Periodic Reporting for period 3 - EditMHC (How MHC-I editing complexes shape the hierarchical immune response)
Reporting period: 2022-01-01 to 2023-06-30
The cells of our body are constantly surveilled by the immune system for signs of disease. To this end, parts of the immune system scan the cells’ surface, on which so-called MHC molecules present fragments of the cellular interior. These fragments, referred to as peptides or antigens, are breakdown products of proteins. Once the immune system detects foreign or aberrant peptides on MHCs, e.g. derived from a virus or abnormal protein, it triggers a response that eventually kills the diseased cells to protect the body from further harm.
For the surveillance system to work, cells have developed a sophisticated machinery not only to transport antigens into the endoplasmic reticulum (ER), a dedicated subcellular compartment, for redirection to the cell surface, but also to select antigens that bind tightly to the presenting MHC molecules and to facilitate antigen loading onto MHCs. A central hub in this machinery is a large assembly termed the peptide-loading complex (PLC). The transporter module of the PLC pumps antigens into the ER, whereas other parts of the PLC inside the ER accelerate selective antigen loading onto MHCs. As long as the MHCs have not found a suitable antigen, they are unstable and must be protected by accompanying proteins called chaperones. Only when firmly bound to their peptide antigen, are MHCs allowed to leave the ER and travel to the cell surface where they display their cargo to the outside world, including patrolling members of the immune system. Given the central role of the PLC in blowing their cover, it is not surprising that several viruses, such as herpes and pox viruses, have evolved means to sabotage the PLC in order to hide from the immune system.
While the key players of antigen presentation have been identified, many crucial aspects of this process remain uncharacterized. The main goal of this project is to contribute to a more comprehensive understanding of antigen presentation, in particular the interplay between the various protein components that enable the immune system to mount a specific response to ailing cells. Vaccine development and the design of novel therapies against infectious diseases, autoimmune disorders, transplant rejection, and cancer will directly benefit from a deeper knowledge of events leading to MHC-mediated immune reactions.
For the surveillance system to work, cells have developed a sophisticated machinery not only to transport antigens into the endoplasmic reticulum (ER), a dedicated subcellular compartment, for redirection to the cell surface, but also to select antigens that bind tightly to the presenting MHC molecules and to facilitate antigen loading onto MHCs. A central hub in this machinery is a large assembly termed the peptide-loading complex (PLC). The transporter module of the PLC pumps antigens into the ER, whereas other parts of the PLC inside the ER accelerate selective antigen loading onto MHCs. As long as the MHCs have not found a suitable antigen, they are unstable and must be protected by accompanying proteins called chaperones. Only when firmly bound to their peptide antigen, are MHCs allowed to leave the ER and travel to the cell surface where they display their cargo to the outside world, including patrolling members of the immune system. Given the central role of the PLC in blowing their cover, it is not surprising that several viruses, such as herpes and pox viruses, have evolved means to sabotage the PLC in order to hide from the immune system.
While the key players of antigen presentation have been identified, many crucial aspects of this process remain uncharacterized. The main goal of this project is to contribute to a more comprehensive understanding of antigen presentation, in particular the interplay between the various protein components that enable the immune system to mount a specific response to ailing cells. Vaccine development and the design of novel therapies against infectious diseases, autoimmune disorders, transplant rejection, and cancer will directly benefit from a deeper knowledge of events leading to MHC-mediated immune reactions.
The PLC is the central hub of MHC molecules for being loaded with antigen on their way to the cell surface. It is composed of several building blocks, each with its own distinct function. The main building block, the transporter associated with antigen processing (TAP), ferries peptide antigens into the ER where they are loaded onto the MHCs. The loading is supported by the remaining building blocks of the PLC. While the constituents of the PLC have been known for quite some time, its architecture has remained undefined. We were able to isolate the PLC from human cells and subject it to a technique called cryogenic electron microscopy (cryo-EM). Cryo-EM makes it possible to visualize tiny protein particles at a resolution close to ten millionth of a millimeter. This allowed us to elucidate the architecture of the PLC assembly: two so-called “editing modules”, each consisting of the MHC and the chaperones tapasin, ERp57, and calreticulin, are positioned around TAP. The chaperones stabilize the MHC while peptides are being loaded and selected. Loading and selection are actively supported by tapasin in a process named peptide proofreading or editing. When a strongly binding peptide antigen has been found, the complex of MHC and antigen can leave the PLC. Thus, the PLC is a highly dynamic machine with a constant turnover of its components.
To study the mechanistic principles of facilitated loading and selection of peptide antigens on MHCs, we turned to a protein termed TAPBPR that is related to tapasin and has the same activity, i.e. it protects empty MHCs and examines peptides for their ability to bind to MHCs. We crystallized empty MHC in complex with TAPBPR and irradiated the crystals using X-rays. This allowed us to see in detail how TAPBPR interacts with unladen MHC: TAPBPR clings to the MHC in such a manner that the MHC’s peptide-binding site is kept in an open state, and only strongly binding peptide antigens are able to free the MHC from TAPBPR’s cuddle. The structure of the TAPBPR-MHC complex also illustrates a striking feature of MHCs: they are highly malleable molecules, and this plasticity is crucial for their physiological function. Using complementary biochemical studies of TAPBPR variants enabled us to dissect the contribution of different structural elements to TAPBPR’s chaperone and proofreading activity.
The TAP transporter of the PLC belongs to a large family of evolutionarily related molecular machines that translocate various substances across cellular membranes. Translocation is coupled to dramatic changes of the machines’ shape, hence only by thoroughly characterizing these changes, one can fully comprehend the mechanism of their activity. In order to investigate the mechanism of TAP, we utilized cryo-EM to study a structural and functional relative of TAP called TmrAB. We were able to take nine different snapshots of the transporter in action, showing all major structural changes. In combination with biochemical experiments, the snapshots substantially advanced our understanding of peptide-translocating molecular machines.
To study the mechanistic principles of facilitated loading and selection of peptide antigens on MHCs, we turned to a protein termed TAPBPR that is related to tapasin and has the same activity, i.e. it protects empty MHCs and examines peptides for their ability to bind to MHCs. We crystallized empty MHC in complex with TAPBPR and irradiated the crystals using X-rays. This allowed us to see in detail how TAPBPR interacts with unladen MHC: TAPBPR clings to the MHC in such a manner that the MHC’s peptide-binding site is kept in an open state, and only strongly binding peptide antigens are able to free the MHC from TAPBPR’s cuddle. The structure of the TAPBPR-MHC complex also illustrates a striking feature of MHCs: they are highly malleable molecules, and this plasticity is crucial for their physiological function. Using complementary biochemical studies of TAPBPR variants enabled us to dissect the contribution of different structural elements to TAPBPR’s chaperone and proofreading activity.
The TAP transporter of the PLC belongs to a large family of evolutionarily related molecular machines that translocate various substances across cellular membranes. Translocation is coupled to dramatic changes of the machines’ shape, hence only by thoroughly characterizing these changes, one can fully comprehend the mechanism of their activity. In order to investigate the mechanism of TAP, we utilized cryo-EM to study a structural and functional relative of TAP called TmrAB. We were able to take nine different snapshots of the transporter in action, showing all major structural changes. In combination with biochemical experiments, the snapshots substantially advanced our understanding of peptide-translocating molecular machines.
By employing cutting-edge methodologies, we have been able to determine the first structure of the native, fully assembled human PLC and to shed light on its highly dynamic nature. Moreover, by solving the structure of the TAPBPR-MHC complex, we could reveal the core mechanistic principles of peptide proofreading on MHC molecules. Finally, the study of TmrAB in a native-like environment made it possible for the first time to delineate the full operational cycle of this kind of transporter and thereby reveal the underpinnings of peptide translocation accelerated by the core component of the PLC. Future studies will concentrate on the cooperation and crosstalk between the various players of antigen presentation. Furthermore, we expect to gain more insights into the numerous strategies of viruses to put the PLC out of action. We are confident that future results of this project will further extend our knowledge of the antigen presentation pathway significantly and help to explore new avenues for therapeutic interventions.