PXD065966-1

PXD065966 is an original dataset announced via ProteomeXchange.

Title	Calcium-loaded acylated segment controls the membrane penetration capacity of repeats-in-toxin cytolysins
Description	Loading of calcium ions into the numerous carboxy-terminus-proximal binding sites in the Repeats in ToXin (RTX) domains drives the cooperative and vectorial folding of RTX β-roll structures involved in cell binding and membrane penetration of RTX cytolysins. Two additional binding sites for calcium ions, coordinated by the side chains of residues D880, D918, and N936, were identified in the structure of the acylated N-terminal cap of the RTX domain of Bordetella pertussis adenylate cyclase toxin (CyaA). Here, we show that this calcium-binding structure plays a key role in membrane insertion of the toxin. An N936L residue substitution did not impact toxin acylation or CR3 receptor binding but disrupted the calcium-dependent fold of the acylated segment and ablated the membrane penetration capacity of the toxin. Similarly, substitution of the corresponding D639 residue of Escherichia coli α-hemolysin (HlyA) abolished its cytolytic capacity. Moreover, disruption of the β-turn structures in the calcium binding sites of the acylated segment of CyaA (G934L) and HlyA (G637L) strongly impaired the cytotoxic capacities of both toxins. On the contrary, a D880L substitution yielded a toxin with an enhanced CR3-independent cell penetration and pore-forming capacity. Hydrogen/deuterium exchange probing revealed that the D880L substitution altered the fold of the acylated segment and stabilized the interaction of its two acylated β-hairpins. Hence, loading the calcium-binding sites in the acylated segment controls its structure and rules the interaction and functional cooperation of its two acylated β-hairpins that initiate penetration of the CyaA polypeptide into the cell membrane.
HostingRepository	PRIDE
AnnounceDate	2026-04-28
AnnouncementXML	Submission_2026-04-28_00:50:46.767.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Sascha Vatic
SpeciesList	scientific name: Escherichia coli; NCBI TaxID: NEWT:562; scientific name: Bordetella pertussis NBRC 107857; NCBI TaxID: NEWT:1216981; scientific name: Bacteria; NCBI TaxID: NCBITaxon:2;
ModificationList	acylated residue
Instrument	maXis

Revision	Datetime	Status	ChangeLog Entry
0	2025-07-09 13:17:34	ID requested
⏵ 1	2026-04-28 00:50:47	announced

10.1016/J.JBC.2026.113079;

submitter keyword: adenylate cyclase toxin, calcium binding sites,RTX toxin, folding

Petr Novak
contact affiliation	The Institute of Microbiology of the Czech Academy of Sciences Head of Group Protein Structure Characterization by Advanced Mass Spectrometry
contact email	pnovac@biomed.cas.cz
lab head
Sascha Vatic
contact affiliation	Biocev MBU CAS
contact email	sascha.vatic@biomed.cas.cz
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD065966
     1. Label: PRIDE project
     2. Name: Calcium-loaded acylated segment controls the membrane penetration capacity of repeats-in-toxin cytolysins