MEMBRANE GLYCOPROTEINS FROM VIRUSES & FROM HUMAN CELLS

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In 1997/98 we collected data at CHESS that allowed us to determine the structures of human endostatin, a potent antiangiogenic factor; HLA-DR2 complexed with Myelin basic peptide antigen, a dominant antigen found in multiple sclerosis patients; human DM, the enzyme that loads antigenic peptides into the kinetic trap of class II MHC molecules; and the structure of a second complex of the class I MHC molecule HLA-A2 complexed with the Tax peptide from HTLV-1 being recognized by a T cell receptor (TCR). All of those studies are either published, submitted, or in the late stage of preparation of a manuscript. We began studies on the Herpes virus glycoprotein gD, the Pox virus chemokine receptor protein P32 and its complex with the chemokine MCP-1, HLA-Cw4 a class I MHC molecule that is the target of a Natural Killer cell receptor whose structure we determined earlier, and the structure of three mutant peptides, an agonist, partial agonist, and a null in complex with HLA-A2 and the same TCR. This last set of crystals will allow us to assess whether any conformational differences in the TCR are induced by peptides that cause different signalling outcomes in T cells. The synchrotron data collected at CHESS remains critical to the progress of a number of projects in our laboratory. 1. Herpes Simplex Virus gD glycoprotein gD285 is a C-terminal truncated form of the envelope glycoprotein gD from HSV-1. We collected 20 degree (test) on a very small crystal (0.1x0.03x0.03um3). The crystal belongs to the orthorhombic space group P222 with cell parameter: a=81 A b=124 A c=130 A. The data are 30% complete between 30 and 3.5
(4704 reflections). The Rsym = 6.7% (15% in the last resolution shell). 2. Poxvirus P32 Chemokine Receptor p32-mcp1 complex. P32 is a secreted soluble protein from Cowpox virus which binds with very high affinity mcp1 (macrophage chemotactic protein-1) and generally all the beta-chemokines. A 3.6
data set of the p32-mcp1 complex was collected on a single crystal. The space group is P6522 (or P6122) with a=b=69.3 A c=454.6
. The data set is 87% complete (overall) with an Rsym of 4.9% (8.5% in the last resolution bin) The data were collected on the F1 beam line on the Q4 CCD. 3. Human T-cell Receptor B7/MHC/Tax peptide complex: CHESS A1 station, ADSC 1K CCD detector: three data sets of human B7 TCR complexed to HLA-A2 Tax peptide were collected. The best set goes to 2.5
resolution. The structure was reported in Immunity. 4.-Human A6 TCR/MHC/Mutant Peptide Complexes: CHESS A1 station, ADSC 1K CCD detector: Two data sets for human A6 TCR complexed with HLA-A2 with mutated peptide, structure determination is underway. The crystals with Y8A peptide give 2.8
data set, the Y5A peptide gave 3.5
data. Refinement is underway. 5. Human Endostatin: One data set from a crystal of human endostatin was collected and goes to 2
. The protein is missing the first 4 amino acids from the N-terminus. One data set of full length human endostatin data was also collected. In this crystal form, the protein has the full sequence, and crystallized in a different space group. The set data goes to 2.9
with 66% completeness at 2.9-3.0
shell and overall completeness is 89%. The structure is solved and paper is being written. 6. HLA-Cw4, the target of the NK cell receptor KIR-1: Crystals of the human class I MHC molecule HLA-Cw4 complexed with the peptide, QYDDAVYKL, diffract to 2.7
resolution. A complete data set has been collected from a single frozen HLA-Cw4 crystal (with the dimension 35 mm x 35 mm x 600 mm) at the A1 beamline in May 1997. 7. HLA-DM the peptide exchange factor that loads class II MHC molecules: Diffraction data were collected at A1 station, ADSC 1K CCD. Two native data sets were collected at cryogenic temperatures (100K) from crystals of HLA-DM alone. The crystals belong to the space group P212121 with cell dimensions a=95.9A, b=106.3A and c=107.3A, and diffract to 2.5
resolution. The first data set (35-2.7A) is 99.3% complete with 8-fold redundancy and overall Rsym of 8.7%. The second one (35-2.5
) is 99.9% complete with 7-fold redundancy and overall Rsym of 6.7%. These data were used for the molecular replacement searching and helped interpretation of the solution. The HLA-DM structure was solved and refined using the data set collected in-house (MAR detector) from crystals having another orthorhombic form (C2221). The paper on this structure has been submitted. 7. HLA-DR2/Myelin Basic Protein Peptide: The MHC class II molecule HLA DR2 has been shown to be associated with susceptibility to the autoimmune disease Multiple Sclerosis (MS) (Wucherpfennig et al). The structure of HLA DR2 complexed with an immunodominant peptide (ENPVVHFFKNIVTPR) from myelin basic protein (MBP) was determined to 2.6
resolution by x-ray crystallography from data collected at CHESS on A1. The structure shows that Phe at position P4 of the MBP peptide binds in a largely hydrophobic pocket interacting with two aromatic side chains Tyrb78 and Pheb26 as well as Glnb70, Alab71, Aspb28 and Argb13. The DR2 P4 pocket most closely resembles the DR4 P4 pocket (Dessen et al), with only residues b71 (Ala in DR2, Lys in DR4) and b13 (Arg in DR2, His in DR4) differing between the two alleles. The polymorphism at position 71 in DR2 appears to be most important in creating the available space for the P4 aromatic side chain of the MBP peptide. Experiments in which the P4 position of the MBP peptide was substituted to different amino acids shows that the DR2 P4 pocket is capable of binding aromatic and positively charged sides chains but cannot tolerate negatively charged side chains (Wucherpfennig et al). The structural basis for this side chain specificity is clear from the structure of the DR2 P4 pocket, which reveals a large predominantly hydrophobic pocket; the presence of negatively charged Aspb28 at the base of the pocket would facilitate the binding of positively charged side chains but be unfavorable for the binding of negatively side chains. The ability of an MHC class II P4 pocket to bind aromatic P4 side chains is unusual and it is this feature of DR2 which is crucial for the binding of the immunodominant MBP peptide. In addition, it has been shown that peptide positions P2 His, P3 Phe and P5 Lys are important for recognition by DR2/MBP specific T cell clones (Wucherpfennig et al). The DR2/MBP crystal structure shows that these peptide sides chains are solvent exposed and available for recognition by T cell receptor. Comparison with the recently published high resolution crystal structures of MHC class I/peptide/TCR complexes (refs)