肌動蛋白結合蛋白L-plastin之功能與結構探討

Abstract

L-plastin是在物種間具有高度保留性的肌動蛋白結合蛋白，能夠使肌動蛋白絲相互交聯形成微絲束。L-plastin主要穩定表達於正常的血細胞以及大多數的癌細胞中，與許多免疫細胞之功能以及惡性腫瘤的發展進程息息相關；L-plastin的微絲束形成活性會受到鈣離子負向的調控，而根據其N端鈣離子結合區的核磁共振結構，目前認為當結構中的EF-hand motifs與鈣離子結合時，就能夠使L-plastin N端與C端之間連接序列中的CaM-binding motif（CBM）序列，從較無構型的型態轉變為穩定的α螺旋結構，進而與N端的EF-hand結構域交互作用，使接收的鈣離子訊號能夠傳遞至肌動蛋白結合功能區，進而調控蛋白形成微絲束之活性；有無鈣離子結合的L-plastin N端結構已經通過核磁共振（Nuclear Magnetic Resonance）被解出來，然而全長的L-plastin蛋白結構至今仍未被發表，因此即使已有了許多相關的研究，L-plastin N端之鈣離子結合功能區與C端之肌動蛋白結合功能區具體交互作用的機制仍有待探討。而為了更加了解L-plastin受鈣離子調控的機制，我們首先利用大腸桿菌表達L-plastin之重組蛋白，透過固定化金屬離子管柱層析以及膠體過濾層析等方式純化之後，將重組蛋白用於後續結構與功能特性上的分析。從純化的圖譜中發現，L-plastin會以單體及雙聚體的型態穩定存在，且兩者間在一周內並不會出現互相轉換的情形，而活性測試與生物物理學方法的測定則顯示，L-plastin之單體的結構更為穩定且具有較佳的微絲束形成活性；等溫滴定量熱法的結果顯示，L-plastin在20°C進行鈣離子滴定試驗時是呈現放熱反應，且單體和雙聚體與鈣離子結合之KD值大約是在μM的等級；為了進一步觀察由鈣離子所介導的結構變化，我們利用L-plastin EF hand與全長蛋白進行了核磁共振的實驗，與預期結果相同，加入鈣離子之後，有許多L-plastin N端EF hand區域之交叉峰訊號出現明顯化學位移的情形，然而鈣離子對於L-plastin C端結構的影響，則需要更進一步的定序分析去證實；除了核磁共振實驗之外，我們亦嘗試透過X光射線蛋白質晶體繞射分析全長L-plastin結構，雖然已成功培養出L-plastin晶體，但目前還無法成功計算出L-plastin之完整結構。綜上所述，本研究透過許多生物物理之方式，進一步探討了L-plastin之功能與其鈣離子調控之機制，希望能夠為未來可能發展之針對L-plastin所進行的藥物設計提供更多的資訊。

L-plastin is an actin-bundling protein, which is expressed in hematopoietic cell lineages and many types of cancer cells. It is critical to many immune cell functions and the invasion and metastasis of cancer cells. The actin-bundling activity of L-plastin is known to be calcium-dependent, where increasing calcium concentrations inhibit the formation of actin-bundles. L-plastin is characterized by EF-hand calcium binding domains and actin-binding domains. A recent study has shown that the CaM-binding motif （CBM）, a part of the linker between the calcium binding domains and actin-binding domains, may be responsible for conveying the calcium signal from the EF-hands to the actin-binding core. The solution structures of calcium-free EF and calcium-bound EF-H5 are determined by NMR （Nuclear Magnetic Resonance） spectroscopy while the structure of full length L-plastin is still remained unclear. To understand how actin-bundling activity the L-plastin is regulated by altering calcium concentration, we use a combination of biochemical and biophysical approaches to deeply investigate the interaction between L-plastin and calcium. The recombinant L-plastin can be expressed in E. coli and purified by immobilized metal affinity chromatography and gel filtration chromatography. Based on the data from size-exclusion chromatography, we find that the recombinant proteins can be monomeric and dimeric forms and a dynamic monomer-dimer equilibrium cannot be detected in a week. According to the functional assay and the biophysical experiments, the monomeric form of L-plastin shows better stability and actin-bundling activity. The ITC results indicate that the interaction between calcium and L-plastin is an exothermic reaction with two binding site events. In order to investigate the structural changes mediated by calcium binding, we perform titration experiments for the EF and full-length constructs. As expected, addition of calcium results in significant chemical shift perturbation in EF-hand domains but the changes of the four C-domains are still elusive without achievements of backbone assignments. We also attempt to solve the full-length structure of L-plastin by X-ray crystallography. Although L-plastin crystals have been produced, the complete structure of L-plastin cannot be determine successfully. Taken together, our findings provide evidences for the function of L-plastin and the mechanism of calcium regulation using biophysical methods, and hope to help the possible drug design for L-plastin in the future.