Crystal structure of lactose and a novel ligand in complex with galectin-8N
(2021) KEMK10 20211Department of Chemistry
- Abstract
- The project’s focus is on the protein galectin-8N and crystallizing it with lactose and a novel ligand, MH1. The question for the project comes from previous work that shows that galectin-8N has an empty pocket on the surface of the protein. A novel ligand was designed to improve the affinity to the protein through potential interactions between the chlorophenyl group in the ligand and a pocket on the surface of the protein containing amino acids Arg66 and Tyr148. First, proven conditions for crystallization of galectin-8N were attempted without any harvestable crystals being produced. Four commercial crystallization screens were then made to map out new galectin-8N crystallization conditions. These were the commercial screens JCSG+ and... (More)
- The project’s focus is on the protein galectin-8N and crystallizing it with lactose and a novel ligand, MH1. The question for the project comes from previous work that shows that galectin-8N has an empty pocket on the surface of the protein. A novel ligand was designed to improve the affinity to the protein through potential interactions between the chlorophenyl group in the ligand and a pocket on the surface of the protein containing amino acids Arg66 and Tyr148. First, proven conditions for crystallization of galectin-8N were attempted without any harvestable crystals being produced. Four commercial crystallization screens were then made to map out new galectin-8N crystallization conditions. These were the commercial screens JCSG+ and PACT, which are complementary, and then two commercial Morpheus screens (first in complex with lactose and the second in complex with the ligand, MH1). An optimization of the crystal hits of JCSG+ was also done. From the first Morpheus screen, crystals in complex with lactose were obtained. Data to 1.2 Å were collected from these crystals at the BioMAX beamline of MAX IV. This structure was solved by molecular replacement with Phaser and refined in phenix.refine. A new space group for galectin-8N in complex with lactose was found, I41. In the second Morpheus screen, co-crystallization with the ligand created well-diffracting crystals which at best had a resolution of 1.3 Å. This structure was also solved by molecular replacement with Phaser and refined in phenix.refine. The crystal was then examined in PyMOL for ligand interactions of MH1. It had overall good electron density and the ligand was in one conformation with full occupancy. The ligand interaction was with amino acids Arg66, Arg52, Arg76, Asn86, Gly149, Glu96, His72 and Trp93. The proposed additional interactions of the ligand with the protein were confirmed, as the Arg66 side chain interacted with the chlorophenyl group and Tyr148 has a ring system that interacts with the phenyl ring at a 90° angle. This gives room for further work with understanding ligand interaction and improving affinity for the N-terminal domain of galectin-8. (Less)
- Popular Abstract
- The first step of drug development is to understand what the cause of the illness is. Then the following question is how the progression of the illness can be stopped and possibly how it can be detected early. Oftentimes what needs to be found is an inhibitor, which is a ligand that binds to the protein causing the illness and stops it from functioning.
An important aspect is to find a ligand that binds strongly and only to the specific protein. To find a ligand that binds in this way, often several different ligands are studied structurally to understand how the interaction looks like between the protein and the ligand. There is a chance of improvement of the affinity and specificity between each ligand based on the structural and... (More) - The first step of drug development is to understand what the cause of the illness is. Then the following question is how the progression of the illness can be stopped and possibly how it can be detected early. Oftentimes what needs to be found is an inhibitor, which is a ligand that binds to the protein causing the illness and stops it from functioning.
An important aspect is to find a ligand that binds strongly and only to the specific protein. To find a ligand that binds in this way, often several different ligands are studied structurally to understand how the interaction looks like between the protein and the ligand. There is a chance of improvement of the affinity and specificity between each ligand based on the structural and biochemical information obtained from the previous ligands studied. In this project, one ligand’s interaction with a disease-causing protein was mapped out.
The protein studied is galectin-8N, a member of the family of galectins. These proteins are interesting because they are involved in a multitude of illnesses in humans including cancer, fibrosis, and heart problems. Galectins are a family of 15 proteins and 10 of these are found in humans. They are highly similar in structure and bind to the same type of galactose-containing carbohydrate groups attached to certain types of proteins.
Galectin-8 is a protein that is interesting to study because it is present in several different tissues in the human body: lungs, liver, kidney, brain and cardiac muscle. Galectin-8 is involved in carcinogenesis (formation of cancer) and cancer within the digestive, reproductive, urinary and hematological systems.
The aim of the project was to crystallize part of galectin-8 called galectin-8N with lactose and a novel synthetic ligand, collect data, and generate a structure to be able to study the interactions between the protein and the ligands. The ligand studied in this project was the latest ligand in a line of ligands with improved selectivity and affinity to galectin-8N. There was a hypothesis from a previous studied ligand that showed that there was a new potential binding pocket and that the two amino acids, Tyr148 and Arg66, in galectin-8N are thought to interact with the ligand.
The crystallization part was done by conducting four screens (tests of conditions that commonly crystalize). The first three screens were with galectin-8N in complex with lactose and in the last galectin-8N was in complex with the novel ligand. After crystallization, a data collection at MAX IV was conducted using the beamline BioMAX. After that a structure was generated.
The lactose-galectin-8N complex has an already known structure and the structure determined was a confirmation of previous research. The more interesting part was when galectin-8N and the novel ligand were in complex and showed a new potential binding site. The complex also had several promising interactions between the ligand and protein. The ligand interacted with the amino acids Tyr148 and Arg66 as expected in the hypothesis.
To conclude, this project gives a deeper understanding for further development of drugs connected to galectin-8N. That gives the opportunity to explore new ligands based on the using the new binding pocket and the two new interactions of the amino acids Tyr148 and Arg66. It leaves room for more studies of the ligand interaction with the protein, to improve ligands selectivity and affinity for galectin-8N to further drug development. (Less)
Please use this url to cite or link to this publication:
http://lup.lub.lu.se/student-papers/record/9057645
- author
- Ströhagen, Noemi LU
- supervisor
-
- Derek Logan LU
- organization
- course
- KEMK10 20211
- year
- 2021
- type
- M2 - Bachelor Degree
- subject
- keywords
- galectin-8N, ligand complex, crystalization, screen, JCSG+, PACT premier, Morpheus, biochemistry
- language
- English
- id
- 9057645
- date added to LUP
- 2021-06-28 16:31:59
- date last changed
- 2021-06-28 16:31:59
@misc{9057645,
abstract = {{The project’s focus is on the protein galectin-8N and crystallizing it with lactose and a novel ligand, MH1. The question for the project comes from previous work that shows that galectin-8N has an empty pocket on the surface of the protein. A novel ligand was designed to improve the affinity to the protein through potential interactions between the chlorophenyl group in the ligand and a pocket on the surface of the protein containing amino acids Arg66 and Tyr148. First, proven conditions for crystallization of galectin-8N were attempted without any harvestable crystals being produced. Four commercial crystallization screens were then made to map out new galectin-8N crystallization conditions. These were the commercial screens JCSG+ and PACT, which are complementary, and then two commercial Morpheus screens (first in complex with lactose and the second in complex with the ligand, MH1). An optimization of the crystal hits of JCSG+ was also done. From the first Morpheus screen, crystals in complex with lactose were obtained. Data to 1.2 Å were collected from these crystals at the BioMAX beamline of MAX IV. This structure was solved by molecular replacement with Phaser and refined in phenix.refine. A new space group for galectin-8N in complex with lactose was found, I41. In the second Morpheus screen, co-crystallization with the ligand created well-diffracting crystals which at best had a resolution of 1.3 Å. This structure was also solved by molecular replacement with Phaser and refined in phenix.refine. The crystal was then examined in PyMOL for ligand interactions of MH1. It had overall good electron density and the ligand was in one conformation with full occupancy. The ligand interaction was with amino acids Arg66, Arg52, Arg76, Asn86, Gly149, Glu96, His72 and Trp93. The proposed additional interactions of the ligand with the protein were confirmed, as the Arg66 side chain interacted with the chlorophenyl group and Tyr148 has a ring system that interacts with the phenyl ring at a 90° angle. This gives room for further work with understanding ligand interaction and improving affinity for the N-terminal domain of galectin-8.}},
author = {{Ströhagen, Noemi}},
language = {{eng}},
note = {{Student Paper}},
title = {{Crystal structure of lactose and a novel ligand in complex with galectin-8N}},
year = {{2021}},
}