Final Report Summary - MEMBRANE PROTEINS (Visualizing the structure and function of elusive membrane receptor proteins of the human cell)
Summary project report: FP7-PEOPLE-2011-CIG - Membrane proteins
One of the great challenges in structural biology has been and remains the study of human membrane proteins. Membrane proteins not only act as exchange gateways of cells for ions, nutrients and drugs, they are also central players in the numerous sophisticated signal transduction pathways of human cells. The fact that over 50% of all drugs in clinical use act through binding to membrane proteins (e.g. G-protein coupled receptors, ion channels, solute transporters) highlights the medical importance of this large family of proteins. Despite their high biological and medical impact, quite limited information is available on the conformational properties of this large biologically important family of proteins as many human membrane proteins have so far not been compatible with powerful and established techniques for structural analysis like X-ray crystallography and NMR spectroscopy. Alternative methods are needed to gain insight into the structure of membrane-bound proteins. In this project we have developed and employed a powerful alternative technology that use mass spectrometry to measure the hydrogen/deuterium exchange (HDX) of proteins in solution. Using this technique, we have sought to examine the structure and interactions of the T-cell receptor (TCR), a ”difficult” membrane receptor protein of key biological function. The ability of the TCR recognize peptide antigen-loaded MHC complexes (pMHC) is pivotal to the human immune response and leads to activation of the T lymphocyte and ultimately killing of the antigen-presenting target cell. The TCR binds peptide antigens through highly variable loop structures, complimentary determining regions (CDR), which provide the structural scaffold for highly specific antigen recognition of foreign antigens. The recognition events taking place at the TCR-pMHC interface are therefore of considerable interest for both fundamental immunology and medical applications, such as potential therapies for autoimmune diseases and cancer.
Main results:
During the first stages of this project, we have refined and developed HDX-MS technology to enable improved analysis of complex proteins like the TCR. Included among such technical developments is a HDX-MS workflow that enables the ability to measure the HDX of individual amide linkages in a protein. Furthermore, we have successfully developed HDX-MS workflows capable of handling the mixtures of protein and lipid and other detergents normally used when studying membrane proteins.
In the later stages, we have used our refined HDX-MS approach to measure the HDX of different soluble TCR ectodomains free in solution and when bound by relevant antigen-loaded MHC complexes and have tracked the effect of antigen binding on the uptake of deuterium into local segments of the receptor. These results are obtained in solution, without any chemical modification to the TCR, antigenic peptide nor MHC molecule in contrast to earlier studies. Initially, we established a protocol for sample preparation and HDX-MS analysis of TCR proteins in the presence and absence of cognate pMHC complexes. We have subsequently successfully measured the HDX of multiple TCR proteins (ectodomain only) with optimized CDR loop sequences for binding the NY-ESO1 cancer testis antigen peptide.
HDX analyses of the TCR free in solution has revealed that both the constant domain and the variable domains in both α- and β-chains has a highly protected core composed of several β-strands of the immunoglobulin fold. Several loops interconnecting these β-strands and segments connecting the variable and constant domains however go fast exchange including the long FG-loop in Cβ and the CDR1, CDR2 and CDR3 loops in both Vα and Vβ. Some β-strands also show pronounced dynamic behavior, in particular the C- and F-strands of Cα (constant region) exchange rapidly and as such differentiated from the other beta-strands of the otherwise protected core of Cα.
We have also been able to visualize experimentally, for the first time, the effect of pMHC binding of the TCR free in solution. Upon binding of pMHC we have observed reduced HDX in several distinct regions of the TCR, directly implicating these regions in pMHC binding. Protection from HDX is observed as expected in the CDR loop structures of the Vα/Vβ domain, known to be responsible for antigen recognition. Interestingly, the magnitude of these reductions in HDX vary significantly from the six CDR loops of the TCR. This provides direct experimental evidence that each loop contribute to different extents to pMHC binding and has provided new knowledge to efforts in our collaborators lab to design new TCR variants with optimal binding of the NY-ESO-1 antigenic peptide. Binding of pMHC, however, also results in reduced deuteration in more remote areas of the TCR. We are currently confirming and investigating the details of this interesting phenomenon further using our HDX-MS approach and Biacore measurements in the lab of the project collaborator.
Based on our demonstrated ability to detect changes in the TCR upon pMHC binding we have also used HDX-MS to study the response of TCR mutants produced by the project collaborator to pMHC binding. In this we have been able to show that TCRs with altered pMHC binding affinities also show different changes in HDX upon pMHC binding. In particular, for some variants we have been able to show a link between reduced binding affinity and the absence of HDX binding effects in distinct CDR loops of the TCR.
Conclusions:
Through work in this project, we have a) developed new HDX-MS methodology to analyze complex protein systems, b) used HDX-MS technology to provide the first comprehensive study of the dynamics of the entire TCR ectodomain upon antigen interaction and identified key regions of the TCR and mutatns thereof that are linked to pMHC interaction.
Impact:
First, this project has advanced the development of technology to study the conformation and interactions of complex protein systems. Secondly, the project has provided detailed insights into the structural origins of the key immunological interaction between TCR and pMHC. Such results will increase our biological understanding of the function of this key receptor of the human immune system and could help motivate the design new TCR variants with attenuated pMHC binding for potential therapeutic use.
One of the great challenges in structural biology has been and remains the study of human membrane proteins. Membrane proteins not only act as exchange gateways of cells for ions, nutrients and drugs, they are also central players in the numerous sophisticated signal transduction pathways of human cells. The fact that over 50% of all drugs in clinical use act through binding to membrane proteins (e.g. G-protein coupled receptors, ion channels, solute transporters) highlights the medical importance of this large family of proteins. Despite their high biological and medical impact, quite limited information is available on the conformational properties of this large biologically important family of proteins as many human membrane proteins have so far not been compatible with powerful and established techniques for structural analysis like X-ray crystallography and NMR spectroscopy. Alternative methods are needed to gain insight into the structure of membrane-bound proteins. In this project we have developed and employed a powerful alternative technology that use mass spectrometry to measure the hydrogen/deuterium exchange (HDX) of proteins in solution. Using this technique, we have sought to examine the structure and interactions of the T-cell receptor (TCR), a ”difficult” membrane receptor protein of key biological function. The ability of the TCR recognize peptide antigen-loaded MHC complexes (pMHC) is pivotal to the human immune response and leads to activation of the T lymphocyte and ultimately killing of the antigen-presenting target cell. The TCR binds peptide antigens through highly variable loop structures, complimentary determining regions (CDR), which provide the structural scaffold for highly specific antigen recognition of foreign antigens. The recognition events taking place at the TCR-pMHC interface are therefore of considerable interest for both fundamental immunology and medical applications, such as potential therapies for autoimmune diseases and cancer.
Main results:
During the first stages of this project, we have refined and developed HDX-MS technology to enable improved analysis of complex proteins like the TCR. Included among such technical developments is a HDX-MS workflow that enables the ability to measure the HDX of individual amide linkages in a protein. Furthermore, we have successfully developed HDX-MS workflows capable of handling the mixtures of protein and lipid and other detergents normally used when studying membrane proteins.
In the later stages, we have used our refined HDX-MS approach to measure the HDX of different soluble TCR ectodomains free in solution and when bound by relevant antigen-loaded MHC complexes and have tracked the effect of antigen binding on the uptake of deuterium into local segments of the receptor. These results are obtained in solution, without any chemical modification to the TCR, antigenic peptide nor MHC molecule in contrast to earlier studies. Initially, we established a protocol for sample preparation and HDX-MS analysis of TCR proteins in the presence and absence of cognate pMHC complexes. We have subsequently successfully measured the HDX of multiple TCR proteins (ectodomain only) with optimized CDR loop sequences for binding the NY-ESO1 cancer testis antigen peptide.
HDX analyses of the TCR free in solution has revealed that both the constant domain and the variable domains in both α- and β-chains has a highly protected core composed of several β-strands of the immunoglobulin fold. Several loops interconnecting these β-strands and segments connecting the variable and constant domains however go fast exchange including the long FG-loop in Cβ and the CDR1, CDR2 and CDR3 loops in both Vα and Vβ. Some β-strands also show pronounced dynamic behavior, in particular the C- and F-strands of Cα (constant region) exchange rapidly and as such differentiated from the other beta-strands of the otherwise protected core of Cα.
We have also been able to visualize experimentally, for the first time, the effect of pMHC binding of the TCR free in solution. Upon binding of pMHC we have observed reduced HDX in several distinct regions of the TCR, directly implicating these regions in pMHC binding. Protection from HDX is observed as expected in the CDR loop structures of the Vα/Vβ domain, known to be responsible for antigen recognition. Interestingly, the magnitude of these reductions in HDX vary significantly from the six CDR loops of the TCR. This provides direct experimental evidence that each loop contribute to different extents to pMHC binding and has provided new knowledge to efforts in our collaborators lab to design new TCR variants with optimal binding of the NY-ESO-1 antigenic peptide. Binding of pMHC, however, also results in reduced deuteration in more remote areas of the TCR. We are currently confirming and investigating the details of this interesting phenomenon further using our HDX-MS approach and Biacore measurements in the lab of the project collaborator.
Based on our demonstrated ability to detect changes in the TCR upon pMHC binding we have also used HDX-MS to study the response of TCR mutants produced by the project collaborator to pMHC binding. In this we have been able to show that TCRs with altered pMHC binding affinities also show different changes in HDX upon pMHC binding. In particular, for some variants we have been able to show a link between reduced binding affinity and the absence of HDX binding effects in distinct CDR loops of the TCR.
Conclusions:
Through work in this project, we have a) developed new HDX-MS methodology to analyze complex protein systems, b) used HDX-MS technology to provide the first comprehensive study of the dynamics of the entire TCR ectodomain upon antigen interaction and identified key regions of the TCR and mutatns thereof that are linked to pMHC interaction.
Impact:
First, this project has advanced the development of technology to study the conformation and interactions of complex protein systems. Secondly, the project has provided detailed insights into the structural origins of the key immunological interaction between TCR and pMHC. Such results will increase our biological understanding of the function of this key receptor of the human immune system and could help motivate the design new TCR variants with attenuated pMHC binding for potential therapeutic use.