Journal:Acta Cryst D:S2059798322004612
From Proteopedia
(Difference between revisions)
| (15 intermediate revisions not shown.) | |||
| Line 8: | Line 8: | ||
The apparent architectural modularity encourages the construction of hybrid mPKSs to generate unnatural polyketides. The cis-AT domains control the acyl unit incorporated into every elongation step and therefore are attractive engineering targets. This highlights the need for more careful consideration of proper AT-ACP interactions in engineering mPKSs. The inherently transient and weak nature is the key challenge in understanding protein-protein interactions between AT and ACP. The weak mutual binding affinity hampers the structural determination of a AT-ACP complex for directly visualization of protein-protein interactions. We reported the crystal structure of a <scene name='91/912928/Cv/7'>cis-AT-ACP complex</scene> (PDB ID: [[7vrs]]) from the 9th extension module of a salinomycin mPKS. SalAT9 is organized into a large α,β-hydrolase subdomain (residues 97−224 and 299−418) and a small ferredoxin-like subdomain (residues 228−294). The N-terminal KS-to-AT linker (residues 1-92) forms a three-stranded antiparallel β-sheet and three α-helices, packing against the AT domain. The transient ''cis''-AT-ACP complex was obtained by using 1,4-bis(maleimido)butane (BMB) as a crosslinking agent. <scene name='91/912928/Cv/5'>The BMB maleimide groups covalently connect the AT and ACP</scene>. The ACP-binding mode in the ''cis''-AT-ACP complex structure is strikingly different from those of the previously reported ''trans''-AT-ACP complex structures. The ACP primarily contacts the large subdomain of the AT in structure of the ''cis''-AT-ACP complex, whereas the ACP primarily contacts the small domain of the AT in the ''trans''-AT-ACP complex structures. The complex structure provides detailed mechanistic insights into the protein-protein interactions between AT and ACP domains of ''cis''-AT mPKSs and could potentially help optimize chimeric ''cis''-AT mPKSs for unnatural polyketides. | The apparent architectural modularity encourages the construction of hybrid mPKSs to generate unnatural polyketides. The cis-AT domains control the acyl unit incorporated into every elongation step and therefore are attractive engineering targets. This highlights the need for more careful consideration of proper AT-ACP interactions in engineering mPKSs. The inherently transient and weak nature is the key challenge in understanding protein-protein interactions between AT and ACP. The weak mutual binding affinity hampers the structural determination of a AT-ACP complex for directly visualization of protein-protein interactions. We reported the crystal structure of a <scene name='91/912928/Cv/7'>cis-AT-ACP complex</scene> (PDB ID: [[7vrs]]) from the 9th extension module of a salinomycin mPKS. SalAT9 is organized into a large α,β-hydrolase subdomain (residues 97−224 and 299−418) and a small ferredoxin-like subdomain (residues 228−294). The N-terminal KS-to-AT linker (residues 1-92) forms a three-stranded antiparallel β-sheet and three α-helices, packing against the AT domain. The transient ''cis''-AT-ACP complex was obtained by using 1,4-bis(maleimido)butane (BMB) as a crosslinking agent. <scene name='91/912928/Cv/5'>The BMB maleimide groups covalently connect the AT and ACP</scene>. The ACP-binding mode in the ''cis''-AT-ACP complex structure is strikingly different from those of the previously reported ''trans''-AT-ACP complex structures. The ACP primarily contacts the large subdomain of the AT in structure of the ''cis''-AT-ACP complex, whereas the ACP primarily contacts the small domain of the AT in the ''trans''-AT-ACP complex structures. The complex structure provides detailed mechanistic insights into the protein-protein interactions between AT and ACP domains of ''cis''-AT mPKSs and could potentially help optimize chimeric ''cis''-AT mPKSs for unnatural polyketides. | ||
| - | SalAT9M large hydrolase subdomain functions as the major binding platform for SalACP9: <scene name='91/912928/Cv/9'>The SalAT9M-ACP9 complex structure, transparent surface representation</scene>. | + | SalAT9M large hydrolase subdomain (in dark turquoise) functions as the major binding platform for SalACP9 (in burlywood): <scene name='91/912928/Cv/9'>The SalAT9M-ACP9 complex structure, transparent surface representation</scene>. |
| + | |||
| + | <scene name='91/912928/Cv/11'>The αII helix of SalACP9 packs against the helix α8 (residues 378-388) of the large hydrolase subdomain</scene> with an angle of 26°. <scene name='91/912928/Cv/15'>The short helix αIII’ of the loop II of SalACP9 packs against the loop C of the small subdomain of SalAT9M</scene>. | ||
| + | |||
| + | In the crystallographic asymmetric unit, a <scene name='91/912928/Cv/19'>SalAT9M-ACP9 complex</scene> and a <scene name='91/912928/Cv/20'>standalone SalAT9M</scene> molecule were observed. <scene name='91/912928/Cv/18'>SalACP9 binding induces SalAT9M to a more open state compared to the standalone SalAT9M’</scene>. | ||
| + | *<scene name='91/912928/Cv/21'>Animation of previous scene</scene>. | ||
| + | <jmol><jmolButton> | ||
| + | <script>if (_animating); anim pause;set echo bottom left; color echo white; font echo 20 sansserif;echo Animation Paused; else; anim resume; set echo off;endif;</script> | ||
| + | <text>Click here to Stop Animation</text> | ||
| + | </jmolButton></jmol> | ||
| + | |||
| + | The KS-to-AT linkers have been shown to functional as a structural element stabilizing the ATs that is expressed as standalone domains. The absence or incompleteness of KS-to-AT linkers in AT constructs may result in insoluble or inactive proteins. The complex structure shows that <scene name='91/912928/Cv/22'>the loop IV (residues 80-86) of the KS-to-AT linker makes contacts with the N-terminal residues of loop I of SalACP9</scene>. | ||
| + | |||
| + | '''PDB references:''' The complex of Acyltransferase and Acyl Carrier Protein Domains from module 9 of Salinomycin Polyketide Synthase, [[7vrs]]; Acyltransferase from the 9th Module of Salinomycin Polyketide Synthase, [[7vt1]]. | ||
<b>References</b><br> | <b>References</b><br> | ||
Current revision
| |||||||||||
This page complements a publication in scientific journals and is one of the Proteopedia's Interactive 3D Complement pages. For aditional details please see I3DC.
