Crystallographic Study Of Hemolin, An Insect Immune Protein Related To Immunoglobulins. Xiao-Dong Su & Pamela Bjorkman, Caltech 156-29, Pasadena, CA 91125
Insects have an efficient defense system against infections. Numerous antibacterial immune proteins induced by bacterial infection have been identified in the giant silk moth, Hyalophora cecropia. One such protein is hemolin, a 48 kDa protein present in low but significant amounts in the hemolymph of cecropia pupae. The concentration of hemolin is increased by a factor of approximately 20 after injection of dead or live bacteria into the pupae. Hemolin binds to an unknown protein component of 125 kDa of the bacterial surface and to another component of hemolymph (Sun et al., 1990). A cDNA clone of hemolin was isolated (Sun et al., 1990), and analysis of the 413 residue sequence revealed that the protein contains four internal repeats characteristic of immunoglobulin-like domains. We are interested in the structural characterization of this protein in order to investigate its immune properties and its relationship to vertebrate members of the immunoglobulin gene superfamily.
A soluble form of hemolin was expressed using a recombinant baculovirus system (L.N. Gastinel, D.E. Vaughn, I. Faye and P.J. Bjorkman, unpublished results). Yields of protein from infected cell supernatants vary between 10-150 mg/liter. Using a cation exchange column as a major purification step, we purified soluble hemolin and crystallized the protein. Hemolin crystals grow in space group P212121 (a = 87.3 A, b = 90.3 A, c = 142.3 A) and diffract to slightly beyond 3.0 A resolution at room temperature. Significant decay of the crystals precludes collection of high quality data at higher resolution (the resolution limit decreases to ~4-5 A after 15 hours exposure using an in-house x-ray source). Conditions for flash cooling the crystals have been obtained, and data can be collected from cryopreserved crystals to 2.5 A resolution at a synchrotron source. The crystal structure was solved by the MIR method using platinum and pressurized xenon derivatives. The structure revealed that each of the four domains has a V-like folding topology. The domains are arranged so that the first two are anti-parallel to the last two in an arrangement resembling a closed horseshoe.
Reference:
Sun, S.-C., Lindstrom, I., Boman, H.G., Faye, I., and Schmidt, O. (1990) Science 250, 1729-1732