BUSCADOR RECOLECTA

(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

Left panel, cartoon representation of the AliD structure (open conformation) using the same color code as Fig 2B. The focus is on the C-terminal α-helix (α19), where positively-charged residues (mainly lysines) are depicted as capped sticks. Right panel, the electrostatic-potential surface of AliD is shown in the same orientation as the left panel. Relevant basic patches are indicated with black arrows. The color key (blue, positive and red, negative) shows the Poisson-Boltzmann electrostatic-potential surface (color bar range ± 68.99 kT/e)., Peer reviewed

(A) Left panel; superposition of the structures of AliD from S. pneumoniae (this work, colored in red) and OppA from S. typhimurium [colored in gray, PDB 1RKM [25]], both in the open conformation (rmsd of 3.18 Å for 349 Cα atoms). Right panel; structure superposition between AliD from S. pneumoniae (this work, colored in red) and OppA from S. typhimurium [colored in gray, PDB 1B4Z [26]], both in the closed conformation (rmsd of 3.20 Å for 347 Cα atoms). Both proteins are represented as cartoon oval. The two crossover connecting regions in AliD are colored yellow and labeled as "c1" and "c2", respectively. (B) Sequence alignment of AliD from S. pneumoniae and OppA from S. typhimurium, generated by T-COFFEE [22] and drawn with ESPript [23]. Sequence identity, estimated with Clustal2.1, is 25,76%. Identities are boxed in black. Similarities are boxed in gray according to physicochemical properties., Peer reviewed

The protein structure is depicted in blue capped sticks. A Mg2+ atom, contributing to crystal packing, is represented as a green sphere. The lower panel provides a detailed view of the boxed area in the upper panel, highlighting electron densities at 1.8 Å resolution., Peer reviewed

ATP-binding cassette (ABC) transport systems are crucial for bacteria to ensure sufficient uptake of nutrients that are not produced de novo or improve the energy balance. The cell surface of the pathobiont Streptococcus pneumoniae (pneumococcus) is decorated with a substantial array of ABC transporters, critically influencing nasopharyngeal colonization and invasive infections. Given the auxotrophic nature of pneumococci for certain amino acids, the Ami ABC transporter system, orchestrating oligopeptide uptake, becomes indispensable in host compartments lacking amino acids. The system comprises five exposed Oligopeptide Binding Proteins (OBPs) and four proteins building the ABC transporter channel. Here, we present a structural analysis of all the OBPs in this system. Multiple crystallographic structures, capturing both open and closed conformations along with complexes involving chemically synthesized peptides, have been solved at high resolution providing insights into the molecular basis of their diverse peptide specificities. Mass spectrometry analysis of oligopeptides demonstrates the unexpected remarkable promiscuity of some of these proteins when expressed in Escherichia coli, displaying affinity for a wide range of peptides. Finally, a model is proposed for the complete Ami transport system in complex with its various OBPs. We further disclosed, through in silico modelling, some essential structural changes facilitating oligopeptide transport into the cellular cytoplasm. Thus, the structural analysis of the Ami system provides valuable insights into the mechanism and specificity of oligopeptide binding by the different OBPs, shedding light on the intricacies of the uptake mechanism and the in vivo implications for this human pathogen., Peer reviewed

Detailed composition for each substrate-binding pocket in AliB:peptide 4 complex., Peer reviewed

Detailed composition for each substrate-binding pocket in AliB:peptide 3 complex., Peer reviewed