Erik Zavala, Stephen Dansereau, Michael J Burke, James M Lipchock, Federica Maschietto, Victor Batista, J Patrick Loria
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引用次数: 0
摘要
PHPT1 是一种组氨酸磷酸酶,通过其催化活性调节真核生物的信号转导。在这里,我们结合溶液核磁共振、分子动力学和生化实验,对 PHPT1 的结构和动力学进行了分析。我们确定了 R78 胍基与 C 端 Y125 上的羧基之间形成的盐桥,它对配体的结合至关重要。通过在 C 端添加一个甘氨酸残基(G126)来破坏该盐桥,会导致催化活性降低,与伪底物对硝基苯磷酸(pNPP)以及活性位点抑制剂苯膦酸(PPA)的结合亲和力下降。我们通过核磁共振化学位移、15N 驰豫测量和分子动力学轨迹分析表明,移除这种盐桥会导致活性位点的结构和动力学发生改变,从而对酶的功能产生重大影响,并证实了这种静电相互作用的重要性。
A salt bridge of the C-terminal carboxyl group regulates PHPT1 substrate affinity and catalytic activity.
PHPT1 is a histidine phosphatase that modulates signaling in eukaryotes through its catalytic activity. Here, we present an analysis of the structure and dynamics of PHPT1 through a combination of solution NMR, molecular dynamics, and biochemical experiments. We identify a salt bridge formed between the R78 guanidinium moiety and the C-terminal carboxyl group on Y125 that is critical for ligand binding. Disruption of the salt bridge by appending a glycine residue at the C-terminus (G126) leads to a decrease in catalytic activity and binding affinity for the pseudo substrate, para-nitrophenylphosphate (pNPP), as well as the active site inhibitor, phenylphosphonic acid (PPA). We show through NMR chemical shift, 15N relaxation measurements, and analysis of molecular dynamics trajectories, that removal of this salt bridge results in an active site that is altered both structurally and dynamically thereby significantly impacting enzymatic function and confirming the importance of this electrostatic interaction.
期刊介绍:
Protein Science, the flagship journal of The Protein Society, is a publication that focuses on advancing fundamental knowledge in the field of protein molecules. The journal welcomes original reports and review articles that contribute to our understanding of protein function, structure, folding, design, and evolution.
Additionally, Protein Science encourages papers that explore the applications of protein science in various areas such as therapeutics, protein-based biomaterials, bionanotechnology, synthetic biology, and bioelectronics.
The journal accepts manuscript submissions in any suitable format for review, with the requirement of converting the manuscript to journal-style format only upon acceptance for publication.
Protein Science is indexed and abstracted in numerous databases, including the Agricultural & Environmental Science Database (ProQuest), Biological Science Database (ProQuest), CAS: Chemical Abstracts Service (ACS), Embase (Elsevier), Health & Medical Collection (ProQuest), Health Research Premium Collection (ProQuest), Materials Science & Engineering Database (ProQuest), MEDLINE/PubMed (NLM), Natural Science Collection (ProQuest), and SciTech Premium Collection (ProQuest).