Protein-protein interactions in "cis-AT" polyketide synthases

Abstract

Covering: up to the end of 2018 Polyketides are a valuable source of bioactive and clinically important molecules. The biosynthesis of these chemically complex molecules has led to the discovery of equally complex polyketide synthase (PKS) pathways. Crystallography has yielded snapshots of individual catalytic domains, di-domains, and multi-domains from a variety of PKS megasynthases, and cryo-EM studies have provided initial views of a PKS module in a series of defined biochemical states. Here, we review the structural and biochemical results that shed light on the protein-protein interactions critical to catalysis by PKS systems with an embedded acyltransferase. Interactions include those that occur both within and between PKS modules, as well as with accessory enzymes.

Fig. 1.

Comparison of metazoan FAS and a PKS modifying region. (A) Porcine FAS crystal structure (PDB 2VZ8). (B) Modifying region of MAS, an iterative PKS (based on PDB 5BP1). The KS (blue), DH (yellow) and ER (beige) domains are dimeric, whereas the AT (green), KR (purple) and ΨMT (orange) domains have no homomeric contacts. The PKS and FAS DH dimers have different overall shapes, but the domain N-termini form the dimer interface in both megasynthases. The PKS KR domain has no contacts with either DH or ER whereas many inter-domain contacts exist in the FAS. The post-AT linker is rendered in red, and KS-AT linker domain is in gray. Helices are rendered as cylinders and β-strands as arrows.

Fig. 2.

Comparison of PikAIII module to an excised KS-AT di-domain. (A) Methylmalonyl-PikAIII (EMDB 5653). Methylmalonyl-ACP is docked at the bottom entrance to the active site. The domains are colored as in Fig. 1, with ACPs in red. (B) KS-AT from DEBS3 module 5 (PDB 2HG4). The linker-AT (gray-green) is differently oriented relative to the KS dimer (blue) in two structures. The post-AT linker (red in B) was not visible in the cryo-EM reconstruction of PikAIII.

Fig. 3.

PKS dimer-forming catalytic domains. (A) KS dimer from DEBS2 module 3 (PDB 2QO3). The catalytic Cys, His and Glu side chains are shown as spheres in atomic colors with gray C atoms. (B) DH dimer from CurK (PDB 3KG9). The catalytic His and Asp side chains are shown as spheres in atomic colors with gray C atoms. (C) ER dimer from MAS (based on PDB 5BP4). The NADPH cofactor is shown as spheres with gray C atoms. (D) TE dimer from PikAIV (PDB 2HFJ). The substrate tunnel in each subunit is rendered as a gray surface. All dimers are viewed approximately along the twofold symmetry axis. Each dimer is colored in the same hue as in Fig. 1, with monomers in different brightness.

Fig. 4.

Small PKS dimerization elements. (A) dd^KS from PikAIV (PDB 3F5H). (B) post-ACP dimerization element from DEBS2 (PSB 1PZQ). (C) post-AT dimerization element from SpnC module 3 (PDB 4IMP). The dimerization elements are colored in the same hue as an adjacent catalytic domain (Fig. 1), with monomers in different brightness. Adjacent domains are shown as circles. The twofold symmetry axes are vertical in these images.

Fig. 5.

ACP interaction with KS and HMGS. The PKS KS and β-branching HMGS are members of the thiolase family. Here they are depicted in identical orientation with the dimer axis vertical on the page with monomers colored in shades of blue (KS) and cyan (HMGS). (A) PikAIII KS dimer with upstream and module ACPs as observed in functional complexes. The active site cavity is shown as a solid surface inside the KS in this composite image from two structures. In the complex with pentaketide-ACP_i-1, the upstream ACP_i-1 from the preceding module (dull red) delivers the pentaketide product of the previous module to a site near the active site side entrance (arrow). In the complex with methylmalonyl-ACP_i, the module ACP_i (bright red) delivers a methylmalonyl building block to the lower active site entrance. (B) HMGS from the curacin A pathway in complex with holo-ACP_D (gold). The holo-ACP Ppant (rendered as sticks with gray C atoms) enters the active site through the side entrance, analogous to the KS side entrance.

Fig. 6.

Fab-KS-AT complex. In the top view, the dimer axis is vertical. In the bottom view, the structure is rotated 45° towards the viewer to illustrate the separation of the AT and the Fab (arrow). Domains within the DEBS2 module 3 KS-AT dimer (PDB 6C9U) are colored as in Fig. 2B, and the Fab heterodimer (V_L-C_L, V_H-C_H) is colored with the variable domains (V_L and C_L) in magenta and the constant domains (V_H and C_H) in cyan.

Fig. 7.

Insertion of catalytic domains between KR structure elements. (A) Composition of canonical cis-AT PKS extension modules. Domains are represented by circles colored as in Fig. 1 and Fig. 2, and the strands of the KR β-zipper as arrows. The schematic illustrates how MT and ER domains, when present, are inserted within the KR. The gray circle is the linker domain between KS and AT, and the post-AT linker peptide is represented as a red line. Docking domains and the small dimerization elements are not depicted. (B) Structure of KR from Plm1 (PDB 4IMP). The two β-zipper strands contribute to the core β-sheets of both KR_S (light purple) and KR_C (dark purple). Approximate insertion points for MT and ER in other modules are indicated by arrows.

Fig. 8.

PKS docking complexes. (A) DEBS2-DEBS3 dock (PDB 1PZR). (B) PikAIII-PikAIV dock (PDB 3F5H). (C) CurG-CurH dock (PDB 4MYY). The dd^KS dimers are in blue and the dd^ACPs in red and mauve with the corresponding KS and ACP domains in schematic form. Polypeptide N and C-termini are labeled.