Complex structure of the acyltransferase VinK and the carrier protein VinL with a pantetheine cross-linking probe

Abstract

Acyltransferases are responsible for the selection and loading of acyl units onto carrier proteins in polyketide and fatty-acid biosynthesis. Despite the importance of protein–protein interactions between the acyltransferase and the carrier protein, structural information on acyltransferase–carrier protein interactions is limited because of the transient interactions between them. In the biosynthesis of the polyketide vicenistatin, the acyltransferase VinK recognizes the carrier protein VinL for the transfer of a dipeptidyl unit. The crystal structure of a VinK–VinL covalent complex formed with a 1,2-bismaleimidoethane cross-linking reagent has been determined previously. Here, the crystal structure of a VinK–VinL covalent complex formed with a pantetheine cross-linking probe is reported at 1.95 Å resolution. In the structure of the VinK–VinL–probe complex, the pantetheine probe that is attached to VinL is covalently connected to the side chain of the mutated Cys106 of VinK. The interaction interface between VinK and VinL is essentially the same in the two VinK–VinL complex structures, although the position of the pantetheine linker slightly differs. This structural observation suggests that interface interactions are not affected by the cross-linking strategy used.

Protein‐Protein Recognition Involved in the Intermodular Transacylation Reaction in Modular Polyketide Synthase in the Biosynthesis of Vicenistatin

“…In the case of VinP2 NDD 4 KS 4 AT 4 C206G/S684G, the reaction with Br‐acetyl pantetheiamide‐VinP1 ACP 3 CDD 3 resulted in the formation of the crosslinked complex. These observations suggest that the Br‐acetoamide group is too reactive toward VinP2 NDD 4 KS 4 AT 4 , resulting in the formation of undesired crosslinked complexes via other nucleophilic residues as reported for the VinK‐VinL crosslinking reaction [28] . By comparison, in the crosslinking reaction of VinP2 NDD 4 KS 4 AT 4 C206G/S684G using Cl‐acetyl pantetheiamide‐VinP1 ACP 3 CDD 3 , almost no crosslinked complex was generated.…”

“…These observations suggest that the Bracetoamide group is too reactive toward VinP2 NDD 4 KS 4 AT 4 , resulting in the formation of undesired crosslinked complexes via other nucleophilic residues as reported for the VinK-VinL crosslinking reaction. [28] By comparison, in the crosslinking reaction of VinP2 NDD 4 KS 4 AT 4 C206G/S684G using Cl-acetyl pantetheiamide-VinP1 ACP 3 CDD 3 , almost no crosslinked complex was generated. Thus, Cl-acetyl pantetheinamide-VinP1 ACP 3 CDD 3 only reacts with the Cys206 catalytic residue, and seems to be suitable for the crosslinking reaction between VinP2 NDD 4 KS 4 and VinP1 ACP 3 CDD 3 .…”

Structural Basis of Transient Interactions of Acyltransferase VinK with the Loading Acyl Carrier Protein of the Vicenistatin Modular Polyketide Synthase

“…Thus, the interface interactions were not affected by the cross-linking strategy used. 8 The VinK−VinL complex structures clearly illustrate how VinK interacts with the acyl donor ACP VinL. However, it is unclear how VinK interacts with VinP1ACP L as an acyl acceptor ACP.…”

“…Asp86 and Val91 of VinP1ACP L are conserved in VinL (Asp35 and Val40) (Figure S10). In the structure of the VinK–VinL complex, Arg299 of VinK forms a bidentate salt bridge with Asp35 of VinL and Met206 of VinK forms hydrophobic contacts with Val40 of VinL, similar to observations for the VinK–VinP1ACP L complex structure. In the structure of the VinK–VinL complex, Arg153 of VinK also forms a bidentate salt bridge with Glu47 of VinL (Figure B).…”

“…We successfully determined the structure of the VinK–VinL complex, which was the first crystal structure of an AT–ACP complex . Recently, we developed a new cross-linking strategy, using synthetic bromoacetamide and chloroacetamide pantetheine probes, and determined the structure of the VinK–VinL complex by using the chloroacetamide pantetheine probe in combination with a Cys mutation of catalytic residue Ser106 of VinK . The VinK–VinL binding interface was essentially the same in the two VinK–VinL complex structures, although the position of the pantetheine linker slightly differed between them.…”