSCADOR RECOLECTA

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

(A) The plots show the frequency distribution of peptide lengths that are found bound to AmiA (left panel) or AliB (right panel). The data are represented as mean ± SE calculated from non-linear Gaussian fit version (full width at half maximum, FWHM) employing the function: (B) Sequence logo analysis of the peptides bound to AmiA (left panel) or AliB (right panel). The sequence logos were generated based on the alignment of the identified peptides bound to each respective protein, taking just into account their starting positions for the alignment. These plots were generated using the WebLogo server (https://weblogo.berkeley.edu/logo.cgi) [29]. (C) Sector graphs comparing the identified bound peptides to AmiA (shown in the upper panel) and AliB (shown in the lower panel). The peptides were categorized according to the biological function exerted by the protein from which they originated., Peer reviewed

(A) 2mFo-DFc electron-density map for peptide 5 (AKTIKITQTR depicted in green caped sticks) for each of the four monomers (monomers A, B, C and D) as observed in the crystallized AmiA:peptide 5 complex. (B) Atomic B factors for peptide 5 (monomer C). The Ligand is represented as capped sticks and colored according to the B factor distribution, ranging from low (blue) to high (red) values. (C) Structural superposition among the peptide 5 molecules (showed in capped sticks) as observed in the AmiA complexes. Lateral chains from AmiA residues that adopt different conformations are labeled. The peptide sequence is indicated and numbered. N-term, amino-terminus; C-term, Carboxy-terminus. Maps contoured at 1.0 σ., Peer reviewed

(A) Ribbon representation of AmiA structure in complex with an unknown peptide from E. coli (in yellow caped sticks) that we further refined as peptide 4. (B) Electron-density map (2mFo-DFc map contoured at 1.0 σ) for the 10-residues long ligand has been traced (yellow caped sticks) assuming the sequence of peptide 4 (AKTIKITQTR). Ligand is presented in a similar orientation as the one found on panel A. Positions for each residue are indicated., Peer reviewed

(A) Cartoon representation of AliB structure (colored green) in complex with an unknown oligopeptide(s) from E. coli modeled as poly-Ala (depicted as yellow capped sticks). (B) Electron-density map (2mFo-DFc map contoured at 1.0 σ) of the unknown ligand in which a nine-residues-long poly-alanine backbone has been traced (depicted as yellow caped sticks). The ligand is presented in a similar orientation to that in panel A. The positions for each alanine residue are indicated. N-term, amino-terminus; C-term, C-terminus., Peer reviewed