The LytA amidase from Streptococcus pneumoniae (pneumococcus) is the major autolysin of this gram-positive pathogen. This cell wall hydrolase is involved in the autolysis of this bacterium either at the end of the stationary phase of growth or in response to beta-lactamic antibiotics. The protein contains an N-terminal, catalytic module, and a C-terminal or choline-binding module (CBM), a polypeptide with a -solenoid fold which is responsible of the attachment of the enzyme to the cell wall through the recognition of choline residues in the teichoic and lipoteichoic acids. LytA is one of the more than 15 members of the pneumococcal choline-binding protein (CBP) family, all of them sharing the presence of a CBM. However, LytA is the only host-encoded CBP that forms dimers through its CBM in response to choline binding, and the only CBP that is secreted without a conventional signal peptide as well. Although this dimeric configuration has been deemed as necessary to acquire the correct topology to attack the peptidoglycan network, this has not been demonstrated and dimerization might also play a particular structural role. Here we have engineered a monomeric variant of LytA by replacing the C-terminal dimerization hairpin of the protein by the C-terminal loop of the monomeric CbpD hydrolase. This results in a monomeric variant with a decreased stability and affinity for choline, although its hydrolytic activities on cell wall preparations in vitro and on LytA-deficient pneumococcal cells in curation experiments in vivo are both comparable to those of the wild-type species.

Conclusions: Dimerization is an unusual feature of -solenoid proteins which has only been observed in the LytA amidase as well as in a few phage-encoded endolysins. Despite this, dimerization is not necessary for the hydrolytic activity of LytA, but suggests instead that this biophysical event might be related to intracellular stability and/or translocation through the membrane without a signal peptide. Moreover, the results pave the way for the design of more stable BB-solenoid polypeptides through the engineering of their oligomeric state.

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

Peer reviewed

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