探討帶有N環的蛋白質酪胺酸磷酸酶: 雙特異性磷酸酶22中形成活性位點的結構見解

The Study of N-loop-containing Protein Tyrosine Phosphatase: Structural Insights into the Active Site Formation of DUSP22

賴治暄(Chih-Hsuan Lai)

指導教授 ： 呂平江

國立清華大學/生命科學院/生物資訊與結構生物研究所/博士(2021年)

摘要

蛋白質酪胺酸磷酸酶 (PTPs)是一種調控細胞內磷酸化路徑的重要酵素，其中多數成員被歸類為基於半胱胺酸進行催化的蛋白質酪胺酸磷酸酶家族 (Cys-based PTPs)。這些Cys-based PTPs與激酶 (kinase) 共同調控細胞的生理活性，其中包含了細胞生長、細胞凋亡、以及免疫反應等。這個家族主要有兩個族群，分別為經典型蛋白質酪胺酸磷酸酶 (classical PTPs)和雙特異性磷酸酶 (DUSPs)。在過去的研究中，classical PTPs被深入的研究，並描述了催化位中的保守環和氫鍵網路對催化反應的重要性。Classical PTPs的活性位由三個保守的環和結構水形成，分別為D環、P環、以及Q環，並生成一個水介導的氫鍵網路。然而，DUSPs中具有複雜的族群分類，且大部分成員的活性位結構都與classical PTPs產生了差異並缺乏探討。為了能更精確的研究DUSPs的活性位結構，本篇研究找出了其中數量最多的活性位結構並發現這些PTPs都具有N環作為結構特徵。在DUSP族群中，這些帶有N環的蛋白質酪胺酸磷酸酶 (N-loop-containing PTPs)佔有45%的成員，並以D環、P環、以及N環組成活性位結構。不同於classical PTPs的地方在於這些環上各自具有一個高保留的胺基酸使用側鏈及主鏈醯胺鍵直接相互生成氫鍵網路，我們將其稱為D-, P-, N-三環交互作用 (DPN-triloop interaction)。
在本篇研究中，DUSP22被作為研究模型來探討DPN-triloop interaction在活性位結構生成的重要性與是否影響催化活性。DUSP22中生成DPN-triloop interaction的胺基酸分別為D環上的D57、P環上的S93、和N環上的N128。D57是一個參與催化反應的胺基酸，但S93和N128的功能未知。這些胺基酸分別以丙胺酸及體細胞突變來替換以進行結構生物學和酵素動力學上的研究。在水溶性結構中，以核磁共振 (NMR)的1H-, 15N-二維異核單量子關聯圖譜 (1H-, 15N-HSQC)觀測發現當對D57、S93、和N128任一點進行突變的時候會連帶的擾動對向參與交互作用的環的構型，並且擾動周圍二級結構上的胺基酸。晶體結構中進一步顯示了S93和N128的突變會造成D環結構變化，而位於D環上的D57則會離開催化位。在酵素動力學的研究中證實，大部分的丙胺酸突變和體細胞突變都會造成催化效率產生102倍以上的下降，顯示出氫鍵網路對活性位生成的重要性。這些結果指出了DPN-triloop interaction為一個生成活性位點的重要構型，並意味著擾動了任何一個環的結構都可能會通過DPN-triloop interaction影響磷酸酶的酵素活性。我們將P127突變為白胺酸來驗證這個理論，結果在1H-, 15N-HSQC圖譜中觀測到DUSP22整體產生明顯的結構變化，並且發現P127L能對催化效率造成近103倍的下降。我們的研究顯示了DPN-triloop interaction穩定了活性位的構型，並將其中的胺基酸置於適合結合受質以進行催化的位置。而這個在N-loop-containing PTPs中的保守氫鍵網路可以在進一步的研究中做為為癌症研究的生物標記，並且也能作為調控磷酸酶活性的目標來研究異位調控 (allosteric regulation)的可能機制。

關鍵字

蛋白質酪胺酸磷酸酶 ； 雙特異性磷酸酶 ； 活性位 ； 氫鍵網路 ； N環 ； 雙特異性磷酸酶22

並列摘要

Protein tyrosine phosphatases (PTPs) play crucial role to hydrolyze the phosphorylated substrates and regulate cell function. More than half of PTPs are belonged to Cysteine-based PTPs (Cys-based PTPs) which use cysteine to catalyze the substrates. Cys-based PTPs cooperate with kinases to regulate phosphorylation signaling pathway in cellular responses, such as cell differentiation, cell proliferation, and immune response. There are two major subfamilies in Cys-based PTPs: Classical PTPs and dual specificity phosphatases (DUSPs). Classical PTPs have been clearly investigated in previous studies, which describe the relationship between conserved loops and hydrogen bonding network in the active site. The active site structures of classical PTPs is consisted of the D-loop, P-loop, Q-loop, and structural water, and it forms a water-mediated hydrogen bonding network. However, DUSPs form the different active site structures from classical PTPs and the forming mechanism is still unclear. To investigate the active site of DUSPs, the conserved structure is searched in this study and we find that the active site structure contains the N-loop is conserved in 45% of DUSPs. The active site structures in N-loop-containing PTPs consist of the D-loop, P-loop, and N-loop. The side chain and backbone amide of conserved residues in each loop directly form a hydrogen bonding network to connect the three loops, and this configuration is described as the D-, P-, N-triloop interaction (DPN-triloop interaction) in this study.
DUSP22 is used as a model system to study whether the DPN-triloop interaction plays crucial role in active site formation. The DPN-triloop interaction in DUSP22 is formed by D57, S93, and N128. D57 participates in catalytic reaction, while the S93 and N128 have unknown effect. These residues are replaced with alanine and somatic mutation to investigate whether the DPN-triloop interaction can affect protein structure and kinetic activity. In solution structure, the 1H-, 15N-HSQC spectra of D57, S93, and N128 mutations indicates that mutation on one residue can perturb the conformation of three loops and the residues in connected secondary structures. The crystal structures show that S93 and N128 mutations induce the conformational change of D-loop, and the catalytic aspartate, D57, leave the catalytic site. In kinetic studies, the mutants indicate that disruption of the DPN-triloop interaction decrease the catalytic efficiency by 102 times. These results reveal that the DPN-triloop interaction is crucial for the active site formation, and perturbing the conformation of one loop can decrease the phosphatase activity through disrupting the DPN-triloop interaction. To verify this theory, the conserved proline in N-loop is mutated to leucine. The conformational change of P127L is observed in 1H-, 15N-HSQC spectrum and declines the catalytic efficiency by nearly 103 times. Our study reveals that the DPN-triloop interaction can stabilize the active site structure and align the active site residues in catalytic favorable site. The DPN-triloop interaction can become the potential biomarkers in further study of cancer development and provide the mechanism for regulating the phosphatase activity through allosteric site.

並列關鍵字

Protein tyrosine phosphatases ； DUSPs ； Active site ； Hydrogen bonding network ； N-loop ； DUSP22

參考文獻

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