Unsuspected pathway of the allosteric transition in hemoglobin

Abstract

Large conformational transitions play an essential role in the function of many proteins, but experiments do not provide the atomic details of the path followed in going from one end structure to the other. For the hemoglobin tetramer, the transition path between the unliganded (T) and tetraoxygenated (R) structures is not known, which limits our understanding of the cooperative mechanism in this classic allosteric system, where both tertiary and quaternary changes are involved. The conjugate peak refinement algorithm is used to compute an unbiased minimum energy path at atomic detail between the two end states. Although the results confirm some of the proposals of Perutz [Perutz MF (1970) Stereochemistry of cooperative effects in haemoglobin. Nature 228:726-734], the subunit motions do not follow the textbook description of a simple rotation of one αβ-dimer relative to the other. Instead, the path consists of two sequential quaternary rotations, each involving different subdomains and axes. The quaternary transitions are preceded and followed by phases of tertiary structural changes. The results explain the recent photodissociation measurements, which suggest that the quaternary transition has a fast (2 μs) as well as a slow (20 μs) component and provide a testable model for single molecule FRET experiments.conformational change | cooperativity | domain motion | protein hinges M any proteins undergo large conformational transitions that are essential for their functions (1-3). The transitions can occur in monomers such as the myosin molecular motor (4-6), in multisubunit complexes such as the chaperone GroEL (7), and in systems such as the flagellar motor of bacteria, which is composed of several hundred proteins (8). Probably the most studied example is the T (tense) to R (relaxed) transition in the vertebrate hemoglobin tetramer (Hb) (9-11), for which the conformational transition increases the efficiency of oxygen transport. Hemoglobin, which is composed of two identical α-and two identical β-subunits, is the paradigm for the development of models of cooperativity and allosteric regulation in proteins (9-13). Phenomenologically, the positive cooperativity involves the increase in the affinity for oxygen of unliganded subunits upon the successive binding of oxygen to other subunits. This is achieved by structural changes within subunits (tertiary changes) and between subunits (quaternary changes). Based on the superposition of the crystallographic structures for the deoxy unliganded T state and the fully liganded R state of the protein, the quaternary T → R transition has been described as involving primarily an approximately 15°rotation of one "dimer" (α 1 β 1 ) relative to the other (α 2 β 2 ) around a virtual axis (Fig. 1A) (9, 14). Together with a small relative translation of the αβ-dimers, this reduces the central cavity between the dimers (a channel along the C2 axis of symmetry), where the heterotropic effector 2,3-bisphosphoglycerate is bound in the T state. An important aspect of these structural ch...

A gating mechanism of pentameric ligand-gated ion channels

“…This quaternary change is followed by primarily tertiary relaxation to reach the fully developed R state. Significant tertiary changes after the quaternary transition are in accord with what has been predicted for hemoglobin (27). Once the twisting is completed, agonist unbinding (here L-glutamate) from the orthosteric site results in a large reorientation or tilting of the extracellular β-sandwiches in the outward direction, which drags the β 1 -β 2 loop into the up position.…”

The Monod-Wyman-Changeux allosteric model accounts for the quaternary transition dynamics in wild type and a recombinant mutant human hemoglobin

“…2 with α ¼ − logðsÞ∕ logðcÞ; the values reported in Table 1 yield α ¼ 0.33 and 0.81 for HbA and HbYQ, respectively. In the case of HbA, the difference between our estimate and the value of α ¼ 0.17 determined by TR-OA data (23) may be traced to the proposed stepwise character of the R-T allosteric transition (10,14,26). Following this idea, Cammarata et al (13) proposed that TR-WAXS essentially monitors a first step of the R-T transition of HbA (namely the αβ dimers rotation and translation), while TR-OA monitors a second slower step associated with a more localized reorganization around the active site; accordingly, the transition states explored by TR-WAXS and TR-OA spectroscopy are not the same (Fig.…”

“…Spectroscopic and crystallographic studies have shown that the αβ dimers rotation is associated with a rearrangement around Trp37(C3)β, located in the so-called "hinge" region. In discussing their conjugate peak refinement calculations, Fischer et al (14) pointed out that the transition of His97(FG4)β gliding over Thr41(C6)α at the switch interface occurred in the main Q 2 phase of the allosteric conformational change. How do the distal side mutations introduced in HbYQ affect the α 1 β 2 interface, where the quaternary transition is associated to the largest changes?…”

“…This estimate implies that dimer rotationthe largest structural change in the R-T allosteric transition-is much faster than 20 μs, the time constant detected by optical spectroscopy (2,3). Quite recently Fischer et al (14) published a computational analysis of Hb dynamics which revealed distinct tertiary and quaternary conformational events; in particular, they reported two sequential quaternary changes (called Q 1 and Q 2 ), which they assumed to be consistent with the time constants at 2 μs (for Q 2 ) and 20 μs (for Q 1 ) reported by Balakrishnan et al (10).…”

Using Markov state models to study self-assembly

“…Many powerful enhanced sampling techniques have been developed to efficiently harvest computational trajectories that include barrier crossings or other rare events (e.g., Refs. [19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34]. However, many of these methods are limited by requiring a priori knowledge of reaction coordinates, one or few pathways to completion, or only several metastable minima.…”