Alterio Vincenzo, Hilvo Mika, Di Fiore Anna, Supuran Claudiu T, Pan Peiwen, Parkkila Seppo, Scaloni Andrea, Pastorek Jaromir, Pastorekova Silvia, Pedone Carlo, Scozzafava Andrea, Monti Simona Maria, De Simone Giuseppina
Istituto di Biostrutture e Bioimmagini-CNR, via Mezzocannone 16, 80134 Naples, Italy.
Proc Natl Acad Sci U S A. 2009 Sep 22;106(38):16233-8. doi: 10.1073/pnas.0908301106. Epub 2009 Sep 14.
Carbonic anhydrase (CA) IX is a plasma membrane-associated member of the alpha-CA enzyme family, which is involved in solid tumor acidification. It is a marker of tumor hypoxia and a prognostic factor in several human cancers. An aberrant increase in CA IX expression in chronic hypoxia and during development of various carcinomas contributes to tumorigenesis through at least two mechanisms: pH regulation and cell adhesion control. Here we report the X-ray structure of the catalytic domain of CA IX in complex with a classical, clinically used sulfonamide inhibitor, acetazolamide. The structure reveals a typical alpha-CA fold, which significantly differs from the other CA isozymes when the protein quaternary structure is considered. Thus, two catalytic domains of CA IX associate to form a dimer, which is stabilized by the formation of an intermolecular disulfide bond. The active site clefts and the PG domains are located on one face of the dimer, while the C-termini are located on the opposite face to facilitate protein anchoring to the cell membrane. A correlation between the three-dimensional structure and the physiological role of the enzyme is here suggested, based on the measurement of the pH profile of the catalytic activity for the physiological reaction, CO(2) hydration to bicarbonate and protons. On the basis of the structural differences observed between CA IX and the other membrane-associated alpha-CAs, further prospects for the rational drug design of isozyme-specific CA inhibitors are proposed, given that inhibition of this enzyme shows antitumor activity both in vitro and in vivo.
碳酸酐酶（CA）IX是α-碳酸酐酶家族中与质膜相关的成员，参与实体瘤的酸化过程。它是肿瘤缺氧的标志物，也是多种人类癌症的预后因素。在慢性缺氧以及各种癌症发生过程中，CA IX表达异常增加，通过至少两种机制促进肿瘤发生：pH调节和细胞黏附控制。在此，我们报道了CA IX催化结构域与一种经典的临床使用的磺酰胺抑制剂乙酰唑胺复合物的X射线结构。该结构揭示了一种典型的α-碳酸酐酶折叠，当考虑蛋白质四级结构时，它与其他碳酸酐酶同工酶有显著差异。因此，CA IX的两个催化结构域缔合形成二聚体，通过分子间二硫键的形成而稳定。活性位点裂隙和PG结构域位于二聚体的一侧，而C末端位于另一侧，以利于蛋白质锚定到细胞膜上。基于对该酶催化生理反应（CO₂水合形成碳酸氢盐和质子）活性的pH谱测量，本文提出了三维结构与该酶生理作用之间的相关性。鉴于抑制该酶在体外和体内均显示出抗肿瘤活性，基于CA IX与其他膜相关α-碳酸酐酶之间观察到的结构差异，提出了同工酶特异性CA抑制剂合理药物设计的进一步前景。
