NMR Lab, National Institute of Immunology, New Delhi, India.
NMR Lab, National Institute of Immunology, New Delhi, India.
J Biol Chem. 2021 Dec;297(6):101394. doi: 10.1016/j.jbc.2021.101394. Epub 2021 Nov 9.
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 GXGXG 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 C- 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 GXGXG motif in regulating acyl chain sequestration, vital for ScACP function.
酿酒酵母酰基辅酶 A 载体蛋白（ScACP）是大型真菌脂肪酸合酶 I（FAS I）复合物的组成部分。ScACP 由两个亚结构域组成：一个保守的 ACP 结构域，与其他 ACP 具有广泛的结构同源性，以及一个独特的结构域。与不隔离酰基链的后生动物 I 型 ACP 不同，ScACP 可以通过一种仍不明确的机制，将生长中的酰基链部分隔离在其疏水性核心内。我们对酰基-ScACP 中间体的研究揭示了螺旋 II 中一个独特的 GXGXG 序列对于 ACP 功能的重要性。当该序列中的三个甘氨酸突变为缬氨酸（G188V/G191V/G195V）时，完全失去了隔离作用，而 G191V 和 G188V/G191V 双突变体显示出更快的酰基链水解速率。同样，将 Thr216 突变为 Ala 改变了疏水性腔的大小，导致 C-链隔离的丧失。结合 NMR 研究和晶体结构的见解，我们表明，螺旋 II 中的三个甘氨酸和螺旋 IV 中的一个苏氨酸有利于构象变化，这反过来又为酰基链隔离产生了空间。此外，我们确定了 ScACP 的主要疏水性腔，位于螺旋 II 和 IV 的羧基端之间。腔的开口位于螺旋 II 的第二和第三圈之间以及环 II 之间。总的来说，该研究强调了 GXGXG 基序在调节酰基链隔离中的新作用，这对 ScACP 的功能至关重要。
