PDBsum entry 2grh

Oxygen storage/transport

PDB id: 2grh

Contents

Protein chains

145 a.a.

Ligands

HEM-CMO ×2

Waters ×241

References listed in PDB file

Key reference

Title

Allosteric action in real time: time-Resolved crystallographic studies of a cooperative dimeric hemoglobin.

Authors

J.E.Knapp, R.Pahl, V.Srajer, W.E.Royer.

Ref.

Proc Natl Acad Sci U S A, 2006, 103, 7649-7654. [DOI no: 10.1073/pnas.0509411103]

PubMed id

16684887

Abstract

Protein allostery provides mechanisms for regulation of biological function at the molecular level. We present here an investigation of global, ligand-induced allosteric transition in a protein by time-resolved x-ray diffraction. The study provides a view of structural changes in single crystals of Scapharca dimeric hemoglobin as they proceed in real time, from 5 ns to 80 micros after ligand photodissociation. A tertiary intermediate structure forms rapidly (<5 ns) as the protein responds to the presence of an unliganded heme within each R-state protein subunit, with key structural changes observed in the heme groups, neighboring residues, and interface water molecules. This intermediate lays a foundation for the concerted tertiary and quaternary structural changes that occur on a microsecond time scale and are associated with the transition to a low-affinity T-state structure. Reversal of these changes shows a considerable lag as a T-like structure persists well after ligand rebinding, suggesting a slow T-to-R transition.

Figure 1.
Fig. 1. Difference Fourier map HbI* (photoproduct) minus HbI-CO at time delays of 5 ns and 60 µs is shown for the entire dimer (A); CD, E, F, and heme regions of subunit A (B); and the Phe F4 of subunit A (C). Fig. 5, which is published as supporting information on the PNAS web site, provides equivalent views for subunit B [the figure was produced with PYMOL (38)]. (A) A ribbon diagram of the HbI-CO dimer (gray) with side chains for His F8 (cyan), Phe F4 (yellow), and key interface water molecules (small cyan spheres) are shown along with the difference Fourier map. The maps are contoured at ±3.5 (blue and red, respectively) for both A and B. Note the concentration of difference density mainly in the immediate heme region and along the F helix at 5 ns. The density distributes toward the interface by 60 µs. Arrows (in cyan) point out the position of two key R-state water molecules in the 5-ns map that show clear negative density as they rapidly respond to the loss of ligand. Removal of these two water molecules is required for the subsequent movement of the heme groups toward the subunit interface. (B) An -carbon trace (gray) for the CD region and E and F helices along with the heme group (salmon), side chains for CD1, CD3, E7, F7, and F8, (cyan) and F4 (yellow) are shown. The photolysis signal at the bound CO position (labeled CO) is highly significant at 5 ns: -14 and -17 for the A and B subunits, respectively. The strong positive feature indicating the iron displacement (labeled Fe) is at +12 and +14 , for the A and B subunits, respectively. Note the extensive structural rearrangement involving the heme group at 5 ns, along with that of the CD region and F helix. (C) Difference electron density is shown for the region around F4 Phe at ±2.5 in blue and red, respectively, along with the atomic model for the liganded (salmon) and unliganded (cyan) structures. Phe F4 undergoes the largest ligand-linked side-chain rearrangement during the R-to-T transition. As the density maps show, this movement has not occurred at 5 ns but is completed by 60 µs after the ligand release.

Figure 3.
Fig. 3. Time-dependent change in the heme iron position. This shift is broken down into components that are perpendicular to the heme plane (blue symbols) and components that are parallel to the heme plane (red symbols). The change is measured as the difference in position relative to the starting R-state position (open diamonds) and ending T-state position (filled diamonds). Flash photolysis causes the heme iron to move 0.4 Å perpendicular to the heme plane while shifting by only 0.15 Å parallel to the plane, away from its starting R-state position and toward the T-state position. The heme iron stays in this vicinity during the nanosecond time domain and moves toward its ending T-state position in the microsecond time domain, synchronously with other structural changes involved in the allosteric transition shown in Fig. 2.

Secondary reference #1

Title

High-Resolution crystallographic analysis of a co-Operative dimeric hemoglobin.

Author

W.E.Royer.

Ref.

J Mol Biol, 1994, 235, 657-681.

PubMed id

8289287