The guanine-nucleotide-exchange factor BopE from Burkholderia pseudomallei adopts a compact version of the Salmonella SopE/SopE2 fold and undergoes a closed-to-open conformational change upon interaction with Cdc42.
The guanine-nucleotide-exchange factor BopE from Burkholderia pseudomallei adopts a compact version of the Salmonella SopE/SopE2 fold and undergoes a closed-to-open conformational change upon interaction with Cdc42.
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DOI：
10.1042/bj20071546
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发表时间：
2008-05-01
影响因子：
4.1
通讯作者：
Bagby, Stefan
中科院分区：
文献类型：
--
作者：
Upadhyay, Abhishek;Wu, Huan-Lin;Williams, Christopher;Field, Terry;Galyov, Edouard E.;van den Elsen, Jean M. H.;Bagby, Stefan
关键词：
BopE是来自类鼻疽伯克霍尔德氏菌的III型分泌蛋白，类鼻疽是一种严重的新发感染的病原体。BopE是Rho GTP酶Cdc 42（细胞分裂周期42）和Rac 1的GEF（鸟嘌呤核苷酸交换因子）。我们用核磁共振波谱法测定了BopE催化结构域（氨基酸78-261）的结构，结果表明BopE 78 -261由两个三螺旋束（α1α4α5和α2α3α6）组成。该折叠类似于BopE同源物SopE和SopE 2所采用的折叠，SopE和SopE 2是来自沙门氏菌的GEF。SopE 78 -240和SopE 269 -240的两个三螺旋束形成“Λ”形的臂，而BopE 78 -261采用更封闭的构象，两个三螺旋束之间具有实质性的相互作用。我们建议，精氨酸和脯氨酸残基是重要的BopE和SopE/E2之间的构象差异。对SopE 78 -240-Cdc 42复合物晶体结构中分子界面的分析表明，在BopE-Cdc 42相互作用中，BopE 78 -261的闭合构象将与Cdc 42开关区域产生空间冲突。这意味着BopE 78 -261必须经历闭合到开放的构象变化以催化鸟嘌呤核苷酸交换。在研究BopE 78 -261-Cdc 42相互作用的NMR滴定中，BopE 78 -261铰链区残基的每个NH出现额外峰表明BopE 78 -261在Cdc 42存在下确实经历了闭合到开放的构象变化。通过有利于开放构象的突变，BopE 78 -261催化效率的显著改善进一步支持了构象变化假说。闭合到开放构象变化的要求解释了与SopE和SopE 2相比，BopE的kcat低10-40倍。
BopE is a type III secreted protein from Burkholderia pseudomallei, the aetiological agent of melioidosis, a severe emerging infection. BopE is a GEF (guanine-nucleotide-exchange factor) for the Rho GTPases Cdc42 (cell division cycle 42) and Rac1. We have determined the structure of BopE catalytic domain (amino acids 78–261) by NMR spectroscopy and it shows that BopE78-261 comprises two three-helix bundles (α1α4α5 and α2α3α6). This fold is similar to that adopted by the BopE homologues SopE and SopE2, which are GEFs from Salmonella. Whereas the two three-helix bundles of SopE78-240 and SopE269-240 form the arms of a ‘Λ’ shape, BopE78-261 adopts a more closed conformation with substantial interactions between the two three-helix bundles. We propose that arginine and proline residues are important in the conformational differences between BopE and SopE/E2. Analysis of the molecular interface in the SopE78-240–Cdc42 complex crystal structure indicates that, in a BopE–Cdc42 interaction, the closed conformation of BopE78-261 would engender steric clashes with the Cdc42 switch regions. This implies that BopE78-261 must undergo a closed-to-open conformational change in order to catalyse guanine nucleotide exchange. In an NMR titration to investigate the BopE78-261–Cdc42 interaction, the appearance of additional peaks per NH for residues in hinge regions of BopE78-261 indicates that BopE78-261 does undergo a closed-to-open conformational change in the presence of Cdc42. The conformational change hypothesis is further supported by substantial improvement of BopE78-261 catalytic efficiency through mutations that favour an open conformation. Requirement for closed-to-open conformational change explains the 10–40-fold lower kcat of BopE compared with SopE and SopE2.