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Quaternary Structure Transitions of Human Hemoglobin

Quaternary Structure Transitions of Human Hemoglobin: An Atomic-Level View of the Functional Intermediate States


Human hemoglobin (HbA) is one of the prototypal systems used to investigate structure–function relationships in proteins. Indeed, HbA has been used to develop the basic concepts of protein allostery, although the atomic-level mechanism underlying the HbA functionality is still highly debated. This is due to the fact that most of the three-dimensional structural information collected over the decades refers to the endpoints of HbA functional transition with little data available for the intermediate states. Here, they report molecular dynamics (MD) simulations by focusing on the relevance of the intermediate states of the protein functional transition unraveled by the crystallographic studies carried out on vertebrate Hbs. Fully atomistic simulations of the HbA T-state indicate that the protein undergoes a spontaneous transition toward the R-state. The inspection of the trajectory structures indicates that the protein significantly populates the intermediate HL-(C) state previously unraveled by crystallography. In the structural transition, it also assumes the intermediate states crystallographically detected in Antarctic fish Hbs. This finding suggests that HbA and Antarctic fish Hbs, in addition to the endpoints of the transitions, also share a similar deoxygenation pathway despite a distace of hundreds of millions of years in the evolution scale. Finally, using the essential dynamic sampling methodology, they gained some insights into the reverse R to T transition that is not spontaneously observed in classic MD simulations.


Human hemoglobin (HbA) deserves a special position in structural biology since it has been the model used to develop the fundaments of protein crystallography.Moreover, myoglobin has been the first protein whose structure has been determined at an atomic level.Even more significantly, HbA has been, and it still is, the prototypal system used to investigate structure–function relationships in proteins. Indeed, the structural characterization of the different functional states of HbA has provided fundamental insights for developing the basic concepts of protein allostery, although the atomic-level mechanism underlying the HbA functionality is still highly debated. Due to the seminal work of Perutz, the structural features of the endpoints of Hb structural transition have been elucidated for more than half a century.These pioneering studies have unveiled that the ligand-bound forms are associated with a rather flexible structure denoted as the R (relaxed) state. On the other hand, the unliganded HbA structure is characterized by a rather rigid, tense, T-state.


Stepwise R–T transition of HbTn as highlighted by crystallographic investigations carried out on the protein. Superimposition of the α1β1 dimer of different HbTn structural states: deoxygenated T-state, TnA, TnB, TnH, and the canonical R-state. Magnifications of the helices A (residue 3–18) and G (residues 99–117) of the β2 subunit are shown to highlight the transition from the T- to the R-state.

Since then, hundreds of HbA structures have been reported in the Protein Data Bank (PDB).These studies have significantly improved the structural sampling of the HbA bound states by showing that the liganded HbA may manifest, in addition to the R-state, in a variety of other relaxed states (R2, R3, RR2) that fall outside the T–R pathway. These findings have initiated an intense debate on the real extension of the Hb functional transition that may go well beyond the T–R pathway and include these other relaxed states. On the other hand, the atomic-level characterizations of intermediate R–T states have proven much more difficult as HbA structures exhibiting intermediate R–T features at the quaternary structure level have been rarely described. The first example has been reported by Schumacher et al. who generated intermediate R–T states through the crosslinking of the β chains of the HbA tetramer. Interesting information on HbA function–structure relationships were also provided by crystallographic structures showing remarkable tertiary structure variations, although confined either to the R- or the T-state. More recently, a unique crystal form of HbA characterized by the presence at the crystalline state of three different quaternary structures has been reported. One of these states, denoted as HL-(C), presents intriguing intermediate features, although it is much closer to the R-state rather than to the T-state.In this scenario, although insightful information on the R–T transition has been obtained using other experimental and computational techniques, the lack of detailed experimental information on intermediate T–R states is one of the most important factors that has so far prevented a full understanding of the HbA functional transition.


(a) RMSD values (computed on the Cα atoms) of the T0 trajectory structures versus the starting T model, the R-state, and the intermediate HL-(C) state . (b) RMSD values computed against the off-pathway structures: R2, RR2 , and R3 . (c) RMSD values computed against the intermediate states identified for HbTn: TnA , TnB , and TnH (C). (d) RMSD values computed against the T-state (black) and the R-state (red) of HbA and against TnH (cyan) in the time interval of 60–100 ns. RMSD values refer to the productive run without the equilibration steps producing the initial drift.


The situation is significantly different for the tetrameric Hbs isolated from Antarctic fish (AntHbs) that share with HbA several functional/structural features despite the fact that AntHbs operates in organisms living in rather extreme conditions. The intrinsic flexibility of AntHbs when studied at temperatures significantly higher than the physiological ones has allowed the visualization of states that exhibit unusual structural properties and/or peculiar oxidation states. Specifically, the structures of Hbs extracted from Trematomus newnesi and Trematomus bernacchii belonging to the Nototheniidae family and from the sub-Antarctic fish Eleginops maclovinus have shown a variety of oxidation (hemichrome, aquomet, pentacoordinated oxidized, etc.) often associated with noncanonical tertiary and quaternary organizations.Altogether, these structures have provided some interesting insights into the possible structural features of the intermediate states Interestingly, for the T. newnesi Hb (HbTn), three distinct intermediate structures (tetramer A, TnA; tetramer B, TnB; and tetramer H, TnH), in addition to the canonical T and R states, have been reported. For HbTn, the overall rotation of one αβ dimer when the other is superimposed is about 11° (15° in the case of HbA). Therefore, taking into account the presence of the three intermediate states, the overall pathway may be dissected into four subtransitions that are separated by approximately 3° rotation of one αβ dimer with respect to the other. In this scenario, to gain further insights into this long-standing issue, they here performed extensive fully atomistic molecular dynamics (MD) simulations on HbA by also checking the relevance of the intermediate species identified for AntHbs to the human counterpart.



Projections of the T0 trajectory structures (a, c) and of the T–R (green) and R–T (orange) trajectory structures generated by the EDS analysis (b, d) in the space defined by some structural probes that are characteristic of the various HbA states. The points corresponding to the structures detected in the pretransition (<75 ns), transition (75–83 ns), and post-transition (>83 ns) time interval of the T0 simulation are colored in gray, cyan, and red, respectively. The representative crystallographic structures of HbA (black circles) and HbTn (black triangles) are also reported. TnB is not reported in panels (a) and (b) as the C-terminal residue of the β-chains (His146) is missing.


Quaternary Structure Transitions of Human Hemoglobin: An Atomic-Level View of the Functional Intermediate States Nicole Balasco, Josephine Alba, Marco D’Abramo, and Luigi Vitagliano Journal of Chemical Information and Modeling 2021 61 (8), 3988-3999 DOI: 10.1021/acs.jcim.1c00315