Jason C Hurlbert | PubMed Health

PubMed2002

Crystallization of HLA-DR4 fused to an immunodominant collagen II peptide implicated in rheumatoid arthritis.

Major histocompatibility complex (MHC) class II molecules are key players in antigen-specific CD4(+) T cell stimulation, despite their lack of discrimination between "self" and foreign antigens. The susceptibility of many individuals to autoimmune diseases is directly attributed to this lack of specificity, as well as to the expression of subclasses of MHC class II molecules. Increased susceptibility to the autoimmune disease rheumatoid arthritis (RA) has been attributed to the expression of the MHC class II alleles HLA-DR1 and HLA-DR4. To define the structural requirements of the HLA-DR4 protein in the autoimmune response of RA, we have crystallized HLA-DR4 with the immunodominant peptide from human collagen II, covalently linked to the N-terminus of the beta-chain [HLA-DR4/hCII(257-273)]. Crystallization time, crystal size, and reproducibility were greatly improved by macroseeding into microdialysis buttons.

Arthritis, Rheumatoid↗

PubMed2002

Differences in the solution structures of the parallel beta-helical pectate lyases as determined by limited proteolysis.

The pectate lyase family of proteins has been shown to fold into a novel domain motif, the right-handed parallel beta-helix. As a means of gaining insight to the solution structure of the pectate lyases, the enzymes were subjected to limited proteolytic digestion by the endoproteases AspN, GluC and trypsin. The effects of proteolytic cleavage on enzymatic activity were determined, and the early products of proteolysis were identified by capillary electrophoresis, MALDI-TOF mass spectrometry and HPLC. A single peptide bond between Lys158 and Asp159 in pectate lyase B (PLb) was cleaved by both AspN and trypsin, with no detectable hydrolysis of PLb by GluC. Pectate lyase E (PLe) was hydrolyzed by trypsin between Lys164 and Asp165, a bond on an analogous loop structure found to be susceptible to proteolytic attack in PLb. AspN and GluC preferentially hydrolyzed peptide bonds (at Asp127 and Glu124, respectively) on another loop extending from the central beta-helical core of PLe. A single beta-strand of the central cylinder of the pectate lyase C (PLc) molecule was susceptible to all three proteases used. These data demonstrate that the most susceptible peptide bonds to proteolytic scission within the native enzymes lie on or near one of the three parallel beta-sheets that compose the core domain motif Despite the proximity of the proteolytic cleavages to the catalytic sites of the enzymes, significant retention of lyase activity was observed after partial proteolysis, indicating preservation of functional tertiary structure in the proteolytic products.

Binding Sites↗