An S S, Marti D N, Carreño C, Albericio F, Schaller J, Llinas M
Department of Chemistry, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213, USA.
Protein Sci. 1998 Sep;7(9):1947-59. doi: 10.1002/pro.5560070910.
The Glu1-Val79 N-terminal peptide (NTP) domain of human plasminogen (Pgn) is followed by a tandem array of five kringle (K) structures of approximately 9 kDa each. K1, K2, K4, and K5 contain each a lysine-binding site (LBS). Pgn was cleaved with CNBr and the Glul-HSer57 N-terminal fragment (CB-NTP) isolated. In addition, the Ile27-Ile56 peptide (L-NTP) that spans the doubly S-S bridged loop segment of NTP was synthesized. Pgn kringles were generated either by proteolytic fragmentation of Pgn (K4, K5) or via recombinant gene expression (rK1, rK2, and rK3). Interactions of CB-NTP with each of the Pgn kringles were monitored by 1H-NMR at 500 MHz and values for the equilibrium association constants (Ka) determined: rK1, Ka approximately 4.6 mM(-1); rK2, Ka approximately 3.3 mM(-1); K4, Ka approximately 6.2 mM-'; K5, K, 2.3 mM(-1). Thus, the lysine-binding kringles interact with CB-NTP more strongly than with Nalpha-acetyl-L-lysine methyl ester (Ka < 0.6 mM(-l), which reveals specificity for the NTP. In contrast, CB-NTP does not measurably interact with rK3. which is devoid of a LBS. CB-NTP and L-NTP 1H-NMR spectra were assigned and interproton distances estimated from 1H-1H Overhauser (NOESY) experiments. Structures of L-NTP and the Glul-Ile27 segment of CB-NTP were computed via restrained dynamic simulated annealing/energy minimization (SA/EM) protocols. Conformational models of CB-NTP were generated by joining the two (sub)structures followed by a round of constrained SA/EM. Helical turns are indicated for segments 6-9, 12-16, 28-30, and 45-48. Within the Cys34-Cys42 loop of L-NTP, the structure of the Glu-Glu-Asp-Glu-Glu39 segment appears to be relatively less defined, as is the case for the stretch containing Lys5O within the Cys42-Cys54 segment, consistent with the latter possibly interacting with kringle domains in intact Glul-Pgn. Overall, the CB-NTP and L-NTP fragments are of low regular secondary structure content-as indicated by UV-CD spectra- and exhibit fast amide 1H-2H exchange in 2H2O, suggestive of high flexibility.
人纤溶酶原（Pgn）的Glu1-Val79 N端肽（NTP）结构域之后是由五个kringle（K）结构组成的串联阵列，每个结构约9 kDa。K1、K2、K4和K5各自包含一个赖氨酸结合位点（LBS）。用溴化氰切割Pgn并分离出Glu1-HSer57 N端片段（CB-NTP）。此外，合成了跨越NTP双S-S桥连环段的Ile27-Ile56肽（L-NTP）。Pgn kringle结构域可通过Pgn的蛋白水解片段化产生（K4、K5），也可通过重组基因表达产生（rK1、rK2和rK3）。通过500 MHz的1H-NMR监测CB-NTP与每个Pgn kringle结构域的相互作用，并确定平衡缔合常数（Ka）的值：rK1，Ka约为4.6 mM-1；rK2，Ka约为3.3 mM-1；K4，Ka约为6.2 mM-1；K5，Ka约为2.3 mM-1。因此，赖氨酸结合kringle结构域与CB-NTP的相互作用比与Nα-乙酰-L-赖氨酸甲酯的相互作用更强（Ka < 0.6 mM-1），这揭示了对NTP的特异性。相比之下，CB-NTP与缺乏LBS的rK3没有可测量的相互作用。对CB-NTP和L-NTP的1H-NMR光谱进行了归属，并通过1H-1H Overhauser（NOESY）实验估计了质子间距离。通过受限动态模拟退火/能量最小化（SA/EM）协议计算了L-NTP和CB-NTP的Glu1-Ile27片段的结构。通过连接两个（亚）结构，然后进行一轮受限SA/EM，生成了CB-NTP的构象模型。6-9、12-1段、28-30和45-48段显示出螺旋转角。在L-NTP的Cys34-Cys42环内，Glu-Glu-Asp-Glu-Glu39段的结构似乎相对不太明确，Cys42-Cys54段内包含Lys50的延伸段也是如此，这与后者可能与完整的Glu1-Pgn中的kringle结构域相互作用一致。总体而言，CB-NTP和L-NTP片段的规则二级结构含量较低——如紫外圆二色光谱所示——并且在2H2O中酰胺1H-2H交换很快，表明具有高灵活性。
