The Stories Tryptophans Tell: Exploring Protein Dynamics of Heptosyltransferase I from Escherichia coli
The Stories Tryptophans Tell: Exploring Protein Dynamics of Heptosyltransferase I from Escherichia coli
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DOI：
10.1021/acs.biochem.6b00850
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发表时间：
2017-02-14
期刊：
影响因子：
2.9
通讯作者：
Taylor, Erika A.
中科院分区：
文献类型：
--
作者：
Cote, Joy M.;Ramirez-Mondragon, Carlos A.;Taylor, Erika A.
作为脂多糖核心区生物合成的一部分，糖基转移酶I催化L-甘油-β-甘露-庚糖与Kdo(2)-Lipid A的加成反应。带有HEPI基因敲除的革兰氏阴性细菌降低了毒力，并增强了对疏水抗生素的敏感性，使HEPI抑制剂的设计引起了人们的兴趣。由于HEPI蛋白动力学部分是限速的，破坏蛋白质动力学可能为抑制HEPI提供新的策略。弄清HEPI的全球机制有望帮助开发内毒素生物合成的抑制剂。在这里，通过结合突变、内源性色氨酸荧光和圆二色谱分析，探讨了参与HEPI催化循环的动态蛋白质重排。利用野生型和突变型HEPI，通过Trp荧光的变化确定了多个动态区。有趣的是，当ODLA与N-末端结构域结合时，C-末端结构域(与ADP-庚糖结合)的色氨酸残基(Trp199和Trp217)处于更疏水性的环境中。这些残基与ADP-庚糖的结合位点相邻(范德华位点上的Trp217与ADP-庚糖腺嘌呤环接触)，这表明这两个结合位点相互作用报告了酶的占据状态。ODLA结合还伴随着HEPI的显著稳定(在ODLA存在的情况下，加热到95℃不能使蛋白质变性)。这些结果表明，底物与HEPI结合的诱导FIT模型的构象重排对催化很重要，这些构象动力学的破坏可能是抑制这种糖基转移酶和其他糖基转移酶的新机制。
Heptosyltransferase I (HepI) catalyzes the addition of L-glycero-beta-Dmanno-heptose to Kdo(2)-Lipid A, as part of the biosynthesis of the core region of lipopolysaccharide (LPS). Gram-negative bacteria with gene knockouts of HepI have reduced virulence and enhanced susceptibility to hydrophobic antibiotics, making the design of inhibitors of HepI of interest. Because HepI protein dynamics are partially rate limiting, disruption of protein dynamics might provide a new strategy for inhibiting HepI. Discerning the global mechanism of HepI is anticipated to aid development of inhibitors of LPS biosynthesis. Herein, dynamic protein rearrangements involved in the HepI catalytic cycle were probed by combining mutagenesis with intrinsic tryptophan fluorescence and circular dichroism analyses. Using wild-type and mutant forms of HepI, multiple dynamic regions were identified via changes in Trp fluorescence. Interestingly, Trp residues (Trp199 and Trp217) in the C-terminal domain (which binds ADP-heptose) are in a more hydrophobic environment upon binding of ODLA to the N-terminal domain. These residues are adjacent to the ADP-heptose binding site (with Trp217 in van der Waals contact with the adenine ring of ADP-heptose), suggesting that the two binding sites interact to report on the occupancy state of the enzyme. ODLA binding was also accompanied by a significant stabilization of HepI (heating to 95 degrees C fails to denature the protein when it is in the presence of ODLA). These results suggest that conformational rearrangements, from an induced fit model of substrate binding to HepI, are important for catalysis, and the disruption of these conformational dynamics may serve as a novel mechanism for inhibiting this and other glycosyltransferase enzymes.