High‐resolution structures of the d‐alanyl carrier protein (Dcp) DltC from Bacillus subtilis reveal equivalent conformations of apo‐ and holo‐forms

Abstract

a b s t r a c t D-Alanylation of lipoteichoic acids plays an important role in modulating the properties of Gram-positive bacteria cell walls. The D-alanyl carrier protein DltC from Bacillus subtilis has been solved in apo-and two cofactor-modified holo-forms, whereby the entire phosphopantetheine moiety is defined in one. The atomic resolution of the apo-structure allows delineation of alternative conformations within the hydrophobic core of the 78 residue four helix bundle. In contrast to previous reports for a peptidyl carrier protein from a non-ribosomal peptide synthetase, no obvious structural differences between apo-and holo-DltC forms are observed. Solution NMR spectroscopy confirms these findings and demonstrates in addition that the two forms exhibit similar backbone dynamics on the ps-ns and ms timescales.

New Structural Data Reveal the Motion of Carrier Proteins in Nonribosomal Peptide Synthesis

“…The secondary and tertiary structures of PCP domains are highly conserved, with only minor deviations from the prototypical four‐helix bundle being documented to date 19, 20, 21, 22, 23, 24, 25, 26, 27. At the level of primary structure, PCPs are more variable,21 which gives rise to variations in local shape and charge distributions of the exposed and buried surfaces.…”

“…This model was based on observations that a PCP domain from the tyrocidine synthetase exists in three different conformations (termed A, A/H, and H) depending on its loaded state 28. However, this has since been dismissed as an artifact of PCP domain excision from the larger synthetase since all other PCP structures elucidated to date adopt the A/H state 19, 20, 21, 22, 23, 24, 25, 26, 29, 30…”

“…Thesecondary and tertiary structures of PCP domains are highly conserved, with only minor deviations from the prototypical four-helix bundle being documented to date. [19][20][21][22][23][24][25][26][27] At the level of primary structure,P CPs are more variable, [21] which gives rise to variations in local shape and charge distributions of the exposed and buried surfaces.T his affects how individual PCPs interact with catalytic partners, [21] especially in trans,w here PCP recognition is crucial to selectivity. [11,20] An early hypothesis to explain the mechanism of NRPSs was based around large changes in PCP tertiary structure as Tiia Kittilä, born in 1984, studied biochemistry at the University of Helsinki, Finland, and completed her M.Sc.…”

Profiling and tandem mass spectrometry analysis of aminoacylated phospholipids in Bacillus subtilis

“…Importantly, the alanylated phosphatidylglycerol is D-alanyl-PG, it is therefore not synthesized from tRNA-carried L-alanyl by a reaction catalyzed by MprF. Instead, another well-known source of activated alanine carried by D-alanine carrier protein DltC in the form of thioester 22, 37 may be the most likely origin.…”

“…Its remote homologues include the acyl-coenzyme A synthetases and firefly luciferases 20 . DltA catalyzes the ATP-driven adenylation of the carboxyl group of D-alanine and the transfer of the activated D-alanyl to the thiol group of 4’-phosphopantetheine which is covalently attached to a serine side chain of D-alanyl carrier protein DltC (~80 amino acid residues) 18, 21, 22 . The functional role has not been firmly established for DltB (~400 amino acid residues), an integral membrane protein.…”