The Role of Molecular Hinges in the Structure and Function of Iron Regulatory Protein I

Iron Regulatory Protein 1 (IRP1) is a bi-functional protein that can act either as a post transcriptional regulator of iron homeostasis genes or as an enzyme (cytosolic aconitase). Crystal structures of IRP1 show that two different conformations are required for RNA binding and enzyme function. Additionally, the crystal structures of IRP1 show that portions of the beta4-alpha4 molecular hinge transition between alpha-helix and beta-turn secondary structures. This secondary structure change corresponds to global conformation changes of IRP1. Elucidations of crystal structures of IRP1 in the cytosolic aconitase and RNA binding conformations greatly expanded knowledge regarding the mechanisms associated with IRP1 structural and functional plasticity. However, because the crystal structure of apo-IRP1 is unavailable and little is known about the structural components involved in conformation change, a comprehensive understanding of the structural and functional plasticity of IRP1 has been precluded. Therefore, the aims of this study were to determine the structure of apo-IRP1 and to determine if the beta4-alpha4 molecular hinge is important in the structure and function of IRP1. The initial hypothesis was that stabilization of the helical structure of the beta4-alpha4 molecular hinge would lock the protein in the closed conformation and prevent RNA binding. This hypothesis was tested using a series of biochemical and biophysical experiments.;Small angle x-ray scattering was utilized to characterize the biophysical properties apo- IRP1 because it provides data about size, shape, molecular structure and distribution of multiple conformational states. The SAXS experiments show that apo-IRP1 is in an open conformation and is predominantly distributed about a radius of gyration of approximately 33.6 +/- .3 A. SAXS experiments also show that mutant forms of apo-IRP1 containing the helix destabilizing mutations D87P and G90A/P92A in the beta4-alpha4 molecular hinge have a decreased radius of gyration compared to the wild type protein. Furthermore, limited proteolysis experiments show that the D87P and the G90A/P92A mutants undergo an alternate cleavage pathway compared to the wild type protein. Together, these results suggest that D87P and G90A/P92A mutations alter the solution conformation of apo-IRP1.;The effects of the mutations on cytosolic aconitase enzyme activity were probed using in vivo and in vitro experiments. Aconitase deficient yeast strains transformed with D87P, G90A and G90A/P92A mutants of cytosolic aconitase had decreased growth rates and were deficient in enzyme activity compared to the wild type protein. More specifically, catalysis of isocitrate was decreased for the G90A, P92A, and G90A/P92A mutants. Interestingly, the D87P mutant was devoid of enzyme activity. The results presented in this study implicate a role for helix destabilizing residues of the beta4-alpha4 hinge in the enzymatic function of cytosolic aconitase.;Analysis of the RNA binding function of the IRP1 hinge mutants revealed that mutants retained their ability to bind RNA with picomolar affinity. Together, these series of experiments shows that hinge mutations neither locked the protein in the closed conformation nor prevented the RNA binding. Rather, the results indicate that the hinge mutations have subtle effects on the structure and enzyme function of IRP1.