Biosynthesis of D-alanyl-lipoteichoic acid: The tertiary structure of apo-D-alanyl carrier protein

The D-alanylation of lipoteichoic acid (LTA) allows the Gram-positive organ ism to modulate its surface charge, regulate ligand binding, and control th e electromechanical properties of the cell wall. The incorporation of D-ala nine into LTA requires the D-alanine:D-alanyl carrier protein ligase (AMP-f orming) (Dcl) and the carrier protein (Dcp), The high-resolution solution s tructure of the 81-residue (8.9 kDa) Dcp has been determined by multidimens ional heteronuclear NMR. An ensemble of 30 structures was calculated using the torsion angle dynamics approach of DYANA, These calculations utilized 3 288 NOEs containing 1582 unique nontrivial NOE distance constraints. Superp osition of residues 4-81 on the mean structure yields average atomic rmsd v alues of 0.43 +/- 0.08 and 0.86 +/- 0.09 Angstrom for backbone and non-hydr ogen atoms, respectively. The solution structure is composed of three a-hel ices in a bundle with additional short 3(10)- and alpha -helices in interve ning loops. Comparisons of the three-dimensional structure with the acyl ca rrier proteins involved in fatty acid, polyketide, and nonribosomal peptide syntheses support the conclusion that Dcp is a homologue in this family. W hile there is conservation of the three-helix bundle fold, Dcp has a higher enthalpy of unfolding and no apparent divalent metal binding site(s), feat ures that distinguish it from the fatty acid synthase acyl carrier protein of Escherichia coli. This three-dimensional structure also provides insight s into the D-alanine ligation site recognized by Dcl, as well as the site w hich may bind the poly(glycerophosphate) acceptor moiety of membrane-associ ated LTA.