During my training period, I determined theatomic structures of three proteins: Phospholipase A2 from Indian cobra, anti-peptide antibody B13A2 (raised against the 19-mer C-terminal helixpeptide of myohemerythrin), and murine MHC Class I Kb. I completed my experimental work on the phospholipase A2 and this work can be summarized as follows. Phospholipase A2 (PLA2) purified from Indian cobra venom (Naja naja naja) was crystallized from ethanol in spacegroup P4 3 2 1 2 with cell dimensions a = b = 88.6 Angstrom, c = 107.4 Angstrom in the presence of Ca2. The X-ray crystal structure was determined to 2.3 Angstrom resolution by molecular replacement techniques using a theoretical model constructed from homologous segments of the bovine pancreatic, porcine pancreatic, and rattlesnake venom crystal structures. The structure was refined to an R-value of 0.174 for 17,542 reflections between 6.0 - 2.3 Angstrom resolution, including 148 water molecules. The 119 amino acid enzyme has an overall architecture strikingly similar to the other PLA2 structures solved to date. Regions implicated in catalysis show the greatest degree of structural homology. Unexpectedly, three monomers were found to occupy the asymmetric unit, and are oriented with their catalytic sites facing the pseudo three-fold axis. Approximately 15% of the solvent accessible surface of each monomer is buried in trimer contacts. The majority of the interactions at the subunit interfaces are made by residues unique to PLA2 sequences from cobra and krait venoms (Group IA). The relevance of this unique trimeric structure to the aggregation observed with cobra venom PLA2 is considered. Future studies carried out by another graduate student, Brent Segelke, in the Xuong and Dennis laboratories will examine substrate analogue and inhibitor complexes with the enzyme. I have also invested considerable research effort pursuing crystallographic studies of the murine MHC Class I Kb antigen in collaboration with the Wilson lab at Scripp's Research Clinic. Progress has been rapid and exciting. Two specific viral peptide Kb complexes were refined during this period (one to 2.3 Angstrom, another to 2.5 Angstrom resolution) and a third is well on its way. This represents the first structure of a murine class I antigen and the first detailed structure analysis of an MHC molecule bound to two different peptides. The results of this project indicate that peptides bind into the Class I membrane distal groove in an extended conformation with a high degree of structural conservation at the N- and C- terminae. Only a small fraction of the molecular surface of the viral peptides are exposed for T-cell recognition after binding into the Class I groove. Small but significant structural differences in the MHC structure itself are observed that are likely to be important in the T-cell recognition of infected cells. Future work on this project will include higher resolution data collection and refinements of the already solved structures, additional peptide complexes, and perhaps the structure determination of the peptide free (empty) MHC molecule.

PUBLICATIONS (resulting from this training, and some recent ones)

Fremont DH, Matsumura M, Stura EA, Peterson PA, Wilson IA. (1992) Crystal structures of two viral peptides in complex with murine MHC class I H-2Kb. Science. 257:919-27.

Matsumura M, Fremont DH, Peterson PA, Wilson IA. (1992) Emerging principles for the recognition of peptide antigens by MHC class I molecules. Science. 257:927-34.

Fremont DH, Anderson DH, Wilson IA, Dennis EA, Xuong NH. (1993) Crystal structure of phospholipase A2 from Indian cobra reveals a trimeric association. Proc Natl Acad Sci U S A. 90:342-6.

Fremont DH, Stura EA, Matsumura M, Peterson PA, Wilson IA. (1995) Crystal structure of an H-2Kb-ovalbumin peptide complex reveals the interplay of primary and secondary anchor positions in the major histocompatibility complex binding groove. Proc Natl Acad Sci U S A. 92:2479-83.

Fremont DH, Hendrickson WA, Marrack P, Kappler J. (1996) Structures of an MHC class II molecule with covalently bound single peptides. Science. 272:1001-4.

Fremont DH, Monnaie D, Nelson CA, Hendrickson WA, Unanue ER. (1998) Crystal structure of I-Ak in complex with a dominant epitope of lysozyme. Immunity. 8:305-17.

Fremont DH, Crawford F, Marrack P, Hendrickson WA, Kappler J. (1998) Crystal structure of mouse H2-M. Immunity. 9:385-93.

Traub LM, Downs MA, Westrich JL, Fremont DH. (1999) Crystal structure of the alpha appendage of AP-2 reveals a recruitment platform for clathrin-coat assembly. Proc Natl Acad Sci U S A. 96:8907-12.

Nelson CA, Fremont DH. (1999) Structural principles of MHC class II antigen presentation. Rev Immunogenet. 1:47-59. Review.

Latek RR, Suri A, Petzold SJ, Nelson CA, Kanagawa O, Unanue ER, Fremont DH. (2000) Structural basis of peptide binding and presentation by the type I diabetes-associated MHC class II molecule of NOD mice. Immunity. 12:699-710.

Kersh GJ, Miley MJ, Nelson CA, Grakoui A, Horvath S, Donermeyer DL, Kappler J, Allen PM, Fremont DH. (2001) Structural and functional consequences of altering a peptide MHC anchor residue. J Immunol. 166:3345-54.

Messaoudi I, LeMaoult J, Metzner BM, Miley MJ, Fremont DH, Nikolich-Zugich J. (2001) Functional evidence that conserved TCR CDR alpha 3 loop docking governs the cross-recognition of closely related peptide:class I complexes. J Immunol. 167:836-43.

Lam J, Nelson CA, Ross FP, Teitelbaum SL, Fremont DH. (2001) Crystal structure of the TRANCE/RANKL cytokine reveals determinants of receptor-ligand specificity. J Clin Invest. 108:971-9.

Arentson E, Faloon P, Seo J, Moon E, Studts JM, Fremont DH, Choi K. (2002) Oncogenic potential of the DNA replication licensing protein CDT1. Oncogene. 21:1150-8.

Facchetti F, Cella M, Festa S, Fremont DH, Colonna M. (2002) An unusual Fc receptor-related protein expressed in human centroblasts. Proc Natl Acad Sci U S A. 99:3776-81.

Fremont DH, Dai S, Chiang H, Crawford F, Marrack P, Kappler J. (2002) Structural basis of cytochrome c presentation by IE(k). J Exp Med. 195:1043-52.

Smith HR, Heusel JW, Mehta IK, Kim S, Dorner BG, Naidenko OV, Iizuka K, Furukawa H, Beckman DL, Pingel JT, Scalzo AA, Fremont DH, Yokoyama WM. (2002) Recognition of a virus-encoded ligand by a natural killer cell activation receptor. Proc Natl Acad Sci U S A. 99:8826-31.

Carayannopoulos LN, Naidenko OV, Fremont DH, Yokoyama WM. (2002) Cutting edge: murine UL16-binding protein-like transcript 1: a newly described transcript encoding a high-affinity ligand for murine NKG2D. J Immunol. 169:4079-83.

Alexander JM, Nelson CA, van Berkel V, Lau EK, Studts JM, Brett TJ, Speck SH, Handel TM, Virgin HW, Fremont DH. (2002) Structural basis of chemokine sequestration by a herpesvirus decoy receptor. Cell. 111:343-56.

Miley MJ, Truscott SM, Yu YY, Gilfillan S, Fremont DH, Hansen TH, Lybarger L. (2003) Biochemical features of the MHC-related protein 1 consistent with an immunological function. J Immunol. 170:6090-8.

Iizuka K, Naidenko OV, Plougastel BF, Fremont DH, Yokoyama WM. (2003) Genetically linked C-type lectin-related ligands for the NKRP1 family of natural killer cell receptors. Nat Immunol. 4:801-7.

Ota N, Brett TJ, Murphy TL, Fremont DH, Murphy KM. (2004) N-domain-dependent nonphosphorylated STAT4 dimers required for cytokine-driven activation. Nat Immunol. 5:208-15.

Miley MJ, Messaoudi I, Metzner BM, Wu Y, Nikolich-Zugich J, Fremont DH. (2004) Structural basis for the restoration of TCR recognition of an MHC allelic variant by peptide secondary anchor substitution. J Exp Med. 200:1445-54.

Nelson CA, Pekosz A, Lee CA, Diamond MS, Fremont DH. (2005) Structure and intracellular targeting of the SARS-coronavirus Orf7a accessory protein. Structure. 13:75-85.

Oliphant T, Engle M, Nybakken GE, Doane C, Johnson S, Huang L, Gorlatov S, Mehlhop E, Marri A, Chung KM, Ebel GD, Kramer LD, Fremont DH, Diamond MS. (2005) Development of a humanized monoclonal antibody with therapeutic potential against West Nile virus. Nat Med. 11:522-30.

Hansen TH, Lybarger L, Yu L, Mitaksov V, Fremont DH. (2005) Recognition of open conformers of classical MHC by chaperones and monoclonal antibodies. Immunol Rev. 207:100-11.

Nybakken GE, Oliphant T, Johnson S, Burke S, Diamond MS, Fremont DH. (2005) Structural basis of West Nile virus neutralization by a therapeutic antibody. Nature. 437:764-9.

Chung KM, Nybakken GE, Thompson BS, Engle MJ, Marri A, Fremont DH, Diamond MS. (2006) Antibodies against West Nile Virus nonstructural protein NS1 prevent lethal infection through Fc gamma receptor-dependent and -independent mechanisms. J Virol. 80:1340-51.

Brett TJ, Legendre-Guillemin V, McPherson PS, Fremont DH. (2006) Structural definition of the F-actin-binding THATCH domain from HIP1R. Nat Struct Mol Biol. 13:121-30.

Kaufmann B, Nybakken GE, Chipman PR, Zhang W, Diamond MS, Fremont DH, Kuhn RJ, Rossmann MG. (2006) West Nile virus in complex with the Fab fragment of a neutralizing monoclonal antibody. Proc Natl Acad Sci U S A. 103:12400-4.

Nybakken GE, Nelson CA, Chen BR, Diamond MS, Fremont DH. (2006) Crystal structure of the West Nile virus envelope glycoprotein. J Virol. 80:11467-74.

Truscott SM, Lybarger L, Martinko JM, Mitaksov VE, Kranz DM, Connolly JM, Fremont DH, Hansen TH. (2007) Disulfide bond engineering to trap peptides in the MHC class I binding groove. J Immunol. 178:6280-9.