Periodic Reporting for period 1 - DynOMIS (Dynamic Origins of MHC class I Selector function)
Reporting period: 2016-09-06 to 2018-09-05
Our health and wellbeing is constantly under threat from infectious pathogens, as well as the ‘enemy-within’ in the form of malignantly transformed cells. Evolution has driven the development of adaptive immunity to identify and eliminate transformed or infected cells. Immune detection requires a constant turnover of cellular proteins to peptides, which are presented at the cell surface by major histocompatibility complex class I molecules (MHC-I) and scanned by cytotoxic T lymphocytes (CTL). A sophisticated system of self–nonself discrimination ensures that CTL only unleash their cytotoxic apparatus when they encounter peptides derived from foreign microbes, or from tumour-associated proteins, hereby ensuring that only non-healthy cells are eliminated.
The peptide presenting function of MHC-I at the cell surface is the result of an equally, if not more important, peptide selecting function in the early secretory pathway. MHC-I molecules acquire peptide cargo soon after their synthesis and assembly with β2-microglobulin in the endoplasmic reticulum (ER), while they are part of a multi-subunit machinery called the peptide-loading complex. Even minor changes in the primary sequence of MHC-I, which do not affect their peptide presenting function, can lead to large differences in their peptide selecting function. Importantly, this cannot be understood by analysing the structures of MHC-I allomorphs in complex with different peptides provided by X-ray crystallography as they are virtually identical.
DynOMIS is the first attempt to link a quantitative cellular level description of a fundamental biological process and its atomistic realization. The proposed methodology integrates computational systems modelling, state-of-the-art molecular dynamics and free energy calculations with information from cellular, biochemical and advanced Nuclear Magnetic Resonance (NMR) experiments in a highly innovative way. Deep understanding of the exact mechanisms that drive peptide selection by MHC-I will enhance the ability to predict immunoprotective epitopes in infections and cancer. This will in turn pave the way for the development of more effective CTL-targeted vaccines and biomarkers to stratify patients’ suitability for immunotherapy, such as checkpoint inhibition in cancer.
The peptide presenting function of MHC-I at the cell surface is the result of an equally, if not more important, peptide selecting function in the early secretory pathway. MHC-I molecules acquire peptide cargo soon after their synthesis and assembly with β2-microglobulin in the endoplasmic reticulum (ER), while they are part of a multi-subunit machinery called the peptide-loading complex. Even minor changes in the primary sequence of MHC-I, which do not affect their peptide presenting function, can lead to large differences in their peptide selecting function. Importantly, this cannot be understood by analysing the structures of MHC-I allomorphs in complex with different peptides provided by X-ray crystallography as they are virtually identical.
DynOMIS is the first attempt to link a quantitative cellular level description of a fundamental biological process and its atomistic realization. The proposed methodology integrates computational systems modelling, state-of-the-art molecular dynamics and free energy calculations with information from cellular, biochemical and advanced Nuclear Magnetic Resonance (NMR) experiments in a highly innovative way. Deep understanding of the exact mechanisms that drive peptide selection by MHC-I will enhance the ability to predict immunoprotective epitopes in infections and cancer. This will in turn pave the way for the development of more effective CTL-targeted vaccines and biomarkers to stratify patients’ suitability for immunotherapy, such as checkpoint inhibition in cancer.
With the aim to characterize the structural dynamics of antigen-bound MHC-I at time scales ranging from picosecond to microseconds we have carried out extended all-atom molecular dynamics simulations of the mouse MHC-I alleles H-2Db and H-2Kb, both in their peptide-free and peptide-loaded states. These data were combined with a wealth of information obtained from NMR experiments employing the H-2Db complex with a Sendai virus nucleoprotein epitope (FAPGNYPAL) to provide the first full characterization of both structure and dynamics of a peptide bound MHC-I system in solution. Our results reveal for the first time that the antigenic peptide samples many more conformational states in solution than those observed by X-ray crystallography, including states with partially dissociated N- and C-terminal regions. This finding has major implications in the selector function of the MHC-I, as well as the interaction of peptide-loaded MHC-I with the chaperones tapasin and tapasin-related (TAPBPR) protein and their recognition by the T-cell receptors on the cell surface.
To elucidate the effect of the N-terminus binding region at the antigen binding groove we employed single-chain trimers of wild type H-2Kb complex with an ovalbumin epitope (SIINFEKL) and three mutants of H-2Kb. Cellular experiments indicated that the three mutations result in greater susceptibility to over-trimming by the ER aminopeptidase ERAP1. Crystallographic analysis of the MHC-I single-chain trimers did not reveal major conformational changes between the wild type and mutant forms. However, free energy calculations revealed that the N-terminus of the bound epitope can partially dissociate with lower energy barriers in the three mutants with respect to the wild type allele. This work allowed us to establish a proof of principle that the ability of ERAP1 to trim MHC I-bound peptide was dependent on an exposed N-terminus.
Within the last part of our work we investigated the dynamics of tapasin and TAPBPR-bound models of MHC-I with partially dissociated antigenic peptides. Free energy calculations for a series of complexes suggested sequence-specific differences between the two chaperone proteins and a potential link to their analogous but distinct biological role. These results can be exploited in the development of optimized prediction methods of immunodominant epitopes for vaccine design.
The results obtained have been disseminated to the scientific community through:
1. Mini-review, open-access article “The Role of Conformational Dynamics in Antigen Trimming by Intracellular Aminopeptidases” published within the research topic “The Importance of Protein Dynamics for function and its relevance to Antigen Processing and Presentation” at Frontiers in Immunology, http://dx.doi.org/10.3389/fimmu.2017.00946(opens in new window)
2. Full article (open access) “The partial dissociation of MHC class I–bound peptides exposes their N terminus to trimming by endoplasmic reticulum aminopeptidase 1” published at Journal of Biological Chemistry, http://dx.doi.org/10.1074/jbc.RA117.000313(opens in new window)
3. An oral presentation at the CompBioMed All-Hands-Meeting 2017 (11-12 April, Barcelona, Spain).
4. A poster presentation at the EMBO workshop on Antigen processing and presentation 2017 (28–31 May, Salamanca, Spain).
5. An oral presentation at the South West Structural Biology Consortium meeting 2017 (3-4 July, Cardiff, UK).
6. An oral presentation at the 68th Congress of the Hellenic Society of Biochemistry and Molecular Biology 2017 (10-12 November, Athens, Greece).
7. A poster presentation at the British Biophysical Society biennial meeting 2018 (11-13 July, Southampton, UK).
8. A poster presentation at the 28th International Conference on Magnetic Resonance in Biological Systems 2018 (19-24 August, Dublin, UK).
Main dissemination actions that target the broad public include participation of the fellow at:
1. Science and Engineering Day, University of Southampton 2017 (18th May)
2. European Researchers’ Night, the “Hellenic Cosmos” Cultural Centre, Athens 2017 (29th September)
3. 1st Summer Camp of the Institute of Biosciences & Applications, NCSR “Demokritos”, Athens 2018 (25th June - 6th July)
To elucidate the effect of the N-terminus binding region at the antigen binding groove we employed single-chain trimers of wild type H-2Kb complex with an ovalbumin epitope (SIINFEKL) and three mutants of H-2Kb. Cellular experiments indicated that the three mutations result in greater susceptibility to over-trimming by the ER aminopeptidase ERAP1. Crystallographic analysis of the MHC-I single-chain trimers did not reveal major conformational changes between the wild type and mutant forms. However, free energy calculations revealed that the N-terminus of the bound epitope can partially dissociate with lower energy barriers in the three mutants with respect to the wild type allele. This work allowed us to establish a proof of principle that the ability of ERAP1 to trim MHC I-bound peptide was dependent on an exposed N-terminus.
Within the last part of our work we investigated the dynamics of tapasin and TAPBPR-bound models of MHC-I with partially dissociated antigenic peptides. Free energy calculations for a series of complexes suggested sequence-specific differences between the two chaperone proteins and a potential link to their analogous but distinct biological role. These results can be exploited in the development of optimized prediction methods of immunodominant epitopes for vaccine design.
The results obtained have been disseminated to the scientific community through:
1. Mini-review, open-access article “The Role of Conformational Dynamics in Antigen Trimming by Intracellular Aminopeptidases” published within the research topic “The Importance of Protein Dynamics for function and its relevance to Antigen Processing and Presentation” at Frontiers in Immunology, http://dx.doi.org/10.3389/fimmu.2017.00946(opens in new window)
2. Full article (open access) “The partial dissociation of MHC class I–bound peptides exposes their N terminus to trimming by endoplasmic reticulum aminopeptidase 1” published at Journal of Biological Chemistry, http://dx.doi.org/10.1074/jbc.RA117.000313(opens in new window)
3. An oral presentation at the CompBioMed All-Hands-Meeting 2017 (11-12 April, Barcelona, Spain).
4. A poster presentation at the EMBO workshop on Antigen processing and presentation 2017 (28–31 May, Salamanca, Spain).
5. An oral presentation at the South West Structural Biology Consortium meeting 2017 (3-4 July, Cardiff, UK).
6. An oral presentation at the 68th Congress of the Hellenic Society of Biochemistry and Molecular Biology 2017 (10-12 November, Athens, Greece).
7. A poster presentation at the British Biophysical Society biennial meeting 2018 (11-13 July, Southampton, UK).
8. A poster presentation at the 28th International Conference on Magnetic Resonance in Biological Systems 2018 (19-24 August, Dublin, UK).
Main dissemination actions that target the broad public include participation of the fellow at:
1. Science and Engineering Day, University of Southampton 2017 (18th May)
2. European Researchers’ Night, the “Hellenic Cosmos” Cultural Centre, Athens 2017 (29th September)
3. 1st Summer Camp of the Institute of Biosciences & Applications, NCSR “Demokritos”, Athens 2018 (25th June - 6th July)
Although different methods over the last 20 years have provided circumstantial evidence that peptide selection function of MHC-I is associated with a conformational intermediate, the structural determinants of the peptide-dependent stabilization mechanism still remain elusive. DynOMIS aim to elucidate the antigen selection mechanisms of MHC-I in the context of the complexity of the cellular environment by characterising the intrinsic plasticity of MHC-I rather than their static structures provide a mechanistic description of antigen selection mechanism, which is expected to furnish reliable biological descriptions that are fundamental to predict peptides that are immunogenic, i.e. those that evoke an immune response. Our results will aid the development and optimization of such predictive algorithms that will play a key role in pharmaceutical drug discovery, novel immunotherapies and personalized medicine.