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Biochemistry

X射线晶体学研究热木体马里蒂玛M42氨基肽TmPep1050的寡聚态过渡

Published: May 13, 2020
doi:
10.3791/61288
, , ,
1Laboratory of Microbiology, Department of Molecular Biology,Université Libre de Bruxelles, 2Labiris Institut de Recherche

Summary

该协议已经开发，以研究TmPep1050的二丁二代过渡，一种M42氨基肽，在结构层面。从蛋白质纯化到 X 射线数据处理，这是一条简单的管道。通过一个案例研究，TmPep1050H60A H307A 变种强调了晶体生成、数据集索引和分子 替代。

Abstract

M42氨基肽形成由12个亚单位构成的功能活性复合物。他们的装配过程似乎由其金属离子辅助因子调节，从而触发了二丁体转换。金属离子结合时，在有源位点和相互作用界面上发生几次结构修改，形成二丁体，促进自组装。为了观察这种修改，在结构研究之前必须分离稳定的寡聚物。这里报道的方法，允许纯化稳定的多德卡派和二甲，TmPep1050，一个M42氨基肽 的T.马里蒂玛，并由X射线晶体学的结构测定。迪姆斯是由多德卡人用溷合剂去除金属离子从多德卡人那里准备的。没有他们的辅助因素，多德卡器变得不那么稳定，在加热时完全分离。通过直接的分子替代方法解决了寡聚结构问题。为了说明该方法，TmPep1050变种的结构，在金属离子结合中完全受损，显示没有进一步分解的二元体到单体。

Introduction

寡聚化是决定许多蛋白质生物功能的主要过程。在大肠杆菌中，估计只有35%的蛋白质是单体1。一些蛋白质，称为形态素，甚至可以采用几种寡聚态，亚基在每个寡聚态2中具有不同的结构。它们的寡聚状态之间的过渡通常是调节蛋白质活性的一种意义，因为每种寡聚状态可能有不同的特定活性或功能。几个例子的形态素已被文献记载，特别是磷脂蛋白原合成酶3，HPr激酶/磷酸酶4，隆蛋白酶5，乳酸脱氢酶6，甘油醛-3-磷酸脱氢酶7，丙酮酸盐激酶8，柠檬酸合成酶9，和核糖酶A10。最近，我们描述了M42氨基肽TmPep1050，另一个酶的例子与形态素样的行为，其活性取决于其寡聚状态11。其寡聚状态之间的过渡由其金属辅助因子进行调解，从而引起亚单位的几次结构修饰。

M42氨基肽家族属于MH氏族12，13，并广泛分布于细菌和阿奇亚14。M42氨基肽是正宗的二核酶，降解肽的长度为35个氨基酸残留物15。他们采用一种奇特的四角体状结构，由12个亚单位形成，其活性位点面向内腔。这种安排通常被描述为对活性的纳米分形，以避免不受控制的蛋白解。M42氨基肽的生理功能可能与蛋白质降解引起的蛋白酶体、水解肽16、17有关。热球菌霍里科希拥有四个M42氨基肽，每个呈现不同但互补的特异性18，19，20，21。单说，由两种不同类型的亚单位制造的异质复杂体在P.horikoshii中描述，暗示了22、23中肽复合物的存在。

文献中对M42氨基肽的几个结构已经描述了11、16、18、19、20、24、25、26。子单元由两个不同的域组成，一个催化域和一个二化域。催化域采用一种共同的α/β折叠保存于整个MH族中，原型催化域为Vibrio蛋白酶27的氨基肽Ap1。二聚化域采用PDZ形折叠16，除了在寡聚化中的作用外，还具有控制内腔11中基板访问和结合的作用。由于基本构建基块是一个二角板，多德卡默通常被描述为六个二角的关联，每个二丁体被放置在四边板16的每一边缘。M42 氨基肽的寡聚化取决于其金属辅助因子的可用性。二氧化二离子，通常为Zn2+和Co2+，在肽结合和水解中催化参与。它们存在于两个不同的绑定站点中，即 M1 和 M2 站点。两个金属离子也驱动和微调寡聚化，如PhTET2，PhTET3，PfTET3和TmPep105011，24，28，29。当金属辅助因子耗尽时，多德卡默分解成二元，如 PhTET2、PhTET3 和 TmPep1050 11、16、28，甚至单体，如 PhTET2和 PfTET324、29。

此处介绍的是用于研究 TmPep1050 寡聚物结构的协议。该协议是一套常见的方法，包括蛋白质纯化、蛋白解活性筛选、结晶、X射线衍射和分子替代。强调了处理金属糖酶、蛋白质寡聚化、蛋白质结晶和分子替代所固有的微妙之处。还提出了一个研究案例，以表明TmPep1050多卡默是否可以进一步分离成单体。为了解决这个问题，TMPep1050变种TmPep1050H60A H307A已经研究过其金属结合位点通过将 His-60（M2位点）和 His-307（M1位点）变异到阿拉残留物而受损。该协议可以容纳研究其他M42氨基肽或任何金属糖酶与形态素一样的行为。

Protocol

1. 重组TmPep1050的生产和纯化 注：下面描述了克隆过程和纯化野生型TmPep1050改编自以前的研究11。或者，克隆可以使用合成基因进行。为了生成 TmPep1050 变种，可以按照以下方法 30 执行站点定向突变，例如，并行协议 （SPRINP） 方法30中的单底反应。纯化协议可用于 TmPep1050 变种。应避免使用 His-tag，因为它干扰金属离子结合。 表达式?…

Representative Results

为了研究在TmPep1050中可能分离成单体的单体，His-60和Sss-307的编码子被使用合成基因的丙氨酸codon所取代。该基因随后在pBAD载体中克隆，用于表达和纯化相应的TmPep1050变种，随后命名为TmPep1050H60A H307A。大小排除色谱（图3B）显示，纯化蛋白质的表观分子量为56kDa（单体分子量为36.0kDa）。据报道，TmPep1050二暗器11的类似明显分子量为52kDa。因此，TmPep1050…

Discussion

此处描述的协议允许在结构层面上理解 TmPep1050 的二代多卡默过渡。该方法之前是用于确定两个TmPep1050寡聚物11的结构的经验。最具挑战性的一步是找到条件，促进多德卡人分离成稳定的暗化器。当添加金属离子辅助因子时，这些条件必须足够温和，才能将二丁二暗器重新关联到多德卡器中。寡聚物的分离也是关键的一步，因为它为结构研究和进一步的生化特性（例如，研究不同…

Disclosures

The authors have nothing to disclose.

Acknowledgements

我们感谢马丁·罗弗斯对本文件进行校对，并提出建设性意见。访问 Proxima 2 光束线 （SOLEIL 同步加速器） 是在块分配组 20151139。

Materials

1,10-phenanthroline Sigma-Aldrich 13, 137-7
Amicon Ultra 0.5 ml Centrifugal Filters Ultracel 30K Merck Millipore UFC503096
Amicon Ultra 15 Centrifugal Filters Ultracel 30K Merck Millipore UFC903024
Benzonase Nuclease Merck Millipore 70664-3
CCP4 N/A visit http://www.ccp4.ac.uk/
cOmplete EDTA-free Roche 5056489001
Coot N/A visit https://www2.mrc-lmb.cam.ac.uk/personal/pemsley/coot/
Crystal Screen I Hampton Research HR2-110
Crystal Screen II Hampton Research HR2-112
DreamTaq Green PCR Master Mix ThermoFisher Scientific K1082
EasyXtal 15-well tool NeXtal 132007
Escherichia coli PPY strain N/A see reference 31
Escherichia coli XL1 blue strain Agilent 200249
Gel Filtration Calibration Kit HMW GE Healthcare Life Sciences 28-4038-42
Gel Filtration Calibration Kit LMW GE Healthcare Life Sciences 28-4038-41
Gel Filtration Standard Biorad 1511901
GeneJET Plasmid Miniprep Kit ThermoFisher Scientific K0503
Index Hampton Research HR2-144
Litholoops Molecular Dimensions
L-leucine-p-nitroanilide Bachem AG 40010720025
Natrix 1 Hampton Research HR2-116
Natrix 2 Hampton Research HR2-117
Neggia plugin Dectris N/A visit https://www.dectris.com/
NeXtal Tubes JCSG Core Suite I NeXtal 130724
NeXtal Tubes JCSG Core Suite II NeXtal 130725
NeXtal Tubes JCSG Core Suite III NeXtal 130726
NeXtal Tubes JCSG Core Suite IV NeXtal 130727
pBAD-TOPO ThermoFisher Scientific K430001
Phenix N/A visit https://www.phenix-online.org/
Phusion High-Fidelity DNA polymerase ThermoFisher Scientific F-530L
Salt RX 1 Hampton Research HR2-107
Salt RX 2 Hampton Research HR2-109
SnakeSkin Dialysis Tubing, 3.5K MWCO ThermoFisher Scientific 88242
Source 15Phe GE Healthcare Life Sciences 17014702
Source 15Q GE Healthcare Life Sciences 17094705
Superdex 200 prep grade GE Healthcare Life Sciences 17104301
Thermotoga maritima MSB8 strain American Type Culture Collection ATCC 43589
TmCD00089984 DNASU Plasmid Repository N/A
XDS N/A visit http://xds.mpimf-heidelberg.mpg.de/
xdsme N/A visit https://github.com/legrandp/xdsme
DOWNLOAD MATERIALS LIST

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Tags

X-ray CrystallographyOligomeric StateThermotoga MaritimaAminopeptidaseTmPep1050Activity AssayApoenzyme PreparationDialysisUltrafiltration

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Dutoit, R., Brandt, N., Van Elder, D., Droogmans, L. X-Ray Crystallography to Study the Oligomeric State Transition of the Thermotoga maritima M42 Aminopeptidase TmPep1050. J. Vis. Exp. (159), e61288, doi:10.3791/61288 (2020).
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