High-Resolution Crystal Structures of Human Hemoglobin with Mutations at Tryptophan 37β: Structural Basis for a High-Affinity T-State,

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Abstract
The high-resolution X-ray structures of the deoxy forms of four recombinant hemoglobins in which Trp37(C3)β is replaced with Tyr (βW37Y), Ala (βW37A), Glu (βW37E), or Gly (βW37G) have been refined and analyzed with superposition methods that partition mutation-induced perturbations into quaternary structure changes and tertiary structure changes. In addition, a new cross-validation statistic that is sensitive to local changes in structure (a “local Rfree” parameter) was used as an objective measure of the significance of the tertiary structure changes. No significant mutation-induced changes in tertiary structure are detected at the mutation site itself for any of the four mutants studied. Instead, disruption of the intersubunit contacts associated with Trp37(C3)β results in (1) a change in quaternary structure at the α1β2 interface, (2) α subunit tertiary structure changes that are centered at Asp94(G1)α−Pro95(G2)α, (3) β subunit tertiary structure changes that are located between residues Asp99(G1)β and Asn102(G4)β, (4) increased mobility of the α subunit COOH-terminal dipeptide, and (5) shortening of the Fe−Nε2His(F8) bond in the α and β subunits of the βW37G and βW37E mutants. In each case, the magnitude of the change in a particular structural parameter increases in the order βW37Y < βW37A < βW37E ≈ βW37G, which corresponds closely to the degree of functional disruption documented in the preceding papers.