Conformational Lability in MHC II Proteins: A Dissertation

MHC II proteins are heterodimeric glycoproteins that form complexes with antigenic peptides in order to elicit a CD4+ adaptive immune response. Even though there have been numerous MHC II-peptide crystal structures solved, there is little insight into the dynamic process of peptide loading. Through biochemical and biophysical studies, it has been shown that MHC II adopt multiple conformations throughout the peptide loading process. At least one of these conformations is stabilized by the MHC II-like homologue, HLA-DM. The main focus of this thesis is to elucidate alternate conformers of MHC II in an effort to better understand the structural features that enable HLA-DM catalyzed peptide loading. In this thesis, two altered conformations of HLA-DR were investigated, one modeled in the absence of peptide using molecular dynamics, and one stabilized by the mutation αF54C. The model for the peptide-free form of HLA-DR1 was derived from a molecular dynamics simulation. In this model, part of the alpha-subunit extended-strand region proximal to the peptide binding groove is folded into the peptide-binding groove such that the architecture of the critical peptide binding pocket, P1, as well as the invariant hydrogen bonding network were maintained. Biochemical studies aimed at validating the predicted structural changes were consistent with the model generated from the simulations. Next, structural studies were carried out on an MHC II mutant, αF54C, which was shown to have unique peptide binding characteristics as well as enhanced susceptibility to HLA-DM. Although this mutation did not affect the affinity for peptide, there was a striking increase in the rate of intrinsic peptide release. Both αF54C and αF54A were over 100-fold more susceptible to HLADM catalyzed peptide release than wild type as well as other mutants introduced along the peptide binding groove. In addition, mutation of the αF54 position results in a higher affinity for HLA-DM, which, unlike wild type, is detectable by surface plasmon resonance. Crystallographic studies resulted in a 2.3 Å resolution structure for the αF54C-Clip complex. There were two molecules in the asymmetric unit, one of which had no obvious deviations from other MHC II-pep complexes and one which had a conformational change as a result of a crystal contact on the αF51 residue, a residue which has been shown to be involved in the HLA-DM/HLA-DR binding interface. The crystal structure of wild type HLA-DR1- Clip was also solved, but did not have the altered conformation even though there was a similar crystal contact at the αF51. These data suggest the altered conformation seen in the mutant structure, results from increased lability in the extended stand region due to the αF54C mutation. As a result of this work, we have developed a new mechanistic model for how structural features of MHC II influence DM mediated peptide release

Baubles, Bangles, and Biotypes: A Critical Review of the use and Abuse of the Biotype Concept

Pest species of insects are notoriously prone to escape the weapons deployed in management efforts against them. This is particularly true in herbivorous insects. When a previously successful tactic fails the insect population has apparently adapted to it and is often considered to be a new or distinct entity, and given the non-formal category ‘biotype’. The entities falling under the umbrella term ‘biotype’ are not consistent either within or between biotypes, and their underlying genetic composition and origins, while generally unknown, are likely heterogeneous within and variable between biotypes. In some cases race or species may be more appropriate referents. Some examples of applications of the concept in the context of host plant resistance are discussed. It is argued here that the term ‘biotype’ and its applications are overly simplistic, confused, have not proved useful in current pest management, and lack predictive power for future management

Model for the Peptide-Free Conformation of Class II MHC Proteins

Background: Major histocompatibility complex proteins are believed to undergo significant conformational changes concomitant with peptide binding, but structural characterization of these changes has remained elusive. Methodology/Principal Findings: Here we use molecular dynamics simulations and experimental probes of protein conformation to investigate the peptide-free state of class II MHC proteins. Upon computational removal of the bound peptide from HLA-DR1-peptide complex, the a50-59 region folded into the P1-P4 region of the peptide binding site, adopting the same conformation as a bound peptide. Strikingly, the structure of the hydrophobic P1 pocket is maintained by engagement of the side chain of Phe a54. In addition, conserved hydrogen bonds observed in crystal structures between the peptide backbone and numerous MHC side chains are maintained between the a51-55 region and the rest of the molecule. The model for the peptide-free conformation was evaluated using conformationally-sensitive antibody and superantigen probes predicted to show no change, moderate change, or dramatic changes in their interaction with peptide-free DR1 and peptide-loaded DR1. The binding observed for these probes is in agreement with the movements predicted by the model. Conclusion/Significance: This work presents a molecular model for peptide-free class II MHC proteins that can help to interpret the conformational changes known to occur within the protein during peptide binding and release, and ca

Crowdsourcing a crisis response for COVID-19 in oncology

Crowdsourcing efforts are currently underway to collect and analyze data from patients with cancer who are affected by the COVID-19 pandemic. These community-led initiatives will fill key knowledge gaps to tackle crucial clinical questions on the complexities of infection with the causative coronavirus SARS-Cov-2 in the large, heterogeneous group of vulnerable patients with cancer

HLA-DO acts as a substrate mimic to inhibit HLA-DM by a competitive mechanism

Mammalian class II major histocompatibility (MHCII) proteins bind peptide antigens in endosomal compartments of antigen-presenting cells. The nonclassical MHCII protein HLA-DM chaperones peptide-free MHCII, protecting it against inactivation, and catalyzes peptide exchange on loaded MHCII. Another nonclassical MHCII protein, HLA-DO, binds HLA-DM and influences the repertoire of peptides presented by MHCII proteins. However, the mechanism by which HLA-DO functions is unclear. Here we have used X-ray crystallography, enzyme kinetics and mutagenesis approaches to investigate human HLA-DO structure and function. In complex with HLA-DM, HLA-DO adopts a classical MHCII structure, with alterations near the alpha subunit\u27s 3(1)(0) helix. HLA-DO binds to HLA-DM at the same sites implicated in MHCII interaction, and kinetic analysis showed that HLA-DO acts as a competitive inhibitor. These results show that HLA-DO inhibits HLA-DM function by acting as a substrate mimic, and the findings also limit the possible functional roles for HLA-DO in antigen presentation

Noble metals strip peptides from class II MHC proteins

Class II major histocompatibility complex (MHC) proteins are essential for normal immune system function but also drive many autoimmune responses. They bind peptide antigens in endosomes and present them on the cell surface for recognition by CD4(+) T cells. A small molecule could potentially block an autoimmune response by disrupting MHC-peptide interactions, but this has proven difficult because peptides bind tightly and dissociate slowly from MHC proteins. Using a high-throughput screening assay we discovered a class of noble metal complexes that strip peptides from human class II MHC proteins by an allosteric mechanism. Biochemical experiments indicate the metal-bound MHC protein adopts a \u27peptide-empty\u27 conformation that resembles the transition state of peptide loading. Furthermore, these metal inhibitors block the ability of antigen-presenting cells to activate T cells. This previously unknown allosteric mechanism may help resolve how gold(I) drugs affect the progress of rheumatoid arthritis and may provide a basis for developing a new class of anti-autoimmune drugs

Identification and characterization of T reg–like cells in zebrafish

Regulatory T (T reg) cells are a specialized sublineage of T lymphocytes that suppress autoreactive T cells. Functional studies of T reg cells in vitro have defined multiple suppression mechanisms, and studies of T reg–deficient humans and mice have made clear the important role that these cells play in preventing autoimmunity. However, many questions remain about how T reg cells act in vivo. Specifically, it is not clear which suppression mechanisms are most important, where T reg cells act, and how they get there. To begin to address these issues, we sought to identify T reg cells in zebrafish, a model system that provides unparalleled advantages in live-cell imaging and high-throughput genetic analyses. Using a FOXP3 orthologue as a marker, we identified CD4-enriched, mature T lymphocytes with properties of T reg cells. Zebrafish mutant for foxp3a displayed excess T lymphocytes, splenomegaly, and a profound inflammatory phenotype that was suppressed by genetic ablation of lymphocytes. This study identifies T reg–like cells in zebrafish, providing both a model to study the normal functions of these cells in vivo and mutants to explore the consequences of their loss