Almog Orna, González Ana, Godin Noa, de Leeuw Marina, Mekel Marlene J, Klein Daniela, Braun Sergei, Shoham Gil, Walter Richard L
Department of Clinical Biochemistry, Faculty of Health Sciences, Ben-Gurion University, Beer Sheva 84105, Israel.
Proteins. 2009 Feb 1;74(2):489-96. doi: 10.1002/prot.22175.
We determine and compare the crystal structure of two proteases belonging to the subtilisin superfamily: S41, a cold-adapted serine protease produced by Antarctic bacilli, at 1.4 A resolution and Sph, a mesophilic serine protease produced by Bacillus sphaericus, at 0.8 A resolution. The purpose of this comparison was to find out whether multiple calcium ion binding is a molecular factor responsible for the adaptation of S41 to extreme low temperatures. We find that these two subtilisins have the same subtilisin fold with a root mean square between the two structures of 0.54 A. The final models for S41 and Sph include a calcium-loaded state of five ions bound to each of these two subtilisin molecules. None of these calcium-binding sites correlate with the high affinity known binding site (site A) found for other subtilisins. Structural analysis of the five calcium-binding sites found in these two crystal structures indicate that three of the binding sites have two side chains of an acidic residue coordinating the calcium ion, whereas the other two binding sites have either a main-chain carbonyl, or only one acidic residue side chain coordinating the calcium ion. Thus, we conclude that three of the sites are of high affinity toward calcium ions, whereas the other two are of low affinity. Because Sph is a mesophilic subtilisin and S41 is a psychrophilic subtilisin, but both crystal structures were found to bind five calcium ions, we suggest that multiple calcium ion binding is not responsible for the adaptation of S41 to low temperatures.
S41是一种由南极杆菌产生的冷适应性丝氨酸蛋白酶，分辨率为1.4 Å；Sph是一种由球形芽孢杆菌产生的嗜温丝氨酸蛋白酶，分辨率为0.8 Å。此次比较的目的是探究多个钙离子结合是否是S41适应极端低温的分子因素。我们发现这两种枯草杆菌蛋白酶具有相同的枯草杆菌蛋白酶折叠结构，两种结构之间的均方根偏差为0.54 Å。S41和Sph的最终模型包括每个枯草杆菌蛋白酶分子结合五个钙离子的钙负载状态。这些钙离子结合位点均与其他枯草杆菌蛋白酶已知的高亲和力结合位点（位点A）不相关。对这两种晶体结构中发现的五个钙离子结合位点的结构分析表明，其中三个结合位点有两个酸性残基的侧链配位钙离子，而另外两个结合位点要么有一个主链羰基，要么只有一个酸性残基侧链配位钙离子。因此，我们得出结论，其中三个位点对钙离子具有高亲和力，而另外两个位点对钙离子具有低亲和力。由于Sph是嗜温枯草杆菌蛋白酶，S41是嗜冷枯草杆菌蛋白酶，但两种晶体结构均发现结合五个钙离子，我们认为多个钙离子结合并非S41适应低温的原因。
