Defining the Structural Basis of Human Plasminogen Binding by Streptococcal Surface Enolase
Defining the Structural Basis of Human Plasminogen Binding by Streptococcal Surface Enolase
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DOI：
10.1074/jbc.m109.004317
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发表时间：
2009-06-19
影响因子：
4.8
通讯作者：
Walker, Mark J.
中科院分区：
文献类型：
--
作者：
Cork, Amanda J.;Jergic, Slobodan;Walker, Mark J.
食肉细菌A群链球菌（GAS）结合并激活人纤溶酶原，促进侵袭性疾病。链球菌表面烯醇化酶（Streptococcal surface enolase， SEN）是一种糖酵解途径酶，是一种经鉴定的GAS纤溶酶原受体。在这里，我们使用质谱（MS）来确认GAS SEN是八聚的，从而验证了基于肺炎链球菌α -烯醇化酶晶体结构的硅模型。研究人员利用定点诱变技术对表面定位的赖氨酸残基（SENK252+255A、SENK304A、SENK334A、SENK344E、SENK435L和SEN Delta 434- 435）进行了研究，以检测它们在维持结构完整性、酶功能和纤溶酶原结合方面的作用。通过圆二色光谱和质谱测定，除SENK344E外，所有突变体都保留了GAS SEN八聚体的结构完整性。然而，离子迁移率质谱显示，与野生型相比，几种突变体的稳定性存在明显差异。酶分析表明，SENK344E失去了α -烯醇化酶活性，SENK334A和SEN Delta 434-435也降低了α -烯醇化酶活性。表面等离子体共振表明，在SENK252+255A、SENK435L和SEN Delta 434-435中，结合人纤溶酶原的能力被取消。因此，位置252、255、434和435的赖氨酸残基在纤溶酶原获得中起协同作用。该研究证明了将硅结构建模与离子迁移率-质谱验证相结合的能力，可以进行复杂蛋白质结构的功能研究。
The flesh-eating bacterium group A Streptococcus ( GAS) binds and activates human plasminogen, promoting invasive disease. Streptococcal surface enolase (SEN), a glycolytic pathway enzyme, is an identified plasminogen receptor of GAS. Here we used mass spectrometry ( MS) to confirm that GAS SEN is octameric, thereby validating in silico modeling based on the crystal structure of Streptococcus pneumoniae alpha-enolase. Site-directed mutagenesis of surface-located lysine residues (SENK252+255A, SENK304A, SENK334A, SENK344E, SENK435L, and SEN Delta 434- 435) was used to examine their roles in maintaining structural integrity, enzymatic function, and plasminogen binding. Structural integrity of the GAS SEN octamer was retained for all mutants except SENK344E, as determined by circular dichroism spectroscopy and MS. However, ion mobility MS revealed distinct differences in the stability of several mutant octamers in comparison with wild type. Enzymatic analysis indicated that SENK344E had lost alpha-enolase activity, which was also reduced in SENK334A and SEN Delta 434-435. Surface plasmon resonance demonstrated that the capacity to bind human plasminogen was abolished in SENK252+255A, SENK435L, and SEN Delta 434-435. The lysine residues at positions 252, 255, 434, and 435 therefore play a concerted role in plasminogen acquisition. This study demonstrates the ability of combining in silico structural modeling with ion mobility-MS validation for undertaking functional studies on complex protein structures.