Crystal Structure of the N-terminal Domain of Ryanodine Receptor from Plutella xylostella

Summary

In this article, we describe the protocols of protein expression, purification, crystallization and structure determination of the N-terminal domain of ryanodine receptor from diamondback moth (Plutella xylostella).

Abstract

Development of potent and efficient insecticides targeting insect ryanodine receptors (RyRs) has been of great interest in the area of agricultural pest control. To date, several diamide insecticides targeting pest RyRs have been commercialized, which generate annual revenue of 2 billion U.S. dollars. But comprehension of the mode of action of RyR-targeting insecticides is limited by the lack of structural information regarding insect RyR. This in turn restricts understanding of the development of insecticide resistance in pests. The diamondback moth (DBM) is a devastating pest destroying cruciferous crops worldwide, which has also been reported to show resistance to diamide insecticides. Therefore, it is of great practical importance to develop novel insecticides targeting the DBM RyR, especially targeting a region different from the traditional diamide binding site. Here, we present a protocol to structurally characterize the N-terminal domain of RyR from DBM. The x-ray crystal structure was solved by molecular replacement at a resolution of 2.84 Å, which shows a beta-trefoil folding motif and a flanking alpha helix. This protocol can be adapted for the expression, purification and structural characterization of other domains or proteins in general.

Introduction

Ryanodine receptors (RyRs) are specific ion channels, which mediate the permeation of Ca²⁺ ions across the sarcoplasmic reticulum (SR) membranes in muscle cells. Therefore, they play an important role in the excitation contraction coupling process. In its functional form, RyR assembles as a homo-tetramer with a molecular mass of >2 MDa, with each subunit comprising of ~5000 amino acid residues. In mammals, there are three isoforms: RyR1- skeletal muscle type, RyR2- cardiac muscle type and RyR3- ubiquitously expressed in different tissues¹.

In insects there is only one type of RyR, which is expressed in....

Protocol

1. Gene Cloning, Protein Expression, and Purification

1. PCR amplify DNA corresponding to protein of interest (residues 1-205 of DBM RyR, Genbank acc. no. AFW97408) and clone into pET-28a-HMT vector by Ligation-Independent Cloning (LIC)²⁵. This vector contains a histidine tag, MBP tag and a TEV protease cleavage site at the N-terminus¹⁵.
   1. Design LIC primers for amplification of target gene with LIC-compatible 5’ extensions:
      .......

Discussion

In this paper, we describe the procedure to recombinantly express, purify, crystallize and determine the structure of DBM RyR NTD. For crystallization, a crucial requirement is to obtain proteins with high solubility, purity and homogeneity. In our protocol, we chose to use pET-28a-HMT vector as it contains a hexahistidine tag and MBP tag, both of which could be utilized for purification to obtain a higher fold purity. Additionally, the MBP tag aids in the solubility of the target protein. We purified the protein by five.......

Materials

Name	Company	Catalog Number	Comments
pET-28a-HMT vector			This modified pET vector contains a hexahistidine tag, an MBP fusion protein and a TEV protease cleavage site at the N-terminus (Lobo and Van Petegem, 2009)
E. coli BL21 (DE3) strain	Novagen	69450-3CN
HisTrapHP column (5 mL)	GE Healthcare	45-000-325
Amylose resin column	New England Biolabs	E8021S
Q Sepharose high-performance column	GE Healthcare	17-1154-01
Amicon concentrators (10 kDa MWCO)	Millipore	UFC901008
Superdex 200 26/600 gel-filtration column	GE Healthcare	28-9893-36
Automated liquid handling robotic system	Art Robbins Instruments	Gryphon
96 Well CrystalQuick	Greiner bio-one	82050-494
Uni-Puck	Molecular Dimensions	MD7-601
Mounted CryoLoop - 20 micron	Hampton Research	HR4-955
CryoWand	Molecular Dimensions	MD7-411
Puck dewar loading tool	Molecular Dimensions	MD7-607
Nano drop	Thermo Scientific	NanoDrop One
Crystal incubator	Molecular Dimensions	MD5-605
X-Ray diffractor	Rigaku	FRX
PCR machine	Eppendorf	Nexus GX2
Plasmid mini-prep kit	Qiagen	27104
Gel extraction kit	Qiagen	28704
SspI restriction endonuclease	NEB	R0132S
T4 DNA polymerase	Novagen	2868713
Kanamycin	Scientific Chemical	25389940
IPTG	Genview	367931
HEPES	Genview	7365459
β-mercaptoethanol	Genview	60242
Centrifuge	Thermo Scientific	Sorvall LYNX 6000
Sonnicator	Scientz	II-D
Protein purification system	GE Healthcare	Akta Pure
Light microscope	Nikon	SMZ745
IzIt crystal dye	Hampton Research	HR4-710
Electrophoresis unit	Bio-Rad	1658005EDU
Shaker Incubator	Zhicheng	ZWYR-D2401
Index crystal screen	Hampton Research	HR2-144
Structure crystal screen	Molecular Dimensions	MD1-01
ProPlex crystal screen	Molecular Dimensions	MD1-38
PACT premier crystal screen	Molecular Dimensions	MD1-29
JCSG-plus crystal screen	Molecular Dimensions	MD1-37