Inflammation-Associated Nitrotyrosination Affects TCR Recognition through Reduced Stability and Alteration of the Molecular Surface of the MHC Complex

Nitrotyrosination of proteins, a hallmark of inflammation, may result in the production of MHC-restricted neoantigens that can be recognized by T cells and bypass the constraints of immunological self-tolerance. Here we biochemically and structurally assessed how nitrotyrosination of the lymphocytic choriomeningitis virus (LCMV)-associated immunodominant MHC class I-restricted epitopes gp33 and gp34 alters T cell recognition in the context of both H-2Db and H-2Kb. Comparative analysis of the crystal structures of H-2Kb/gp34 and H-2Kb/NY-gp34 demonstrated that nitrotyrosination of p3Y in gp34 abrogates a hydrogen bond interaction formed with the H-2Kb residue E152. As a consequence the conformation of the TCR-interacting E152 was profoundly altered in H-2Kb/NY-gp34 when compared to H-2Kb/gp34, thereby modifying the surface of the nitrotyrosinated MHC complex. Furthermore, nitrotyrosination of gp34 resulted in structural over-packing, straining the overall conformation and considerably reducing the stability of the H-2Kb/NY-gp34 MHC complex when compared to H-2Kb/gp34. Our structural analysis also indicates that nitrotyrosination of the main TCR-interacting residue p4Y in gp33 abrogates recognition of H-2Db/gp33-NY complexes by H-2Db/gp33-specific T cells through sterical hindrance. In conclusion, this study provides the first structural and biochemical evidence for how MHC class I-restricted nitrotyrosinated neoantigens may enable viral escape and break immune tolerance

Crystal structures of designed armadillo repeat proteins: Implications of construct design and crystallization conditions on overall structure

Designed armadillo repeat proteins (dArmRP) are promising modular proteins for the engineering of binding molecules that recognize extended polypeptide chains. We determined the structure of a dArmRP containing five internal repeats and 3rd generation capping repeats in three different states by X-ray crystallography: without N-terminal His6 -tag and in the presence of calcium (YM5 A/Ca(2+) ), without N-terminal His6 -tag and in the absence of calcium (YM5 A), and with N-terminal His6 -tag and in the presence of calcium (His-YM5 A/Ca(2+) ). All structures show different quaternary structures and superhelical parameters. His-YM5 A/Ca(2+) forms a crystallographic dimer, which is bridged by the His6 -tag, YM5 A/Ca(2+) forms a domain-swapped tetramer, and only in the absence of calcium and the His6 -tag, YM5 A forms a monomer. The changes of superhelical parameters are a consequence of calcium binding, because calcium ions interact with negatively charged residues, which can also participate in the modulation of helix dipole moments between adjacent repeats. These observations are important for further optimizations of dArmRPs and provide a general illustration of how construct design and crystallization conditions can influence the exact structure of the investigated protein

Structures of designed armadillo repeat proteins binding to peptides fused to globular domains

Designed armadillo repeat proteins (dArmRP) are α-helical solenoid repeat proteins with an extended peptide binding groove that were engineered to develop a generic modular technology for peptide recognition. In this context, the term "peptide" not only denotes a short unstructured chain of amino acids, but also an unstructured region of a protein, as they occur in termini, loops, or linkers between folded domains. Here we report two crystal structures of dArmRPs, in complex with peptides fused either to the N-terminus of Green Fluorescent Protein or to the C-terminus of a phage lambda protein D. These structures demonstrate that dArmRPs bind unfolded peptides in the intended conformation also when they constitute unstructured parts of folded proteins, which greatly expands possible applications of the dArmRP technology. Nonetheless, the structures do not fully reflect the binding behavior in solution, that is, some binding sites remain unoccupied in the crystal and even unexpected peptide residues appear to be bound. We show how these differences can be explained by restrictions of the crystal lattice or the composition of the crystallization solution. This illustrates that crystal structures have to be interpreted with caution when protein-peptide interactions are characterized, and should always be correlated with measurements in solution

Structure and Energetic Contributions of a Designed Modular Peptide-Binding Protein with Picomolar Affinity

Natural armadillo repeat proteins (nArmRP) like importin-α or β-catenin bind their target peptides such that each repeat interacts with a dipeptide unit within the stretched target peptide. However, this modularity is imperfect and also restricted to short peptide stretches of usually four to six consecutive amino acids. Here we report the development and characterization of a regularized and truly modular peptide-specific binding protein, based on designed armadillo repeat proteins (dArmRP), binding to peptides of alternating lysine and arginine residues (KR)n. dArmRP were obtained from nArmRP through cycles of extensive protein engineering, which rendered them more uniform. This regularity is reflected in the consistent binding of dArmRP to (KR)-peptides, where affinities depend on the lengths of target peptides and the number of internal repeats in a very systematic manner, thus confirming the modularity of the interaction. This exponential dependency between affinity and recognition length suggests that each module adds a constant increment of binding energy to sequence-specific recognition. This relationship was confirmed by comprehensive mutagenesis studies that also reveal the importance of individual peptide side chains. The 1.83 Å resolution crystal structure of a dArmRP with five identical internal repeats in complex with the cognate (KR)5 peptide proves a modular binding mode, where each dipeptide is recognized by one internal repeat. The confirmation of this true modularity over longer peptide stretches lays the ground for the design of binders with different specificities and tailored affinities by the assembly of dipeptide-specific modules based on armadillo repeats

Crystal structure of a fungal protease inhibitor from Antheraea mylitta

Indian tasar silk is produced by a wild insect called Antheraea mylitta. Insects do not have any antigen-antibody mediated immune system like vertebrates but they produce a wide variety of effector proteins and peptides possessing potent antifungal and antibacterial activity to combat microbial attack. Antheraea mylitta expresses a fungal protease inhibitor AmFPI-1, in the hemolymph that inhibits alkaline protease of Aspergillus oryzae for protection against fungal infection. AmFPI-1 is purified from the hemolymph, crystallized and the structure is solved using the single isomorphous replacement with anomalous scattering (SIRAS) method to a resolution of 2.1 Å. AmFPI-1 is a single domain protein possessing a unique fold that consists of three helices and five β strands stabilized by a network of six disulfide bonds. The reactive site of AmFPI-1 is located in the loop formed by residues 46-66, wherein Lys54 is the P1 residue. Superimposition of the loop with reactive sites of other canonical protease inhibitors shows that reactive site conformation of AmFPI-1 is similar to them. The structure of AmFPI-1 provides a framework for the docking of a 1:1 complex between AmFPI-1 and alkaline protease. This study addresses the structural basis of AmFPI-1's specificity towards a fungal serine protease but not to mammalian trypsin and may help in designing specific inhibitors against fungal proteases