The BB-solenoid fold is present in proteins from several bacterial species, ranging from food-related microorganisms (e.g. Leuconostoc citreum) to pathogens (Clostridioides difficile, Streptococcus mutans or S. pneumoniae). The most studied representatives of this family are the choline-binding modules (CBMs) that are part of the pneumococcal choline-binding proteins (CBPs), and that are responsible for their binding to the bacterial cell wall, where they carry out their biological function. The standard three-dimensional structure of the CBMs is based on four elements: i) B-hairpin repeats; ii) linker loops; iii) N- and C-terminal tags usually involved in dimerization or configuring a non-standard choline-binding site, and iv) aromatic residues in or near the choline binding sites (CBSs). Previous results have demonstrated that B-hairpins are robust structures able to fold autonomously, and that the loops are essential to keep B-hairpins in place. Here we have checked the importance of termini tags and of a fully conserved inner "fourth" aromatic residue in contact with other three aromatics which are directly involved in choline binding. Protein engineering was employed to generate site-specific mutants targeting the non-standard CBSs in the C-terminal tag of the pneumococcal CbpD choline-binding module, as well as the four inner aromatics in the corresponding module of the major pneumococcal autolysin, LytA. Structure, stability and ligand affinity was assessed by circular dichroism and folding thermodynamics. As a result, all mutations generated natively folded and functional proteins but with a severe decrease in stability and in affinity for choline.

Conclusions: The BB-solenoid fold of CBMs can be thought as a sort of molecular "spring" with a tendency to elasticity unless it is kept in place by terminal tags and inner aromatic residues. This flexibility may however play a biological role in the pneumococcal physiology, as it will be discussed in the communication. Moreover, our results also pave the way for the engineering and biotechnological application of BB-solenoid proteins.

Grants PID2022-139209OB-C21 and PID2022-139209OB-C22 funded by MICIU/AEI/ 10.13039/501100011033.

Peer reviewed

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