Mochalkin I, Cheng B, Klezovitch O, Scanu A M, Tulinsky A
Department of Chemistry, Michigan State University, East Lansing 48824, USA.
Biochemistry. 1999 Feb 16;38(7):1990-8. doi: 10.1021/bi9820558.
The kringle modules of apolipoprotein(a) [apo(a)] of lipoprotein(a) [Lp(a)] are highly homologous with kringle 4 of plasminogen (75-94%) and like the latter are autonomous structural and functional units. Apo(a) contains 14-37 kringle 4 (KIV) repeats distributed into 10 classes (1-10). Lp(a) binds lysine-Sepharose via a lysine binding site (LBS) located in KIV-10 (88% homology with plasminogen K4). However, the W72R substitution that occurs in rhesus monkeys and occasionally in humans leads to impaired lysine binding capacity of KIV-10 and Lp(a). The foregoing has been investigated by determining the structures of KIV-10/M66 (M66 variant) in its unliganded and ligand [epsilon-aminocaproic acid (EACA)] bound modes and the structure of recombinant KIV-10/M66R72 (the W72R mutant). In addition, the EACA liganded structure of a sequence polymorph (M66T in about 42-50% of the human population) was reexamined (KIV-10/T66/EACA). The KIV-10/M66, KIV-10/M66/EACA, and KIV-10/T66/EACA molecular structures are highly isostructural, indicating that the LBS of the kringles is preformed anticipating ligand binding. A displacement of three water molecules from the EACA binding groove and a movement of R35 bringing the guanidinium group close to the carboxylate of EACA to assist R71 in stabilizing the anionic group of the ligand are the only changes accompanying ligand binding. Both EACA structures were in the embedded binding mode utilizing all three binding centers (anionic, hydrophobic, cationic) like plasminogen kringles 1 and 4. The KIV-10/T66/EACA structure determined in this work differs from one previously reported [Mikol, V., Lo Grasso, P. V. and, Boettcher, B. R. (1996) J. Mol. Biol. 256, 751-761], which crystallized in a different crystal system and displayed an unbound binding mode, where only the amino group of EACA interacted with the anionic center of the LBS. The remainder of the ligand extended into solvent perpendicular to the kringle surface, leaving the hydrophobic pocket and the cationic center of the LBS unoccupied. The structure of recombinant KIV-10/M66R72 shows that R72 extends along the ligand binding groove parallel to the expected position of EACA toward the anionic center (D55/D57) and makes a salt bridge with D57. Thus, the R72 side chain mimics ligand binding, and loss of binding ability is the result of steric blockage of the LBS by R72 physically occupying part of the site. The rhesus monkey lysine binding impairment is compared with that of chimpanzee where KIV-10 has been shown to have a D57N mutation instead.
脂蛋白(a)[Lp(a)]中载脂蛋白(a)[apo(a)]的kringle结构域与纤溶酶原的kringle 4高度同源(75 - 94%)，并且与后者一样是自主的结构和功能单位。Apo(a)包含14 - 37个kringle 4 (KIV)重复序列，分为10类(1 - 10)。Lp(a)通过位于KIV - 10中的赖氨酸结合位点(LBS)与赖氨酸 - 琼脂糖结合(KIV - 10与纤溶酶原K4的同源性为88%)。然而，恒河猴中出现且偶尔在人类中出现的W72R替换会导致KIV - 10和Lp(a)的赖氨酸结合能力受损。通过确定KIV - 10/M66( M66变体)在其未结合配体和结合配体[ε - 氨基己酸(EACA)]模式下的结构以及重组KIV - 10/M66R72( W72R突变体)的结构，对上述情况进行了研究。此外，还重新研究了一种序列多态性(约42 - 50%的人群中为M66T)的EACA结合结构(KIV - 10/T66/EACA)。KIV - 10/M66、KIV - 10/M66/EACA和KIV - 10/T66/EACA的分子结构高度同构，表明kringle的LBS在预期配体结合之前就已形成。从EACA结合凹槽中置换出三个水分子以及R35的移动使胍基靠近EACA的羧酸盐以协助R71稳定配体的阴离子基团是配体结合伴随的唯一变化。两种EACA结构均采用嵌入结合模式，利用了所有三个结合中心(阴离子、疏水、阳离子)，就像纤溶酶原kringle 1和4一样。本研究中确定的KIV - 10/T66/EACA结构与之前报道的[Mikol, V., Lo Grasso, P. V.和Boettcher, B. R. (1996) J. Mol. Biol. 256, 751 - 761]不同，后者在不同的晶体系统中结晶并显示出未结合的结合模式，其中只有EACA的氨基与LBS的阴离子中心相互作用。配体的其余部分垂直于kringle表面延伸到溶剂中，使LBS的疏水口袋和阳离子中心未被占据。重组KIV - 10/M66R72的结构表明，R72沿着配体结合凹槽平行于EACA的预期位置向阴离子中心(D55/D57)延伸，并与D57形成盐桥。因此，R72侧链模拟配体结合，结合能力的丧失是由于R72物理占据了部分位点而对LBS造成空间位阻的结果。将恒河猴的赖氨酸结合损伤与黑猩猩的进行了比较，在黑猩猩中KIV - 10已被证明存在D57N突变。
