Three Camelid VHH domains in complex with porcine pancreatic α-amylase -: Inhibition and versatility of binding topology
Three Camelid VHH domains in complex with porcine pancreatic α-amylase -: Inhibition and versatility of binding topology
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DOI：
10.1074/jbc.m202327200
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发表时间：
2002-06-28
影响因子：
4.8
通讯作者：
Cambillau, C
中科院分区：
文献类型：
--
作者：
Desmyter, A;Spinelli, S;Cambillau, C
骆驼科动物产生缺乏轻链和CH 1结构域的功能性抗体。因此，此类重链抗体的抗原结合片段包含在一个单一结构域中，即骆驼科重链抗体VH（VHH）。在这里，我们报告的三个单峰骆驼VHH域的结构与猪胰腺α-淀粉酶的复合物。两个VHH结合的催化位点外，不抑制或仅部分抑制淀粉酶活性。第三个是AMD 9，与活性位点缝隙相互作用，是一个强的淀粉酶抑制剂（Ki = 10 nM）。与其他蛋白质淀粉酶抑制剂的复合物相比，淀粉酶保持其天然结构。被淀粉酶覆盖的VHH的水可及表面积在850和1150埃之间（2），该值类似于或甚至大于在蛋白质和经典抗体之间的复合物中观察到的值。这些值当然可以达到，因为令人惊讶的高程度的框架残基参与VHH与淀粉酶的相互作用。参与抗原识别的框架残基占掩埋表面的25-40%。AMD 9的抑制相互作用主要涉及其互补决定区（CDR）2环，而CDR 3环较小，并且肯定不像cAb-Lys 3（一种VHH抑制性溶菌酶）那样突出。AMD 9抑制淀粉酶，尽管它在催化残基的直接可及范围之外;因此，预期抑制VHH也可能针对蛋白酶引起。这些结果说明了VHH结构域作为蛋白质结合剂和酶抑制剂的多功能性和效率，并且是支持其用作抗糖尿病药物的论据。
Camelids produce functional antibodies devoid of light chains and CH1 domains. The antigen-binding fragment of such heavy chain antibodies is therefore comprised in one single domain, the camelid heavy chain antibody VH (VHH). Here we report on the structures of three dromedary VHH domains in complex with porcine pancreatic a-amylase. Two VHHs bound outside the catalytic site and did not inhibit or inhibited only partially the amylase activity. The third one, AMD9, interacted with the active site crevice and was a strong amylase inhibitor (K-i = 10 nM). In contrast with complexes of other proteinaceous amylase inhibitors, amylase kept its native structure. The water-accessible surface areas of VHHs covered by amylase ranged between 850 and 1150 Angstrom(2), values similar to or even larger than those observed in the complexes between proteins and classical antibodies. These values could certainly be reached because a surprisingly high extent of framework residues are involved in the interactions of VHHs with amylase. The framework residues that participate in the antigen recognition represented 25-40% of the buried surface. The inhibitory interaction of AMD9 involved mainly its complementarity-determining region (CDR) 2 loop, whereas the CDR3 loop was small and certainly did not protrude as it does in cAb-Lys3, a VHH-inhibiting lysozyme. AMD9 inhibited amylase, although it was outside the direct reach of the catalytic residues; therefore it is to be expected that inhibiting VHHs might also be elicited against proteases. These results illustrate the versatility and efficiency of VHH domains as protein binders and enzyme inhibitors and are arguments in favor of their use as drugs against diabetes.