The successful establishment of most microbial pathogens depends upon their ability to multiply within the host environment. Iron is essential for growth and a mechanism for obtaining this nutrient from the host environment is an essential prerequisite for infection. This research proposal will investigate the iron-acquisition mechanism of the related pathogens N. gonorrhoeae and N. meningitidis and H. influenza by determining the X-ray crystal structure of the pathogens' 37 kD ferric binding protein (FBP). Neisseria gonorrhoeae and Neisseria meningitidis are closely related pathogens which remain wide-spread health problems. In the United States alone the average number of gonococcal infections reported annually between 1983 and 1990 was 800,000. FBP is believed to be a central participant in the uptake of iron from the host environment, without which the bacterium cannot survive. Obtaining the structure of FBP may ultimately lead to the rational design of therapeutic agents active against these and other pathogens which use FBP to obtain growth essential iron. Increasing evidence indicates that FBP shares a common fold and Fe-binding mechanism with lactoferrin, transferrin and bacterial periplasmic binding proteins. Studies of FBP can be directly compared with these proteins, all of which have crystallographic structures available.
Specific Aims i nclude: 1) Structure of FBP from N. gonorrhoeae. 2) Structure of FBP from N. meningitidis. 3) Structure of FBP from H. influenza. 4) Mechanism of reversible iron binding. 5) Similarity to other known structures of bacterial periplasmic proteins. 6) Similarity to lactoferrin and transferrin. 7) Structures of functional mutations. 8) Design of iron- transport inhibitors. We have successfully initiated crystallographic studies of FBP. Diffraction quality crystals that diffract to 2.5 angstroms have been grown and characterized from N. gonorrhoeae (space group P4/1, a=b=110, c=200, 6/a.u.) and 1.6 angstroms resolution crystals for H. influenza FBP (space group P2/1/2/1 2, a=132.9, b=52.3, c=41.3 angstroms, 1/a.u.). A molecular replacement study using human lactoferrin, transferrin, and sulfate binding protein, has been undertaken in parallel with a search for heavy atom derivatives. Heavy atom compounds that produce significant changes in the diffraction pattern have been found and promise to yield isomorphous heavy atom derivatives. A tentative molecular replacement solution for H. influenza FBP has been determined.
