Structural basis for activity regulation and substrate preference of clostridial collagenases G, H, and T.
Structural basis for activity regulation and substrate preference of clostridial collagenases G, H, and T.
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DOI：
10.1074/jbc.m112.448548
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发表时间：
2013-07-12
期刊：
影响因子：
--
通讯作者：
Brandstetter H
中科院分区：
文献类型：
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作者：
Eckhard U;Schönauer E;Brandstetter H
背景：细菌胶原酶降解胶原底物效率高，但特异性不同。结果：新发现的钙位点、天冬氨酸开关和构象选择性过滤器调节底物进入这些胶原酶的活性位点。结论：底物识别位点的动态变化和锌的占用共同调节了酶的活性。意义：晶体结构为理解和优化同种异构体依赖性胶原酶活性提供了合理的框架。梭状芽胞杆菌胶原酶是迄今为止生物圈中最主要的蛋白质降解效率最高的酶之一。在这里，我们展示了三种梭菌胶原酶异构体（ColG， ColH和ColT）的肽酶的晶体结构。非配体结构和配体结构的比较揭示了四元亚畴动力学。在无配体的ColH结构中，这种球形动力学是由与催化锌结合的天冬氨酸开关运动调节的。我们进一步确定了靠近催化锌的钙结合位点。这两种离子都是充分发挥活性所必需的，这就解释了为什么钙会严重影响梭状菌胶原酶的酶活性。我们的研究进一步揭示了靠近活性位点的环因此可以作为特征底物选择性过滤器。这些元素解释了这些酶独特的肽解和胶原解活性，并为设计具有定制底物特异性的胶原酶和抑制剂提供了合理的框架。
Background: Bacterial collagenases degrade collagen substrates with high efficiency yet varying specificity. Results: The newly identified calcium site, aspartate switch, and conformational selectivity filter regulate substrate access to the active sites of these collagenases. Conclusion: The unanticipated dynamics of the substrate recognition sites plus zinc occupancy combine to tune the enzymatic activity. Significance: The crystal structures provide a rational framework to understand and optimize the isoform-dependent collagenase activities. Clostridial collagenases are among the most efficient enzymes to degrade by far the most predominant protein in the biosphere. Here we present crystal structures of the peptidases of three clostridial collagenase isoforms (ColG, ColH, and ColT). The comparison of unliganded and liganded structures reveals a quaternary subdomain dynamics. In the unliganded ColH structure, this globular dynamics is modulated by an aspartate switch motion that binds to the catalytic zinc. We further identified a calcium binding site in proximity to the catalytic zinc. Both ions are required for full activity, explaining why calcium critically affects the enzymatic activity of clostridial collagenases. Our studies further reveal that loops close to the active site thus serve as characteristic substrate selectivity filter. These elements explain the distinct peptidolytic and collagenolytic activities of these enzymes and provide a rational framework to engineer collagenases with customized substrate specificity as well as for inhibitor design.