Subject(s)
Information Dissemination , Publishing , Research , Software , Access to Information , Disclosure , Editorial Policies , Public Policy , Research Support as TopicABSTRACT
We have determined high-resolution crystal structures of the complexes of HLA-A2 molecules with two modified immunodominant peptides from the melanoma tumor-associated protein Melan-A/Melanoma Ag recognized by T cells-1. The two peptides, a decamer and nonamer with overlapping sequences (ELAGIGILTV and ALGIGILTV), are modified in the second residue to increase their affinity for HLA-A2. The modified decamer is more immunogenic than the natural peptide and a candidate for peptide-based melanoma immunotherapy. The crystal structures at 1.8 and 2.15 A resolution define the differences in binding modes of the modified peptides, including different clusters of water molecules that appear to stabilize the peptide-HLA interaction. The structures suggest both how the wild-type peptides would bind and how three categories of cytotoxic T lymphocytes with differing fine specificity might recognize the two peptides.
Subject(s)
Antigens, Neoplasm/chemistry , HLA-A2 Antigen/chemistry , Neoplasm Proteins/chemistry , Amino Acid Sequence , Antigens, Neoplasm/metabolism , Binding Sites , Crystallography, X-Ray , HLA-A2 Antigen/metabolism , Humans , MART-1 Antigen , Macromolecular Substances , Melanoma/immunology , Models, Molecular , Neoplasm Proteins/metabolism , Peptide Fragments/chemistry , Peptide Fragments/metabolism , Pliability , Protein Binding , Protein Conformation , T-Lymphocytes, Cytotoxic/immunology , WaterABSTRACT
BACKGROUND: The bacterial phosphoenolpyruvate: sugar phosphotransferase system (PTS) is responsible for the binding, transmembrane transport and phosphorylation of numerous sugar substrates. The system is also involved in the regulation of a variety of metabolic and transcriptional processes. The PTS consists of two non-specific energy coupling components, enzyme I and a heat stable phosphocarrier protein (HPr), as well as several sugar-specific multiprotein permeases known as enzymes II. In most cases, enzymes IIA and IIB are located in the cytoplasm, while enzyme IIC acts as a membrane channel. Enzyme IIAlactose belongs to the lactose/cellobiose-specific family of enzymes II, one of four functionally and structurally distinct groups. The protein, which normally functions as a trimer, is believed to separate into its subunits after phosphorylation. RESULTS: The crystal structure of the trimeric enzyme IIAlactose from Lactococcus lactis has been determined at 2.3 A resolution. The subunits of the enzyme, related to each other by the inherent threefold rotational symmetry, possess interesting structural features such as coiled-coil-like packing and a methionine cluster. The subunits each comprise three helices (I, II and III) and pack against each other forming a nine-helix bundle. This helical bundle is stabilized by a centrally located metal ion and also encloses a hydrophobic cavity. The three phosphorylation sites (His78 on each monomer) are located in helices III and their sidechains protrude into a large groove between helices I and II of the neighbouring subunits. A model of the complex between phosphorylated HPr and enzyme IIAlactose has been constructed. CONCLUSIONS: Enzyme IIAlactose is the first representative of the family of lactose/cellobiose-specific enzymes IIA for which a three-dimensional structure has been determined. Some of its structural features, like the presence of two histidine residues at the active site, seem to be common to all enzymes no overall structural homology is observed to any PTS proteins or to any other proteins in the Protein Data Bank. Enzyme IIAlactose shows surface complementarity to the phosphorylated form of HPr and several energetically favourable interactions between the two molecules can be predicted.
Subject(s)
Bacterial Proteins , Lactococcus lactis/enzymology , Lactose/metabolism , Phosphoenolpyruvate Sugar Phosphotransferase System/chemistry , Phosphoenolpyruvate Sugar Phosphotransferase System/metabolism , Amino Acid Sequence , Crystallography, X-Ray , Metals/chemistry , Metals/metabolism , Methionine , Models, Molecular , Molecular Sequence Data , Protein Conformation , Protein Structure, Secondary , Protein Structure, Tertiary , Sequence Homology, Amino Acid , Staphylococcus aureus/enzymologyABSTRACT
Lactose-specific enzyme IIA of the phosphoenol:pyruvate-dependent sugar phosphotransferase system from Lactococcus lactis has been crystallized in phosphate buffer. The crystals belong to space group P4(1)2(1)2 or its enantiomorph P4(3)2(1)2 with unit-cell axes a = b = 90.9 and c = 82.4 A. The packing parameter (Matthews parameter) V(m) of 2.48 A(3) Da(-1) is consistent with one trimer per asymmetric unit and non-crystallographic threefold symmetry has been confirmed by calculating a self-rotation function. The crystals diffract X-rays to at least 2.3 A resolution, are stable in an X-ray beam and are therefore appropriate for structure determination. Native data to 2.3 A resolution have been collected using a MAR image-plate system at a synchrotron source. One isomorphous heavy-atom derivative has been identified and the presence of an isomorphous signal in the data has been confirmed by Patterson methods.
