ABSTRACT
A collaborative project between two Structural Proteomics In Europe (SPINE) partner laboratories, York and Oxford, aimed at high-throughput (HTP) structure determination of proteins from Bacillus anthracis, the aetiological agent of anthrax and a biomedically important target, is described. Based upon a target-selection strategy combining ;low-hanging fruit' and more challenging targets, this work has contributed to the body of knowledge of B. anthracis, established and developed HTP cloning and expression technologies and tested HTP pipelines. Both centres developed ligation-independent cloning (LIC) and expression systems, employing custom LIC-PCR, Gateway and In-Fusion technologies, used in combination with parallel protein purification and robotic nanolitre crystallization screening. Overall, 42 structures have been solved by X-ray crystallography, plus two by NMR through collaboration between York and the SPINE partner in Utrecht. Three biologically important protein structures, BA4899, BA1655 and BA3998, involved in tRNA modification, sporulation control and carbohydrate metabolism, respectively, are highlighted. Target analysis by biophysical clustering based on pI and hydropathy has provided useful information for future target-selection strategies. The technological developments and lessons learned from this project are discussed. The success rate of protein expression and structure solution is at least in keeping with that achieved in structural genomics programs.
Subject(s)
Bacillus anthracis/genetics , Proteomics/methods , Bacillus cereus/genetics , Bacterial Proteins , Cloning, Molecular , Computational Biology , Crystallization , Crystallography, X-Ray , DNA, Bacterial/genetics , Escherichia coli/genetics , Escherichia coli/metabolism , Genetic Vectors , Magnetic Resonance Spectroscopy , RNA, Transfer/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Robotics , Spores, Bacterial/genetics , SulfurtransferasesABSTRACT
Human natural killer cells and a subset of T cells express a repertoire of killer cell immunoglobulin receptors (KIRs) that recognize major histocompatibility complex (MHC) class I molecules. KIRs and T cell receptors (TCRs) bind in a peptide-dependent manner to overlapping regions of peptide-MHC class I complexes. KIRs with two immunoglobulin domains (KIR2Ds) recognize distinct subsets of HLA-C alleles. Here we use surface plasmon resonance to study the binding of soluble forms of KIR2DL1 and KIR2DL3 to several peptide-HLA-Cw7 complexes. KIR2DL3 bound to the HLA-Cw7 allele presenting the peptide RYRPGTVAL with a 1:1 stoichiometry and an affinity (K(d) approximately 7 microM at 25 degrees C) within the range of values measured for other cell-cell recognition molecules, including the TCR. Although KIR2DL1 is reported not to recognize the HLA-Cw7 allele in functional assays, it bound RYRPGTVAL/HLA-Cw7, albeit with a 10-20-fold lower affinity. TCR/peptide-MHC interactions are characterized by comparatively slow kinetics and unfavorable entropic changes (Willcox, B. E., Gao, G. F., Wyer, J. R. , Ladbury, J. E., Bell, J. I., Jakobsen, B. K., and van der Merwe, P. A. (1999) Immunity 10, 357-365), suggesting that binding is accompanied by conformational adjustments. In contrast, we show that KIR2DL3 binds RYRPGTVAL/HLA-Cw7 with fast kinetics and a favorable binding entropy, consistent with rigid body association. These results indicate that KIR/peptide-MHC class I interactions have properties typical of other cell-cell recognition molecules, and they highlight the unusual nature of TCR/peptide-MHC recognition.
Subject(s)
Histocompatibility Antigens Class I/metabolism , Killer Cells, Natural/immunology , Oligopeptides/metabolism , Receptors, Antigen, T-Cell/metabolism , Receptors, Immunologic/metabolism , Amino Acid Sequence , Base Sequence , DNA Primers , Humans , Kinetics , Protein Binding , Receptors, Antigen, T-Cell/chemistry , Receptors, Immunologic/chemistry , Receptors, KIR , Receptors, KIR2DL1 , Receptors, KIR2DL3 , Surface Plasmon Resonance , ThermodynamicsABSTRACT
The human leukocyte antigen class I allele HLA-B27 is a major histocompatibility complex (MHC) antigen that is strongly associated with the spondyloarthritic group of human rheumatic diseases, the most common of which is ankylosing spondylitis. Although the mechanism underlying this disease association remains unknown, numerous theories have been proposed. Much more is known of the natural role of HLA-B27 in binding and presenting antigenic peptides to T cells. The 'arthritogenic peptide hypothesis' suggests that the role of HLA-B27 in disease relates to its specificity for binding certain peptides. Recently, it has also been shown that HLA-B27 has an unusual cell biology and can adopt a novel homodimeric structure. In this review, a molecular model of the HLA-B27 homodimer is presented and the possible pathogenic significance of such a structure is discussed.
