Kojima Shuichi, Iwahara Akane, Hisano Yuri, Yanai Hideyuki
Institute for Biomolecular Science, Gakushuin University, Mejiro, Tokyo 171-8588, Japan.
Protein Eng Des Sel. 2007 May;20(5):211-7. doi: 10.1093/protein/gzm013. Epub 2007 Apr 16.
We previously demonstrated that Pleurotus ostreatus proteinase A inhibitor 1 (POIA1) could function as an intramolecular chaperone of subtilisin BPN', as in the case of the propeptide of subtilisin BPN', and that its Phe44 --> Ala mutant, which lost its tertiary structure, could not assist the refolding of subtilisin BPN'. In this study, we examined the effects of hydrophobic amino acid substitutions at other sites and substitutions of Phe44 with other hydrophobic residues on the structure and functions of POIA1. These mutations were introduced into POIA1cm that had been obtained by the substitution of the C-terminal six residues of POIA1 with those of the propeptide of subtilisin BPN'. When Ile32 or Ile64 was substituted with Ala, the tertiary structure of the resultant mutant was markedly destroyed, and the activities as a protease inhibitor and an intramolecular chaperone were significantly lowered. Among the position 44 mutants, the Phe44 --> Val mutant was a much less effective intramolecular chaperone with conversion to a digestible inhibitor, possibly owing to destruction of the tertiary structure. On the other hand, the Phe44 --> Leu or Ile mutant maintained its tertiary structure, and hence could function as a more effective intramolecular chaperone than the Phe44 --> Val mutant. Furthermore, since the Phe44 --> Leu mutant was a more susceptible inhibitor than POIA1cm, the halo formed around a colony of Bacillus cells transformed with a plasmid encoding this mutant was larger than others. These results clearly show the close relationship between the tertiary structure and functions of POIA1 as a protease inhibitor and an intramolecular chaperone, and that a combination of such inhibitory properties and intramolecular chaperone activity of POIA1 might affect the diameter of the halo formed around Bacillus colonies in vivo.
我们之前证明，平菇蛋白酶A抑制剂1（POIA1）可作为嗜热栖热菌蛋白酶BPN'的分子内伴侣，就像嗜热栖热菌蛋白酶BPN'的前肽一样，并且其苯丙氨酸44突变为丙氨酸的突变体失去了三级结构，无法协助嗜热栖热菌蛋白酶BPN'重折叠。在本研究中，我们研究了其他位点的疏水氨基酸取代以及苯丙氨酸44被其他疏水残基取代对POIA1结构和功能的影响。这些突变被引入到POIA1cm中，POIA1cm是通过将POIA1的C端六个残基替换为嗜热栖热菌蛋白酶BPN'前肽的相应残基而获得的。当异亮氨酸32或异亮氨酸64被丙氨酸取代时，所得突变体的三级结构被显著破坏，作为蛋白酶抑制剂和分子内伴侣的活性显著降低。在44位突变体中，苯丙氨酸44突变为缬氨酸的突变体作为分子内伴侣的效果要差得多，并且转变为可消化的抑制剂，这可能是由于三级结构的破坏。另一方面，苯丙氨酸44突变为亮氨酸或异亮氨酸的突变体保持了其三级结构，因此可以作为比苯丙氨酸44突变为缬氨酸的突变体更有效的分子内伴侣发挥作用。此外，由于苯丙氨酸44突变为亮氨酸的突变体比POIA1cm更容易被抑制，用编码该突变体的质粒转化的芽孢杆菌细胞菌落周围形成的晕圈比其他菌落的更大。这些结果清楚地表明了POIA1作为蛋白酶抑制剂和分子内伴侣的三级结构与功能之间的密切关系，并且POIA1的这种抑制特性和分子内伴侣活性的组合可能会影响体内芽孢杆菌菌落周围形成的晕圈直径。
