W0467

The Structure of a Proteolytically Modified Immunoglobulin Light-chain Dimer, Bence-Jones KWR Protein. Debora L. Makino, Steven B. Larson, Agnes Henschen-Edman, Alexander McPherson. The Univ. of California, Irvine, CA 92697.

Approximately 40 years ago, large amounts of immunoglobulin light-chain dimers were collected from the urine of an Australian patient (KWR) afflicted with Multiple Myeloma disease. These light-chains, stored lyophilized during this time, and named Bence-Jones KWR, are covalently linked through a disulfide bridge. Bence-Jones KWR crystallizes at room temperature between pH 8.0 and 8.5 with 1.5M ammonium sulfate as precipitant. Within the first few weeks, tetragonal crystals with marginal X-ray diffraction properties, and frequently disordered, were obtained. After eight to ten months hexagonal prismatic crystals of a proteolytically modified form of the protein were found. Those were well ordered, and diffracted to 2.8Å using synchrotron radiation (Lawrence Berkeley National Laboratory) at cryogenic temperature. Data analysis indicated the hexagonal prismatic crystals to belong to space group P3₁21 with cell edges of a=b=153.52Å and c=93.95Å. There are two Bence-Jones dimers per asymmetric unit. The molecular replacement method was used to solve the structure. Elbow angles of 131.6^o for one molecule and 134.5^o for the second molecule were found optimal. The two Bence-Jones dimers in the asymmetric unit stack parallel to the c-axis. After several rounds of refinement, a Fourier map was generated using RESOLVE. Substantial regions of the constant domain in each dimer were weak or absent in the electron density map. The initial R and R-free values, calculated using a resolution range of 15.0-2.9 Å and sigma cut-off of 4.0, were 27.0% and 39.7%, respectively. The residuals decreased to R=20.7% and R-free=34.7% after occupancy and B-factor refinements. These further identified the parts of the constant domains that were either very disordered or absent. It has been noted that crystals grown only after several months of incubation are often constituted of products obtained from protein degradation processes. Therefore, it is likely that proteolysis ultimately allowed Bence-Jones dimers to arrange themselves in a more stable manner, transitioning from poorly diffracting tetragonal crystals to trigonal crystals having improved diffraction properties.