ABSTRACT
GldL is an inner-membrane protein that is essential for the function of the type IX secretion system (T9SS) in Flavobacterium johnsoniae. The complex that it forms with GldM is supposed to act as a new rotary motor involved in the gliding motility of the bacterium. In the context of structural studies of GldL to gain information on the assembly and function of the T9SS, two camelid nanobodies were selected, produced and purified. Their interaction with the cytoplasmic domain of GldL was characterized and their crystal structures were solved. These nanobodies will be used as crystallization chaperones to help in the crystallization of the cytoplasmic domain of GldL and could also help to solve the structure of the complex using molecular replacement.
Subject(s)
Bacterial Proteins/immunology , Bacterial Secretion Systems/immunology , Flavobacterium/chemistry , Single-Domain Antibodies/chemistry , Animals , Camelus , Crystallography, X-Ray , Kinetics , Models, Molecular , Protein Domains , Protein Multimerization , Scattering, Radiation , ThermodynamicsABSTRACT
Odorant-binding proteins (OBPs), as they occur in insects, form a distinct class of proteins that apparently has no closely related representatives in other animals. However, ticks, mites, spiders and millipedes contain genes encoding proteins with sequence similarity to insect OBPs. In this work, we have explored the structure and function of such non-insect OBPs in the mite Varroa destructor, a major pest of honey bee. Varroa OBPs present six cysteines paired into three disulphide bridges, but with positions in the sequence and connections different from those of their insect counterparts. VdesOBP1 structure was determined in two closely related crystal forms and appears to be a monomer. Its structure assembles five α-helices linked by three disulphide bridges, one of them exhibiting a different connection as compared to their insect counterparts. Comparison with classical OBPs reveals that the second of the six α-helices is lacking in VdesOBP1. Ligand-binding experiments revealed molecules able to bind only specific OBPs with a moderate affinity, suggesting that either optimal ligands have still to be identified, or post-translational modifications present in the native proteins may be essential for modulating binding activity, or else these OBPs might represent a failed attempt in evolution and are not used by the mites.
Subject(s)
Insect Proteins/chemistry , Receptors, Odorant/chemistry , Varroidae/chemistry , Amino Acid Sequence , Animals , Binding Sites , Conserved Sequence , Crystallography, X-Ray , Cysteine/chemistry , Fluorescent Dyes/metabolism , Kinetics , Ligands , Models, Molecular , Phylogeny , Protein Binding , Protein Conformation , Protein Folding , Sequence Alignment , Sequence Homology, Amino AcidABSTRACT
OBPs and CSPs are small soluble proteins used by organisms as shuttle to transport odorant molecules between air and the membrane-embedded receptors. Deciphering the interactions of these proteins with their ligands at a molecular level may give clue on the function and specificity of the olfactory chain. To reach this goal, protein crystallography is very helpful with more than hundred entries available in the protein data bank (PDB). In this chapter, we present the peculiarities of OBPs and CSPs concerning their crystallization and 3D structure determination by X-ray diffraction.
Subject(s)
Odorants , Receptors, Odorant , Carrier Proteins , Crystallography, X-Ray , Insect Proteins/metabolism , Phylogeny , Receptors, Odorant/genetics , Receptors, Odorant/metabolismABSTRACT
Producing intact recombinant membrane proteins for structural studies is an inherently challenging task due to their requirement for a cell-lipid environment. Most of the procedures developed involve isolating the protein by solubilization with detergent and further reconstitutions into artificial membranes. These procedures are highly time consuming and suffer from further drawbacks, including low yields and high cost. We describe here an alternative method for rapidly obtaining recombinant cell-surface membrane proteins displayed on extracellular vesicles (EVs) derived from cells in culture. Interaction between these membrane proteins and ligands can be analyzed directly on EVs. Moreover, EVs can also be used for protein structure determination or immunization purposes.
Subject(s)
Extracellular Vesicles/metabolism , Membrane Proteins/isolation & purification , Recombinant Proteins/isolation & purification , 5'-Nucleotidase/immunology , Cloning, Molecular , Cryoelectron Microscopy , Detergents/chemistry , Dynamic Light Scattering , Extracellular Vesicles/immunology , Extracellular Vesicles/ultrastructure , GPI-Linked Proteins/immunology , HEK293 Cells , Humans , Ligands , Mass Spectrometry , Membrane Proteins/genetics , Membrane Proteins/metabolism , Microscopy, Electron , Plasmids/geneticsABSTRACT
PorM is a membrane protein that is involved in the assembly of the type IX secretion system (T9SS) in Porphyromonas gingivalis, a major bacterial pathogen that is responsible for periodontal disease in humans. In the context of structural studies of PorM to better understand T9SS assembly, four camelid nanobodies were selected, produced and purified, and their specific interaction with the N-terminal or C-terminal part of the periplasmic domain of PorM was investigated. Diffracting crystals were also obtained, and the structures of the four nanobodies were solved by molecular replacement. Furthermore, two nanobodies were used as crystallization chaperones and turned out to be valuable tools in the structure-determination process of the periplasmic domain of PorM.
