Modifying the Thioester Linkage Affects the Structure of the Acyl Carrier Protein

Sztain, Terra; Patel, Ashay; DJ, Lee; Davis, Thomas B.; McCammon, J. Andrew; Burkart, Michael D.

doi:10.1002/anie.201903815

Cited by 15 publications

(19 citation statements)

References 29 publications

(26 reference statements)

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“…To gain a better understanding of the structural implications of the observed chemical shift trends, we performed MD simulations of AcpP carrying every biological chain length of E. coli FAB, except for the C8 simulation which we've previously reported (25) and further analyzed here. Five independent simulations of 500 ns for each acyl chain length were carried out, all modeled from the crystal structure of heptanoyl-AcpP (Protein Data Bank [PDB] ID code 2FAD) (20).…”

Section: Molecular Dynamics Simulations Reveal Structural Signatures and Correlated Motionsmentioning

confidence: 99%

Decoding allosteric regulation by the acyl carrier protein

Sztain

Bartholow

et al. 2021

Proc. Natl. Acad. Sci. U.S.A.

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Enzymes in multistep metabolic pathways utilize an array of regulatory mechanisms to maintain a delicate homeostasis [K. Magnuson, S. Jackowski, C. O. Rock, J. E. Cronan, Jr, Microbiol. Rev. 57, 522–542 (1993)]. Carrier proteins in particular play an essential role in shuttling substrates between appropriate enzymes in metabolic pathways. Although hypothesized [E. Płoskoń et al., Chem. Biol. 17, 776–785 (2010)], allosteric regulation of substrate delivery has never before been demonstrated for any acyl carrier protein (ACP)-dependent pathway. Studying these mechanisms has remained challenging due to the transient and dynamic nature of protein–protein interactions, the vast diversity of substrates, and substrate instability [K. Finzel, D. J. Lee, M. D. Burkart, ChemBioChem 16, 528–547 (2015)]. Here we demonstrate a unique communication mechanism between the ACP and partner enzymes using solution NMR spectroscopy and molecular dynamics to elucidate allostery that is dependent on fatty acid chain length. We demonstrate that partner enzymes can allosterically distinguish between chain lengths via protein–protein interactions as structural features of substrate sequestration are translated from within the ACP four-helical bundle to the protein surface, without the need for stochastic chain flipping. These results illuminate details of cargo communication by the ACP that can serve as a foundation for engineering carrier protein-dependent pathways for specific, desired products.

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Section: Molecular Dynamics Simulations Reveal Structural Signatures and Correlated Motionsmentioning

confidence: 99%

Decoding allosteric regulation by the acyl carrier protein

Sztain

Bartholow

et al. 2021

Proc. Natl. Acad. Sci. U.S.A.

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“…To function as a carrier for acyl groups, an apo Tm-ACP should be converted to the holo form, where the phosphopantetheine linker is covalently connected to the side chain of S40 in the conserved DSL motif [12][13][14]. Both tertiary structures of the holo and apo forms of Tm-ACP were determined by NMR spectroscopy and X-ray crystallography, respectively (see associated statistics in Table 2; Table 3).…”

Section: Tertiary Structure Of Tm-acpmentioning

confidence: 99%

“…The overall completeness of assignment was 93% and the quality factor (Q) of residual dipolar coupling (RDC) data was calculated as 39.52% using Q = rms(RDC measured -RDC calculated )/rms(RDC measured ) [36]. Tm-ACP consists of four α-helices (helix I (4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19), helix II (40)(41)(42)(43)(44)(45)(46)(47)(48)(49)(50)(51)(52)(53)(54), helix III (60-65), helix IV (69-80)) connected by a long α 1 α 2 loop and two shorter loops. To accommodate the acyl chains, the hydrophobic cavities of ACPs can be expanded by protruding helix III outward [1,28,37].…”

Section: Tertiary Structure Of Tm-acpmentioning

confidence: 99%

“…ACPs share highly conserved structures, including Asp-Ser-Leu (DSL) motifs at the N-terminus of helix II. A phosphopantetheine group is attached to the Ser residue (purple box in Figure 1a) by a phosphodiester linkage, and the free thiol group at the other end of the phosphopantetheine group can form a thioester bond with acyl groups [11][12][13][14]. The key structural features of various type II ACPs have been reported [15][16][17][18][19][20][21][22][23].…”

Section: Introductionmentioning

confidence: 99%

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Structural Characterization of an ACP from Thermotoga maritima: Insights into Hyperthermal Adaptation

Lee

Jang

Jeong

et al. 2020

IJMS

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Thermotoga maritima, a deep-branching hyperthermophilic bacterium, expresses an extraordinarily stable Thermotoga maritima acyl carrier protein (Tm-ACP) that functions as a carrier in the fatty acid synthesis system at near-boiling aqueous environments. Here, to understand the hyperthermal adaptation of Tm-ACP, we investigated the structure and dynamics of Tm-ACP by nuclear magnetic resonance (NMR) spectroscopy. The melting temperature of Tm-ACP (101.4 • C) far exceeds that of other ACPs, owing to extensive ionic interactions and tight hydrophobic packing. The D59 residue, which replaces Pro/Ser of other ACPs, mediates ionic clustering between helices III and IV. This creates a wide pocket entrance to facilitate the accommodation of long acyl chains required for hyperthermal adaptation of the T. maritima cell membrane. Tm-ACP is revealed to be the first ACP that harbor an amide proton hyperprotected against hydrogen/deuterium exchange for I15. The hydrophobic interactions mediated by I15 appear to be the key driving forces of the global folding process of Tm-ACP. Our findings provide insights into the structural basis of the hyperthermal adaptation of ACP, which might have allowed T. maritima to survive in hot ancient oceans. 1 Thermotoga maritima ACP; 2 Pseudothermotoga thermarum ACP; 3 Thermus aquaticus ACP; 4 Enterococcus faecalis ACP; 5 Brucella melitenis ACP; 6 Escherichia coli ACP; 7 Vibrio harveyi ACP; 8 The isoelectric points (pI) of bacterial ACPs were calculated by ProtParam tool (https://web.expasy.org/protparam) [35].

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“…maleimidocaproyl conjugation, Diels–Alder ligation) have been used to generate some S‐palmitoylated protein analogues, the products contained non‐native structural motifs . To date, there has been no study on the preparation of native S‐palmitoylated membrane proteins by chemical ligation methods …”

Section: Introductionmentioning

confidence: 99%

Chemical Synthesis of Native S‐Palmitoylated Membrane Proteins through Removable‐Backbone‐Modification‐Assisted Ser/Thr Ligation

et al. 2020

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The preparation of native S‐palmitoylated (S‐palm) membrane proteins is one of the unsolved challenges in chemical protein synthesis. Herein, we report the first chemical synthesis of S‐palm membrane proteins by removable‐backbone‐modification‐assisted Ser/Thr ligation (RBMGABA‐assisted STL). This method involves two critical steps: 1) synthesis of S‐palm peptides by a new γ‐aminobutyric acid based RBM (RBMGABA) strategy, and 2) ligation of the S‐palm RBM‐modified peptides to give the desired S‐palm product by the STL method. The utility of the RBMGABA‐assisted STL method was demonstrated by the synthesis of rabbit S‐palm sarcolipin (SLN) and S‐palm matrix‐2 (M2) ion channel. The synthesis of S‐palm membrane proteins highlights the importance of developing non‐NCL methods for chemical protein synthesis.

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Modifying the Thioester Linkage Affects the Structure of the Acyl Carrier Protein

Cited by 15 publications

References 29 publications

Decoding allosteric regulation by the acyl carrier protein

Decoding allosteric regulation by the acyl carrier protein

Structural Characterization of an ACP from Thermotoga maritima: Insights into Hyperthermal Adaptation

Chemical Synthesis of Native S‐Palmitoylated Membrane Proteins through Removable‐Backbone‐Modification‐Assisted Ser/Thr Ligation

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