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

Abstract

The high-resolution X-ray structures of the deoxy forms of four recombinant hemoglobins in which Trp37(C3)beta is replaced with Tyr (betaW37Y), Ala (betaW37A), Glu (betaW37E), or Gly (betaW37G) 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)beta results in (1) a change in quaternary structure at the alpha1beta2 interface, (2) alpha subunit tertiary structure changes that are centered at Asp94(G1)alpha-Pro95(G2)alpha, (3) beta subunit tertiary structure changes that are located between residues Asp99(G1)beta and Asn102(G4)beta, (4) increased mobility of the alpha subunit COOH-terminal dipeptide, and (5) shortening of the Fe-Nepsilon2His(F8) bond in the alpha and beta subunits of the betaW37G and betaW37E mutants. In each case, the magnitude of the change in a particular structural parameter increases in the order betaW37Y < betaW37A < betaW37E approximately betaW37G, which corresponds closely to the degree of functional disruption documented in the preceding papers.

Crystal Structures of Beryllium Fluoride-free and Beryllium Fluoride-bound CheY in Complex with the Conserved C-terminal Peptide of CheZ Reveal Dual Binding Modes Specific to CheY Conformation

“…reflects the magnitude of a main chain displacement vector that is a measure of individual atomic displacement vectors with summations carried out over a short peptide of three contiguous residues in the protein. 41 Concerted structural changes result in constructive addition of these vectors while uncorrelated differences lower the magnitude of these vectors. − species is fully occupied.…”

“…Superpositions of atomic models were accomplished by a previously described least-squares based iterative superposition procedure 41 that is similar to the "sievefit" program 66 to define a "static core" of main chain atoms for CheY molecules, achieved by omitting residues with atomic displacements greater than twice the r.m.s.d. value.…”

Interaction of CheY with the C-Terminal Peptide of CheZ

“…Structural analyses. Least-square superpositions of atomic models and estimates of differences in tertiary structure by ␦ 3 -rmsd analyses were performed as previously described (13,15). Peptide-CheY contact analyses were performed using the software programs CNS 1.1 (6) and iMOLTALK version 2.0 (10) and the protein-protein interaction server (http://www.biochem.ucl.ac.uk/bsm/PP /server/).…”

Cys-93-ββ-Succinimidophenyl Polyethylene Glycol 2000 Hemoglobin A

“…Stripped Bis-Mal-PEG2000 HbA was then concentrated to ϳ100 mg/ml and was frozen and stored in liquid nitrogen until being used for crystallization. Crystals of the deoxy-Bis-Mal-PEG2000 HbA were grown at room temperature according to a batch method with the addition of a seeding step as described previously by Kavanaugh et al (17). Specifically, the crystallization conditions were 10 mM potassium phosphate at pH 7.0, 100 mM potassium chloride, 3 mM sodium dithionite, and 10ϳ12% PEG 6000.…”