A GX2GX3G motif facilitates acyl chain sequestration by Saccharomyces cerevisiae acyl carrier protein

Garima Garima,Rashima Prem,Usha Yadav,Monica Sundd

doi:10.1016/j.jbc.2021.101394

Garima Garima, Rashima Prem + Show 2 more

Open Access

https://doi.org/10.1016/j.jbc.2021.101394

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Abstract

Saccharomyces cerevisiae acyl carrier protein (ScACP) is a component of the large fungal fatty acid synthase I (FAS I) complex. ScACP comprises two subdomains: a conserved ACP domain that shares extensive structural homology with other ACPs and a unique structural domain. Unlike the metazoan type I ACP that does not sequester the acyl chain, ScACP can partially sequester the growing acyl chain within its hydrophobic core by a mechanism that remains elusive. Our studies on the acyl-ScACP intermediates disclose a unique 188GX2GX3G195 sequence in helix II important for ACP function. Complete loss of sequestration was observed upon mutation of the three glycines in this sequence to valine (G188V/G191V/G195V), while G191V and G188V/G191V double mutants displayed a faster rate of acyl chain hydrolysis. Likewise, mutation of Thr216 to Ala altered the size of the hydrophobic cavity, resulting in loss of C12- chain sequestration. Combining NMR studies with insights from the crystal structure, we show that three glycines in helix II and a threonine in helix IV favor conformational change, which in turn generate space for acyl chain sequestration. Furthermore, we identified the primary hydrophobic cavity of ScACP, present between the carboxyl end of helix II and IV. The opening of the cavity lies between the second and third turns of helix II and loop II. Overall, the study highlights a novel role of the GX2GX3G motif in regulating acyl chain sequestration, vital for ScACP function.

Highlights

Six β-subunits, three on either side of a central disc formed by six α-subunits [1, 2]
Saccharomyces cerevisiae acyl carrier protein (ScACP) helix III displays a turn between Lys 200 and Thr 204, i + 4 residues apart in the solution structure (PDB 2ML8) [16]
The conserved DSL motif of most type I and II Acyl carrier protein (ACP) is substituted with a KST sequence in ScACP

Summary

Results

S. cerevisiae acyl carrier protein (ScACP) comprises two sub domains, (a) a canonical ACP domain (Ala138-Gly219) homologous to most type I and type II ACPs, and (b) a structural domain (Ala220-Leu302) found in fungi and a few bacterial species. In C12-ScACP, a noticeable change was observed (−88 and 11), suggesting a 180 flip of the Gly 195 backbone These TALOS+ predictions were substantiated by ScACP crystal (2UV8, 6QL9) and cryoEM structure (6U5U), where Gly 195 and other loop II residues display a remarkably different conformation (Fig. 4B). A remarkable decrease in the magnitude of chemical shift change was observed in C12-G191VScACP (single mutant), compared with C12-wild type ScACP. Cavity #5 formed by Leu 187, Leu 190, Gly 191, Gly 195-Pro 198, Pro 201, Leu 209, and Phe 213 is in compliance with our C8-ScACP chemical shift perturbation data and glycine mutagenesis studies The opening of this cavity lies between helix II and loop II.

B C12-G191VScACP

Discussion

Cavity 4

Experimental procedures