Pierik, A. J. | bioRxiv Science

bioRxiv2021

A repurposed, non-canonical cytochrome c, chaperones calcium binding by PilY1 for type IVa pili formation

Type IVa pili (T4aP) are versatile bacterial cell surface structures that undergo extension/adhesion/retraction cycles powered by the cell envelope-spanning T4aP machine. In this machine, a complex composed of four minor pilins and PilY1 primes T4aP extension and is also present at the pilus tip mediating adhesion. Similar to many other bacteria, Myxococcus xanthus contains multiple minor pilins/PilY1 sets that are incompletely understood. Here, we report that minor pilins and PilY1 (PilY1.1) of cluster_1 form priming and tip complexes contingent on a non-canonical cytochrome c (TfcP) with an unusual His/Cys heme ligation and calcium. We provide evidence that TfcP is unlikely to participate in electron transport and has been repurposed to promote calcium binding by PilY1.1 at low calcium concentrations, thereby stabilising PilY1.1 and enabling T4aP function in a broader range of calcium concentrations. These results identify a novel function of cytochromes c and illustrate how incorporating an accessory factor expands the environmental range under which the T4aP system functions.

microbiology↗

bioRxiv2020

Exchange of a single amino acid residue in the cryptophyte phycobiliprotein lyase GtCPES expands its substrate specificity

Cryptophyte algae are among the few eukaryotes that employ phycobiliproteins (PBP) for light harvesting during oxygenic photosynthesis. In contrast to the cyanobacterial PBP that are organized in large membrane-associated super complexes, the phycobilisomes, those from cryptophytes are soluble within the chloroplast thylakoid lumen. Their light-harvesting capacity is due to covalent linkage of several open-chain tetrapyrrole chromophores (phycobilins). Guillardia theta utilizes the PBP phycoerythrin PE545 with 15,16-dihydrobiliverdin (DHBV) in addition to phycoerythrobilin (PEB) as chromophores. Thus far, the assembly of cryptophyte PBPs is not yet completely understood but involves the action of PBP-lyases as shown for cyanobacterial PBP. PBP-lyases facilitate the attachment of the chromophore in the right configuration and stereochemistry. Here we present the functional characterization of eukaryotic S-type PBP lyase GtCPES from G. theta. We show GtCPES mediated transfer and covalent attachment of PEB to the conserved Cys82 of the acceptor PBP {beta}-subunit (PmCpeB) of Prochlorococcus marinus MED4. Based on the previously solved crystal structure, the GtCPES binding pocket was investigated using site-directed mutagenesis. Thereby, amino acid residues involved in phycobilin binding and transfer were identified. Interestingly, exchange of a single amino acid residue Met67 to Ala extended the substrate specificity to phycocyanobilin (PCB) likely by enlarging the substrate-binding pocket. Variant GtCPES_M67A binds both PEB and PCB forming a stable, colorful complex in vitro and in vivo produced in Escherichia coli. GtCPES_M67A is able to mediate PCB transfer to Cys82 of PmCpeB. Based on our data we postulate that a single amino acid residue determines the bilin-specificity of phycoerythrin S-type lyases but that additional factors regulate hand over to the target protein.

biochemistry↗