All allosteric proteins are design as oligomeric assemblies of subunits (or discrete domains) with spatially distinct ligand binding sites. A comprehensive set of structural and functional data on the individual ligation states of an allosteric protein is a prerequisite to developing an accurate stereochemical model that fully describes its mechanism of action. In the case of hemoglobin, Ackers and co-workers have taken at large step toward this ambitious goal by determining for the first time a complete set of thermodynamic relationships between all of hemoglobin's ten ligation states. These data have revealed a """"""""molecular code mechanism"""""""" which states that binding of the first ligand to hemoglobin (a tetramer composed of two alphabeta dimers) increases the ligand affinity of the adjacent unliganded subunit on the liganded alphabeta dimer, but it does not increase (or increases to much smaller degree depending on the type of ligand) the affinity of the two unliganded subunits on the opposite alphabeta dimer. For this to be true, a dimer's alpha and beta subunits must be linked to a common region of quaternary constraint. We have formulated an initial stereochemical model that identifies such a region, and in collaboration with the other members of this Program Project, are now in a position to test the model's validity and, hopefully, refine the model. The model describes how specific residues propagate ligation-induced changes in heme structure to subunit interfaces and thereby disrupt the """"""""hinge"""""""" region of an alphabeta-alphabeta interface. Specifically, the ligand- induced transition (between the deoxy, or T quaternary structure, and other T-like structures) includes large tertiary structure changes to the alpha F helix and COOH terminus, a 2 degrees bending of each alphabeta dimer, and a approximately 5.5 degrees rotation of one alphabeta dimer relative to the other alphabeta dimer. We will test and extend the proposed stereochemical model by 1) determining the structures of specific site-directed mutant hemoglobins that are predicted to alter the ligand-induced tertiary structure changes to the alpha F helix and a COOH terminus, 2) carrying out a mutational screen to uncover the stereochemical mechanism of ligand- induced alphabeta dimer bending, and 3) determine the energetically accessible structures of hemoglobin's intermediate ligation species with a strategy that we have successfully used to determine the wide range for quaternary structures of fully liganded hemoglobin. This research should result in new insights into hemoglobin's molecular mechanism that may provide for better understanding and treatment of hemoglobinopathies and for the design of improved blood substrates.
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