Detecting Transient Binding in HATH Domain of HDGF by NMR Paramagnetic Relaxation Enhancement

林尚錡

指導教授 ： 蘇士哲

國立清華大學/生命科學院/生物資訊與結構生物研究所/碩士(2010年)

摘要

A new protein growth factor, Hepatoma-derived growth factor (HDGF), was discovered from medium of hepatoma cell line and was identified in a broad range of tissues. Exogenously supplied HDGF enables to enter cells and be transported into nucleus. The process further promotes mitogenic process and enhances cell proliferation. The N-terminal conserved HATH domain is provided to be responsible for HDGF internalization through binding to membrane surface heparan sulfate (HS). There are well-known examples of dimerized growth factors with active function, i.e. fibroblast growth factor, thus we are interested in identifying the intermolecular interaction between HATH domains under the HS-presence or absence conditions. In the study, we examined the interaction by adopting paramagnetic relaxation enhancement (PRE) method. It provides a straightforward measurement to determine the distance between the paramagnetic nitroxide center and the affected nuclei by site-directed spin labeling on cysteine residue (Cys12). PRE profile established from 1H-15N HSQC measurements was not fully equal to the theoretically calculated PRE result derived from the determined structural coordinate. Differences happen in regions of N- and C-terminus, loop2 and loop3, where higher relaxation enhancement was observed in experiment. Examination from the sample mixture of 15N-labeled HATH and nitroxide spin labeling HATH dissected the effect derived from intermolecular interaction, where significant intermolecular PRE effect is observed in the regions of conflict, such as N- and C-terminus, loop2 and loop3, indicating domain contact through the particular areas. Meanwhile the presence of heparin polysaccharide even enhanced the PRE effect. Our result implies the possibility of presence of transient interaction between HATHs in solution, although no biochemical study ever reported a stable dimer. Considering the result of heparin fragment longer than 10-12 residues, twice the size of a heparin-binding site in one HATH domain, effectively blocking the HATH mediated internalization, the domain transient binding might regulate HATH dimer formation for HS binding and, in addition, manipulate the domain orientation lining on a polysaccharide chain.

關鍵字

肝癌衍生生長因子 ； 電子順磁的弛緩增強 ； 核磁共振 ； 瞬間作用

並列摘要

肝癌衍生生長因子 (HDGF) 從肝癌細胞株的培養液被發現而且已在許多組織中被發現。細胞外提供的 HDGF 能夠被胞吞進入細胞質中，且可被運送到細胞核內進一步促進細胞的增生，位於蛋白質 N 端的 HATH domain 能藉由結合細胞膜上的硫酸乙酰肝素 (heparan sulfate) 來進行胞吞作用，使得 HDGF 能夠進入細胞。先前已經知道許多生長因子能以形成二聚體的方式作用，例如: 纖維細胞生長因子 (FGF)，因此我們研究在 HATH domain 在水溶液下是否具有形成二聚體的能力。在研究中我們使用電子順磁的弛緩增強 (PRE) 方法來觀測這個作用。藉由在原有的半胱氨酸 12 的位置接上電子順磁分子 (spin label)，來測量順磁中心及觀察原子核間的距離。結果發現，在 1H-15N HSQC 光譜下的 PRE 實驗，計算出的結果不完全與理論計算的結果一致，其中 PRE 的效應在N端、C端、loop2 和 loop3 的區域，實驗值明顯高於理論值。在實驗利用混合15N-labeled HATH 和 nitroxide spin labeling HATH 可以進一步區分出分子間的作用。藉由此實驗，我們瞭解實驗值與理論值不吻合的原因來自 HATH domain 具有分子間的作用，同時肝素分子會進一步加強了這個 PRE 效應。雖然目前沒有生化方法測量到穩定的二聚體，但從較靈敏的 PRE 計算，我們的結果顯示水溶液中HATH domain 具有瞬間作用的能力。目前已知道 heparin 片段超過10-12 residues， 能有效的阻止 HATH 進入細胞的作用，這個大小剛好是 HATH domain Heparin 結合區的兩倍大小，這個 HATH 分子間結合的能力指出了與 heparin 結合時形成二聚體的可能。

並列關鍵字

hHDGF ； PRE ； NMR ； transient binding

參考文獻

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