Structural and bioinformatic characterization of an Acinetobacter baumannii type II carrier protein

Abstract

Microorganisms produce a variety of natural products via secondary metabolic biosynthetic pathways. Two of these types of synthetic systems, the nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs), use large modular enzymes containing multiple catalytic domains in a single protein. These multidomain enzymes use an integrated carrier protein domain to transport the growing, covalently bound natural product to the neighboring catalytic domains for each step in the synthesis. Interestingly, some PKS and NRPS clusters contain free-standing domains that interact intermolecularly with other proteins. Being expressed outside the architecture of a multi-domain protein, these so-called type II proteins present challenges to understand the precise role they play. Additional structures of individual and multi-domain components of the NRPS enzymes will therefore provide a better understanding of the features that govern the domain interactions in these interesting enzyme systems. The high-resolution crystal structure of a free-standing carrier protein from Acinetobacter baumannii that belongs to a larger NRPS-containing operon, encoded by the ABBFA_003406-ABBFA_003399 genes of A. baumannii strain AB307-0294, that has been implicated in A. baumannii motility, quorum sensing and biofilm formation, is presented here. Comparison with the closest structural homologs of other carrier proteins identifies the requirements for a conserved glycine residue and additional important sequence and structural requirements within the regions that interact with partner proteins.

Keywords: Acinetobacter baumannii; acyl carrier proteins; biofilm formation; motility; natural product biosynthesis; nonribosomal peptide synthetase; peptidyl carrier proteins.

Figure 1

(a) The NRPS cluster from A. baumannii (with the gene nomenclature from both the AB307-0294 and ATCC17978/M2 strains). Protein sizes and proposed functions are included. (b) The genes are organized in a polycistronic operon containing eight genes (grey) preceded by a transcriptional regulatory protein (white). The sequences of the a3404 gene and protein are shown, with the carrier protein phosphopantetheinylation motif in red. (c) A ribbon diagram of A3404 highlights the four primary helices, α1–α4, and the long turn between the first two helices that contains two single-turn 3₁₀-­helices. Ser40, the site of phosphopantetheinylation, is shown in a stick representation.

Figure 2

The 14 structures of closest homologs as identified by the DALI server were compared with A3404. (a) Sequence alignment of the homologous proteins. The first three columns represent the rank in the DALI scoring, the type of protein and the PDB code. Proteins in the top half of the alignment are from NPRS or PKS clusters, while proteins in the bottom half are ACPs from fatty-acid synthesis and transport. The pantetheinylation motif is highlighted in yellow; helices α2 and α3 are shaded in pink. In the alignment, acidic amino acids are red, while basic residues are blue. (b) A ribbon diagram of A3404 (red) is superimposed on the top two closest homologs of each of the three carrier protein types. The same orientation is used in all panels and the helix designations are shown in the top left panel. The two PKS acyl carrier proteins 2liu and 2ju2 are shown in light blue. Two NRPS PCP domains, the type II 4i4d and the type I 3tej, are shown in green. Two acyl carrier proteins (1x3o and 4dxe) are shown in yellow. In all structures, the serine residue at the start of helix α2 is shown in a stick representation.