組合蛋白(integrins)是一群由a及b次單位所組合而成的穿膜蛋白受體，它們在生理上所參與的功能，包括：細胞之間的黏合、細胞的遷移以及訊息的傳遞...等等。很多源自於體內或非源自於體內的配體，大多使用RGD(Arg-Gly-Asp)、LDV(Leu-Asp-Val)或是其他一小段的胺基酸序列，以做為和組合蛋白結合的位置。去組合蛋白(disintegrins)是從蛇毒毒液中所得到的小蛋白，通常含有RGD的胺基酸序列並富含有胺基酸Cys。去組合蛋白會和組合蛋白的b1及b3次單位結合。Rhodostomin(簡稱Rho)是個容易和血小板上的組合蛋白aIIbb3結合，而抑制血小板凝集的去組合蛋白；它擁有68個胺基酸，在其胺基酸位置48-53的PRGDMP序列是它的活性功能區間。由過去細胞黏合及血小板凝集實驗的分析結果，我們發現當把胺基酸P48突變成胺基酸Ala後，其抑制組合蛋白a5b1的能力增加了4.4倍，而當把胺基酸D51突變成胺基酸Glu後，其抑制組合蛋白的能力下降了863倍，但是在NMR結構上的分析，並未發現這兩個突變蛋白與Rho有結構上的明顯差異。經由蛋白質骨架動力學的研究後，我們發現位於RGD功能區間的胺基酸R49及D51具有蛋白質動力學特質的差異。Rho的胺基酸R49及D51比突變蛋白P48A相同的胺基酸具有1.4及1.5倍高的R2遲緩參數，造成了model-free參數Rex及e的不同；Rho的胺基酸R49及D51之Rex值分別為1.39及2.94 s-1，相對的，突變蛋白P48A則不具Rex值。然而，Rho的胺基酸R49及D51之te值分別為0.11及0.11 ns，這相對於突變蛋白P48A的0.77及0.73 ns是較低的。由這樣子的結果，似乎指出：突變蛋白P48A的胺基酸R49及D51，因為具有較多的te值，而且不具有Rex值，因而使得P48A對於組合蛋白a5b1具有較好的抑制能力。我們也發現突變蛋白D51E胺基酸E51的R2遲緩參數比Rho高出1.13倍，使的其Rex值成為4.09 s-1，這比Rho胺基酸D51的Rex值高出了1.4倍；因此，胺基酸D51 Rex值的高低，對於Rho的功能活性似乎扮演重要角色。我們也使用NMR的方法，決定會和組合蛋白avb3專一性結合之突變蛋白G50L的立體結構；從結構上的分析結果，發現突變蛋白G50L除了L50的側鏈位向與Rho有較大的差異以外，G50L與Rho具有類似的立體摺疊。在決定突變蛋白G50L的蛋白質骨架動力學後，發現Rho胺基酸G50及突變蛋白G50L胺基酸L50的S2值，分別為0.63及0.75；而Rho胺基酸G50及突變蛋白G50L胺基酸L50之的te值，分別為0.81及0.07 ns。然而，突變蛋白G50L胺基酸L50具有0.48 s-1的Rex值，但Rho胺基酸G50不具有Rex值。Rho及突變蛋白G50L胺基酸D51的Rex值，則分別為2.94及1.61 s-1。從這些結果可以知道，在RXD功能區間的胺基酸R及D之蛋白質骨架動力學性質與構形，對於調控和組合蛋白結合的親和力及專一性扮演重要的角色。除此之外，為了探討去組合蛋白延長的C端對於去組合蛋白的結構與功能活性之相關性，我們表現純化並利用NMR的方法決定了三個Rho C端延長突變蛋白的立體結構，包括：P48A/M52D/P53M-NPHKGPAT (DM-NPHKGPAT), P48A/M52N-NRFH (NP-NRFH),以及P48A/M52W/P53N-NPWNG (WN-NPWNG)。經過NMR圖譜的分析，發現P48A/M52W/P53N-NPWNG (WN-NPWNG)這個C端延長突變蛋白的胺基酸D51會和C端胺基酸WNG有相互作用，這代表C端區域的胺基酸WNG，可能成為另一個辨識組合蛋白的位置。統整這些結果，我們推測：去組合蛋白RGD功能區間及C端區域胺基酸的蛋白質動力學特質及構形，對於和組合蛋白的結合扮演了重要角色。

Integrins are a/b heterodimeric receptors that are involved in cell adhesion, mobility, and signal transduction. Many physiological and non-physiological integrin ligands utilize RGD, LDV, or a short sequence as a key structural component for their integrin-binding site. Disintegrins are a family of RGD-containing and cysteine-rich proteins isolated from snake venoms. They interact with the b1 and b3 families of integrins. Rhodostomin (Rho) is a disintegrin that inhibits the platelet aggregation by blocking integrin aIIbb3 of platelets. Rho consists of 68 amino acids with a PRGDMP sequence at positions of 48-53. Functional analysis of Rho and its mutants using cell adhesion and platelet aggregation assays, we found that the mutation of P48 to A caused a 4-fold increase in its activity to integrin a5b1; however, the mutation of D51 to E caused an 863-fold decrease in activity. Interestingly, our NMR structural analysis showed that there is no structural difference in their 3D structures. In contrast, we found dynamic differences in the R and D residues of their RGD motif. The values of R2 relaxation parameter for the residues R49 and D51 of Rho were 1.4- and 1.5-folds higher than those of the P48A mutant, resulting in the differences in Rex and te. The Rex values of R49 and D51 residues of Rho were 1.39 and 2.94 s-1, respectively. In contrast, no Rex values were obtained for P48A mutant. The te values of R49 and D51 residues of Rho were 0.11 and 0.11 ns, respectively. Their values were less than those of P48A mutant, which were 0.77 and 0.73 ns, respectively. These findings indicate that high te and no Rex of the P48A mutant result in its high activity in integrin a5b1. We also found that the R2 value of the E51 residue of the D51E mutant was 1.13-fold higher than that of Rho. The resulting Rex value of the E51 residue was 4.09 s-1, which is 1.4-fold higher than that of the D51 residue in Rho. Our finding also indicates that low Rex of D51 residue in Rho plays an important role in its activity. In structural study, we determined 3D structure of the G50L mutant, an integrin avb3-specific mutant, by NMR spectroscopy. Structural analysis of the G50L mutant showed that it has the same tertiary fold as Rho, except for the chemical property difference between G50 and L50. Our dynamics analysis showed that the S2 values of G50 in Rho and L50 in the G50L were 0.63 and 0.75, respectively. The te values of G50 in Rho and L50 in the G50L were 0.81 and 0.07 ns, respectively. In contrast, the L50 residue of G50L mutant has the Rex values of 0.48 s-1, and no Rex was found in the G50 residue of Rho. The Rex values of the residue D51 of Rho and the G50L mutant were 2.94 and 1.61 s-1, respectively. In this study, we showed that the dynamic properties and conformation of R and D residues in RXD motif play an important role in modulating its activity and selectivity for integrins. In order to study the effects of the elongated C-terminal regions on structure and function relationships of disintegrins, we expressed and determined 3D structures of three C-terminal mutants of Rho, including P48A/M52D/P53M-NPHKGPAT (DM-NPHKGPAT), P48A/M52N-NRFH (NP-NRFH), and P48A/M52W/P53N-NPWNG (WN-NPWNG) by NMR spectroscopy. Structural analysis of the P48A/M52W/P53N-NPWNG (WN-NPWNG) mutant showed that the D51 residue interacted with the elongated C-terminal WNG sequence, suggesting that the C-terminal WNG tail may serve as an additional recognition site for integrin. In conclusion, our findings indicate that the dynamics properties and conformations of the RGD motif and C-terminal region in disintegrins play important roles in interacting with integrins.

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